CIISA CONGRESS 2018

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ACKNOWLEDGEMENTS

Book Editors

António Duarte Luis Lopes da Costa

Scientific Commission Alexandre Leitão António Duarte Carlos Fontes José Prates Luisa Mateus Rui Bessa

Organizing Commitee Alexandre Trindade António Duarte Catarina Costa Idalina Camões Luis Lopes da Costa

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Foreword and welcome message

CIISA, the Centre for Interdisciplinary Research in Animal Health, is celebrating its 25-year jubilee in 2018. Within this commemoration, and together with its management institution, the Faculty of Veterinary Medicine of the University of Lisbon, CIISA launched its Congress 2018 - “Exploring the boundaries of animal, veterinary and

biomedical sciences”.

The Congress Plenary Sessions present internationally renowned keynote speakers, belonging to CIISA’s External Advisory Board and partner and collaborative institutions with which CIISA is currently enrolled in research networks, consortiums and projects. The Congress Oral Communications and Poster Presentation Sessions are addressed to showcase CIISA’s scientists research, with special attention to the Master and PhD students and Post-Docs. In this regard, the Congress attributes two awards, the CIISA Congress PhD Award and the CIISA Congress Master Award, to the best works presented by PhD and Master students, respectively.

The Congress is organized in four thematic sessions - Biomedical Sciences, Animal Science, Biotechnology and Animal Health – which represent main scientific areas of fundamental and applied research at CIISA, addressing multi and interdisciplinary concepts such as One Health or From Farm To Fork.

It is in this rich atmosphere of enthusiastic interdisciplinary sharing of experiences that I welcome you to CIISA’s Congress 2018, expecting that at the end you may take with you an enjoyable and profitable scientific memory.

Luís Lopes da Costa

CIISA Coordinator

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KEYNOTE SPEAKERS

Session 1 – Biomedical Sciences – One Medicine

Castanho MARB, Veiga AS. Hidden functions in the capsid protein of Dengue virus: biological relevance and drug development opportunities. 13

Rodrigues CMP. Biomarkers and targets in metabolic liver disease. 14

Session 2 – Animal Science and Production, Food Technology, Quality and Security – From farm to fork McAllister T. New approaches to the enzymatic enhancement of ruminal fibre digestion. 16 Toldrá F, Gallego M, Reig M, Aristoy M-C, Mora L. Bioactive peptides from animal by-products. 17

Session 3 – Biotechnology and Genetics Gilbert HJ. The prebiotic potential of complex carbohydrates in mammalian gut bacterial communities. 19 Romão MJ. The impact of structural biology in deciphering enzymatic mechanisms. 20 Session 4 – Animal Health – One Health Caniça M. Antibiotic resistance and One Health perspective 22 Hemphill A. Novel drugs and drug targets for the treatment of toxoplasmosis and other diseases caused by cyst-forming apicomplexan parasites. 23

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SESSION 1 Oral Communications - CIISA Post-Docs and Junior Researchers Aguiar SI, Gaspar MM, André A, Martins D, Ferreira M, Carvalheiro M, Neves V, Coelho S, Dias J, Carrapiço B, Gano L, Galamba J, Nobre R, Almeida L, Tavares L, Gonçalves J, Castanho M, Aires-da-Silva F. sdAb-immunoliposomes for brain targeting and drug delivery 25

Murta D, Batista M, Pinto M, Silva E, Trindade A, Duarte A, Lopes-da-Costa L. Dll4 and Jagged1 signaling are relevant players in mammalian spermatogenesis and sperm maturation 26 Catita J, Valença A, Ramos D, Bonet A, Nacher V, Navarro M, Carretero A, Mendes-Jorge L, Ruberte J. The role of Sirt1 overexpression in vascular retinopathies 27 Poster Presentations - CIISA PhD Students P1. Gameiro A, Almeida F, Correia J, Ferreira F. Characterization of Her2 single variants and evaluation of the anti-tumor properties of tyrosine kinase inhibitors in feline mammary carcinoma cell lines. 29

P2. Valença A, Ramos D, Catita J, Bonet A, Nacher V, Navarro M, Carretero A, Mendes-Jorge L, Ruberte J.

TIM2: a new membrane receptor for H-ferritin in the mouse retina. 30 P3. Mendonça L, Trindade A, Carvalho C, Badenes M, Gigante J, Duarte A. Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition, reduction of cancer stem cells and tumour cell intravasation. 31 P4. Diniz P, Batista M, Silva MF, Mateus L, Lopes-da-Costa L, Silva E. JAGGED1 and JAGGED2 participation in bovine sperm acrosome reaction. 32 P5. Amaral A, Fernandes C, Lukasik K, Szóstek-Mioduchowska A, Rebordão MR1, Baclawska A, Morazzo S, Pinto-Bravo P, Skarzynski DJ, Ferreira-Dias G. Sivelestat and noscapine modulate collagen transcription and prostaglandin secretion in mare endometrium. 33 6. Zúquete ST, Santa C, Santos D, Pereira da Fonseca I, Manadas B, Alfaro-Cardoso L. Artificial tick-feeding system as a tool to investigate new approaches in tick control 34

7. Francisco S, Delgado ILS, Felix-Stortz J, Jimenez-Ruis E, Meissner M, Zabala JC, Leitão A, Soares H, Nolasco S. Tubulin Cofactor B: a key player in Toxoplasma gondii host cell invasion. 35 SESSION 2 Oral Communications - CIISA Post-Docs and Junior Researchers

Alves SP, Bessa RJB. New insights on the ruminal biohydrogenation pathways of unsaturated fatty acids. 37

Madeira MS, Alfaia CM, Pires VMR, Lopes PA, Prates JAM. Feeding strategies to improve fat partitioning and meat quality in pigs: from genetic background to dietary protein and amino acids. 38 Fradinho MJ, Fernandes R, Botelho M, Francisco P, Martin-Rosset W, Santos AS, Ferreira LMM, Ferreira-Dias G, Bessa RJB, Caldeira RM. Effect of two feeding levels on growth and development of the Lusitano foal. 39 Poster Presentations - CIISA PhD Students P8. Borges A, Krivorotova T, Sereikaite J, Gama LT, Fernandes MH, Fernandes MJ, Barreto A, Fraqueza MJ. Combined effect of nisin loaded pectin nanoparticles and high hydrostatic pressure on the extension of alheira’s shelf life. 41

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P9. Pereira G, Silva MF, Carneiro C, Duarte A, Mateus L, Lopes da Costa L, Silva E. A putative new Campylobacter species potentially associated with Bovine Genital Campilobacteriosis 42 P10. Silva MF, Pereira G, Duarte A, Mateus L, Lopes da Costa L, Silva E. Evaluation of molecular diagnostic methods for detection of Campylobacter fetus subsp. venerealis in bovine preputial samples. 43 P11. Grilo M, Sousa-Santos C, Tavares L, Robalo JI, Oliveira M. Portuguese nase (Iberochondrostoma lusitanicum) Aeromonas spp. biosurveillance program – filling a gap in endangered freshwater fish species conservation. 44 P12. Pessoa VP, Marti E, Branco Ferreira M, Tilley P. Study of insect bite hypersensivity (IBH) in Lusitano horses in Portugal – preliminary contribution to the development of immunotherapy. 45

13. Dentinho MTP, Paulos K, Francisco A, Belo AT, Jerónimo E, Almeida J, Santos-Silva J, Bessa RJB. The use of Cistus ladanifer condensed tannin extract as additive to improve the soybean meal protein efficiency in lamb diets. 46

14. Tomás A, Palma R, Rebelo MT, Pereira da Fonseca I. Chewing lice of wild birds from Portugal: neglected group of ectoparasites 47

15. Fortunato R, Saavedra M, Nunes R, Nardozi B, Moreira O, Murta D. Use of insect meal for total replacement of soybean cake in laying hens 48 SESSION 3 Oral Communications - CIISA Post-Docs and Junior Researchers Bule P, Alves VD, Israeli-Ruimy V, Smith SP, Gilbert HJ, Najmudin S, Bayer EA, Fontes CMGA. Assembly of Ruminococcus flavefaciens cellulosome involves novel single binding mode cohesin-dockerin interactions. 50

Amaral AJ, Marthey S, Hoffman A, Lagnel J, Haack F, Pokharel K, Palasca O, Seemann S, Arya A, Gama LT, Groenen MAM, Kantanen J, Crooijmans RPMA, Giuffra E, Stadler PF, Anton C, Madsen O, Gorodkin J. Broadening the miRNA catalogue in livestock species: a contribution to the functional annotation of animal genomes consortium. 51 Pimenta J, Pires VM, Nolasco S, Castelo-Branco P, Apolónio JD, Marques CC, Lopes-da-Costa L, Prates JA, Azevedo ARA, Fonseca EA, Pereira RMLN. Gene silencing (siRNA) of Bos taurus prion-like genes, and its relation to progesterone (P4) and 17E-estradiol (E2) concentrations. 52 Poster Presentations - CIISA PhD Students P16. Freitas FB, Frouco G, Martins C, Ferreira F. African swine fever virus (ASFV) encodes for QP509L and Q706L, two RNA helicases, essential for viral replication. 54 P17. Coelho D, Lopes PA, Cardoso V, Ponte P, Madeira MS, Alfaia CM, Bandarra N, Fontes CMGA, Prates JAM. A High-Throughput approach to assess microalgae cell wall disruption by enzymatic digestion. 55

P18. André A, Dias J, Aguiar S, Oliveira S, Ministro J, Gano L, Correia J, Tavares L, Aires-da-Silva F. A novel functional strategy for the development of highly specific antibody drug conjugates for non-Hodgkin’s lymphoma.

56 P19. Silva B, Marto J, São Braz B, Delgado E, Gonçalves LMD. Development of a hyaluronic acid and chitosan nanoparticulated system for topical ocular delivery of erythropoietin. 57

P20. Carinhas JG, Djokovic D, Fernandes AC, Trindade A, Carmona D, Duarte A, Tavares AT. A potential role of the dll4 / notch signaling pathway in the stabilization of atherosclerotic plaques. 58 P21. Cunha E, Janela R, São Braz B, Moreira da Silva J, Tavares L, Veiga AS, Oliveira M. Mutant prevention concentration of nisin towards canine oral enterococci 59 P22. Belas A, Aboim C, Leal R, Mendes CI, Marques C, Carrico JA, Pomba C. Characterization of gut microbiome of healthy companion animals from households and shelters. 60

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SESSION 4 Oral Communications - CIISA Post-Docs and Junior Researchers Trindade A, Pedrosa AR, Graça JL, Carvalho S, Peleteiro MC, Duarte A. Developmental versus oncogenic Notch function in the prostate. 62 Marcelino E, Morais J, Lopes A, Santos D, Novo C, Leitão A. Besnoitia besnoiti protein disulfide isomerase: inhibition of its catalytic activity and of tachyzoite host cell invasion. 63 Brás, JLA, Sequeira AF, Fernandes VO, Abecassis DMA, Vincentelli R, Fontes CMGA High-throughput expression of marine animal venom toxins in Escherichia coli to generate libraries of recombinant venom peptides for drug discovery 64

Poster Presentations CIISA PhD Students P23. Santos R, Gomes D, Tavares L, Veiga AS, Oliveira M. Antimicrobial activity of the antimicrobial peptide pexiganan against a diabetic foot infection Pseudomonas aeruginosa strain. 66 P24. Santos M, Alexandre-Pires G, Pereira MA, Marques CS, Gomes J, Correia J, Gomes L, Rodrigues AV, Basso A, Reisinho A, Meireles J, Santos-Gomes GM, Pereira da Fonseca I. Miltefosine and meglumine antimoniate treatments combined with allopurinol restore lymphokine normal levels in canine leishmaniosis 67 P25. Madeira S, Osório H, Gaspar N, Boinas F. Species composition of the mosquito fauna in the Lisbon zoological garden 68 P26. Dias J, Aguiar S, Pereira D, Andre A, Gano L, Correia J, Carrapico B, Rutgen B, Malho R, Peleteiro MC, Gonçalves J, Rodrigues C, Gil S, Tavares L, Aires-da-Silva F. The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma 69 P27. Marques C, Belas A, Menezes J, Aboim C, Cavaco-Silva P, Trigueiro G, Telo da Gama L, Pomba C. Companion animals as reservoirs of Klebsiella pneumoniae 70 P28. Simões J, Sales Luís J , Tilley P. Contribution to the staging of severe equine asthma syndrome (EAS) in the field using lung function testing 71

P29. Canejo-Teixeira R, Almiro PA, Serpell JA, Baptista LV, Niza MMRE Canine Behavioural Assessment and Research Questionnaire (C-BARQ): study of its factorial structure in European Portuguese 72

Posters Master students contest M30. Santos H, Puerta B, Pestana J, Alfaia CM, Lordelo M, Prates JAM, Fontes CMGA. Assessing the capacity of lysozyme into improve the nutritive value of Spirulina-containing diets for broilers. 74 M31. Andrade JC, Henriques AR. Genetic relatedness of Listeria monocytogenes isolates from a ready-to-eat meat-based food producing industry with a history of persistent contamination. 75 M32. Bernardo R, Henriques AR. Assessing Listeria monocytogenes growth in ready-to-eat chicken salads as a function of temperature. 76 M33. Pires D, Grade A, Ruano F, Afonso F. Production of bivalve molluscs and associated risk factors. 77 M34. Rocha A, Alves S, Bessa RJB, Fraqueza MJ. Occurrence and characterization of White Striping in broilers breast meat in a Portuguese slaughterhouse. 78

M35. Ribeiro DM, Madeira MS, Martins CF, Kilminster T, Scanlon T, Oldham C, Greeff J, Freire JPB, Prates JAM, Mourato MP, Almeida AM. Mineral and amino acid profiling of muscle and liver of Damara, Dorper and Australian Merino lambs under restricted feeding. 79

M36. Rico I, Dias J, Andre A, Aguiar SI, Gaspar MM, Gil S, Aires-da-Silva F. Development and optimization of liposomes encapsulated with potent cytotoxic compounds for immunotherapy. 80

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M37. Vasconcelos AM, Carmo B, Ferreira B, Viegas I, Carvalho MG, Ferreira A, Amaral AJ. IsomiR Window: A platform for analysing small-RNA-seq data of mammalian species in an integrative and user-friendly manner.

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M38. Dordio M, Nunes, T, Beck R, Pereira da Fonseca I, Gomes J. Molecular survey of vector Borne Diseases in two groups of domestic dogs from Southern Portugal. 82

M39. Martins A, Basso MA, Mateus L. Renal injury in bitches diagnosed with pyometra. 83 M40. Barros C, Duarte A, Brito T, Rosa T, Magalhães M, Tavares L, Lamas L. Equine piroplasmosis: hematological and inflammatory biomarkers changes in horses with clinical and subclinical disease. 84

M41. Varandas C, Cartaxeiro C, Delgado E, Gil S. Expression of pro-inflammatory and Th1 cytokines in canine allergic conjunctivitis. 85

M42. Trigo da Roza F , Cunha E , Tavares L, Oliveira M, Lamas LP. Biocide and antibiotic cross-resistance in equine medicine: is there a link? 86

M43. Evangelista F, Pena MV, Meissner M, Luís J, Palomero A, Arias MS, Sánchez-Andrade R, Torres MI, Gomes L, Madeira de Carvalho LM, Paz-Silva A. New administration formula of parasiticide fungi spores to prevent infection by gastrointestinal nematodes in pasturing horses. 87

M44. Santos I, Vicente G, Nunes T, São Braz B. Splenic hemangiosarcoma in dog: association between erythrocyte parameters and the prognosis. 88

M45. Cardoso M, Prata I, Rebelo I, Nunes T, Carneiro C, Bexiga R. ESBL resistance genes in fecal E. coli of calves fed waste milk with antimicrobial residues. 89

M46. Nunes M, Delgado E. Effects of methadone on intraocular pressure in dogs and cats. 90 M47. Dourado R, Soares B, Alves I; Mateus L. Hemogram of pregnant Labrador Retriever bitches. 91 M48. Rosa R, Branco V, Iglésias L, Pissarra H, Niza M, Mestrinho L. The role of alpha-1 acid glycoprotein in post operatory monitoring of chronic gingivostomatitis in the cat: an exploratory study. 92 M49. Fernandes M, Vicente G, Nunes T, São Braz B. Evaluation of adverse effects – increase in serum creatinin and/or urea, in cats undergoing doxorubicin administration. 93 Posters 50. Gomes D, Rego P, Reis S, Carvalho S, Dias S, Santos R, Mendes JJ, Peleteiro MC, Tavares L, Veiga AS, Oliveira M. Diffusion of a nisin-biogel on a Diabetic Foot Ulcer collagen model. 95 51. Pinto M, Batista M, Diniz P, Murta D, Silva E, Lopes-da-Costa L, Torres. A. Wnt-Prostaglandin Pathways interaction in Murine Embryo Development. 96 52. Ruivo M, Crespo MTB, Barreto AS, Semedo-Lemsaddek T. Portuguese traditional PDO cheeses: characterization of the autochthonous microbiome. 97

53. Dias S, Rodrigues C, Veloso MG, Nunes TP, Cota JB. Pulmonary pathological processes and animal welfare in pigs. 98

54. Aires A, Barreto AS, Semedo-Lemsaddek T. Antimicrobial activity of essential oils against bacteria of the oral microbiota. 99

55. Costa J, Steinmetz A, Delgado E. Ocular brachycephalic syndrome. 100

56. Martins S, Resende AP, Delgado E. Medical therapeutic approaches to corneal ulcers in dogs and cats: a prospective study of 112 cases. 101

57. Camarão AAR, Boinas F, Quan M. Development and analytical validation of a group-specific real-time RT-PCR assay for the detection of the Simbu serogroup orthobunyaviruses. 102 58. Prata S, Aguiar S, Gomes J, Almeida V, Bexiga R, Tavares L, Aires da Silva F, Gil S. Canine model for sepsis: a contribution to the classification and stratification of septic patients. 103

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59. Catarino P, Hofmann-Lehmann R, Meireles J, Nunes T, Tavares L, Duarte A. Feline hemoplasmas: evaluation of specific antibodies for molecular and cytological diagnostic. 104

60. Machado IC, Cunha E, Gomes J, Tavares L, Almeida V, Gil S. Frequency of companion animals infectious diseases at a veterinary hospital isolation unit. 105 61. Delgado ILS, Tavares A, Francisco S, Coelho J, Santos D, Leitao A, Soares H, Nolasco S. The Toxoplasma gondii MOB1 protein is involved in the regulation of the parasite’s replication rate. 106 62. Abade dos Santos F, Duarte M, Peleteiro MC. Establishment of patterns for histopathological lesions and viral loads in vaccinated and non-vaccinated wild rabbits, all positive for rabbit haemorrhagic disease virus. 107

63.Baptista E, Crespo MTB, Barreto AS, Semedo-Lemsaddek T. Microbiological and physico-chemical features of artesanal Portuguese PDO cheeses. 108 64.Valente P, Carvalho S, Peleteiro MC, Correia J, Duarte A, Pomba C. Comparative study of two methods in the molecular diagnosis of lymphoma in dogs. 109 65. Semedo-Lemsaddek T, Touret T, Oliveira MM. Putative probiotic lactic acid bacteria isolated from sauerkraut fermentations. 110 66. Sousa S, Martins CL, van Harten S. Ruminal health and performance of feedlot fed Nellore cattle supplemented with live yeast and lipids. 111

67. Pestana JM, Puerta B, Lopes PA, Santos H, Alfaia CM, Madeira MS, Lemos JPC, Lordelo M, Prates JAM. The effect of Arthrospira sp. microalga as a feed supplement on growth performance, pigmentation and meat quality in broiler chickens. 112

68. Almeida F, Gameiro A, Ferreira F. Unveiling new histone deacetylase- and microtubule-based strategies for treatment of feline mammary tumors. 113 69. Ramilo DW, Monteiro C, Carreira M, Pereira da Fonseca I, Cardoso L. First report of Neotrombicula inopinata infestation in domestic cats from Portugal. 114

70. Waap H, Oliveira U, Gomes J, Schares G. Prevalence of antibodies to Neospora spp. in horses in Portugal.

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71. Gomes J, Costa M, Pereira da Fonseca I. Molecular detection of Theileria species infecting sheep: a retrospective study. 116

72. Alho AM, Kayikci F, Shahi Barogh B, Rosa H, Tomás J, Rocha H, Madeira de Carvalho LM, Fuehrer HP. Equine piroplasmosis by Theileria equi in military horses from Portugal. 117

73. Alexandre-Pires G, Dias-GuerreiroT, Palma-Marques J, Mourata-Gonçalves P, Valério-Bolas A, Gabriel A, Pereira da Fonseca I, Sousa-Silva M, Santos-Gomes G. Topographic study of Trypanosoma brucei parasites.

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74. Guerreiro O, Alves SP, Soldado D, Cachucho L, Francisco A, Costa M, Duarte MF, Santos-Silva J, Jerónimo E, Bessa RJB. Use of Cistus ladanifer condensed tannins to improve the nutritional value of ruminant edible fat. 119

75. Martins CF, Matzapetakis M, Ribeiro DM, Prates JAM, Almeida AM, Freire JPB. Spirulina in piglet diets: effects on growth, feed conversion and muscle, liver and intestinal metabolomics profiles using NMR (nuclear magnetic resonance). 120 76. Fontes MA, Viegas I, Madureira L, Nunes LC, Santos JML. Beef consumption in the near future: what do we need to change? 121 77. Francisco AE, Santos-Silva J, Portugal P, Alves SP, Bessa RJB. Relationships between rumen protozoa, biohydrogenation and bioactive fatty acids deposition on lamb muscle. 122

78. Santos-Silva J, Francisco A, Dentinho MT, Portugal P, Almeida J, Jerónimo E, Soldado D, Alves SP, Bessa RJB. Valrumeat - How to reconcile the intensive production of ruminants with the production of meat with high nutritional value. 123 79. Alfaia CM, Madeira MS, Pestana JM, Toldra F, Prates JAM. Edible animal by-products as a source of valuable compounds with potential sustainable applications. 124

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80. Quaresma M, Antunes I, Montoito M, Jardim A, Bandarra N, Carvalho A, Santos C, Roseiro C. The composition of the lipid and proteic fractions of dried salted cod obtained from the Atlantic cod (Gadus morhua) and the Pacific cod (Gadus macrocephalus). 125 81. Mendes-Jorge M, Lucas S, Valença A, Mendes MG, Ferraz C, Mateus L, George Stilwell G, Brito-Paes V. Response to local pain in Brava de Lide cows. 126

82. Bressan MC, Amaral AJ, Almeida J, Bettencourt C, Moreira O, Sá J, Gama-Carvalho M, Bessa R, Gama LT. Genome-wide association analysis and gene interaction networks to identify genes associated with carcass traits, meat quality and fatty acid profiles. 127 83. Centeno MSJ, Pires VR, Fontes CMGA. Biochemical characterization of a bi-functional glycoside hydrolase family 5 from Ruminococcus flavefaciens. 128 84. Alves VD, Cameron K, Bule P, Smith SP, Gilbert HJ, Bayer EA, Najmudin S, Fontes CMGA. A structural-based approach to unravel all protein-protein interactions supporting Acetivibrio cellulolyticus cellulosome assembly. 129 85. Lopes PA, Martins R, Silva IV, Madeira MS, Prates JAM, Soveral G. Modulation of aquaporins gene expression by n-3 long-chain polyunsaturated fatty acids (LCPUFA) lipid structures in white and brown adipose tissue from hamsters. 130 86. Brás JLA, Ribeiro D, Romão MJ, Carvalho AL, Chai W, Liu Y, Feizi T, Prates JAM, Ferreira LMA, Palma AS, Fontes CMGA. The CBMomes of cellulolytic bacteria colonizing different ecological niches present distinct carbohydrate specificities. 131 87. Pires VMR, Correia M, Centeno MSJ, Bastos J, Alves VD, Ferreira LMA, Fontes CMGA. The mechanism by which family 56 Carbohydrate-Binding Modules recognize complex β-1,3-glucans. 132 88. Correia JJ, Lamas L, Cosinha C, Mousinho C, Carneiro C, Oliveira M, Peleteiro MC, Madeira de Carvalho L. Larval cyatostominosis in domestic (Equus caballus) and wild equids (Equus quagga) in Portugal: pathology and epidemiology in outbreaks with a high mortality rate. 133

89. Madeira de Carvalho LM, Alho AM, Breuer H, Shahi Barogh B, Zittra C, Harl J, Caetano I, Brazio E, Mira M, Fuehrer HP. Hemoparasites of birds of prey and other avian hosts kept in a Wildlife Rehabilitation Center in Lisbon, Portugal. 134 90. Delgado E. Ocular sequelae of heminopter bites in dogs - 6 clinical cases. 135 91.Tilley P, Simoes J, Sales Luis, JP. Effects of a 15o variation in poll flexion during riding on the respiratory system and on behaviour in pure blood Lusitano high dressage horses. 136 92. Fraqueza MJ, Chaves A, Rebelo C, Goncalves S, Martins A, Fernandes MH, Fernandes MJ, Barreto AS. Bioprotective solution against pathogens: selection of protective starter cultures for meat products. 137 93. Laranjo M, Potes ME, Véstia J, Rodrigues S, Agulheiro-Santos AC, Charneca R, Fraqueza MJ, Elias M. Effect of genotype and salt reduction on the quality of traditional Portuguese sausages. 138 94. Barreto AS, Félix V, Semedo-Lemsaddek T. Isolation of bioactive compounds from Arbutus unedo “medronheiro” and evaluation of antimicrobial properties against bacteria from food related environments. 139

95. Gama LT, Pavão AL, Amaral AJ. Estimation of inbreeding using pedigree or genomic information in Ramo Grande cattle. 140 96. Peleteiro MC, Borges I, Carvalho S, Pissarra H, Carvalho T. Correlation between histological grading and KIT expression patterns in eighty canine mast cell tumors. 141

97. Pereira da Fonseca I, Diz S, Loução C, Santos M, Rebelo MT. Diptera fauna associated with cat corpses’s decomposition as a possible entomological forensic tool. 142 98. Lança A, Nikonov V, Ramos A, Tavares L, Bernardo F, Oliveira M. Dogs and Cats dermatophytosis in Portugal: a 16-year study. 143

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99. Carvalho S, Pissarra H, Afonso F, Luís MR, Correia J, Ferreira da Silva J, Peleteiro MC. The experience of forensic pathology of the Laboratory of Pathological Anatomy of the Faculty of Veterinary Medicine, University of Lisbon (2015-2018). 144 100. Fernandes MJ, Alves SP, Alfaia CM, Skrbic BD, Zivancev JR, Bessa RJB, Fraqueza MJ. Chemical hazards screening in dry fermented sausages from Mediterranean influence countries. 145 101. Andre LA, Pires VMR, Fontes CMGA. The ability of Ruminococcus albus and Ruminococcus flavefaciens to share important components of their cellulosome systems. 146

Keynote Speakers

16-11

Session 1

Biomedical Sciences – One Medicine 09:30 – 10:10 - Miguel Castanho

10:10 – 10:50 - Cecília Rodrigues

Chairperson – Luís Lopes da Costa

Session 2

Animal Science and Production, Food Technology, Quality and Security – From farm to fork 14:30 - 15:10 - Tim McAllister

15:10 – 15:50 - Fidel Toldrá

Chairperson – Rui Caldeira 17-11

Session 3

Biotechnology and Genetics 09:00 – 09:40 - Harry Gilbert

09:40 – 10:20 - Maria João Romão

Chairperson – Carlos Fontes

Session 4

Animal Health – One Health 14:00 – 14:40 – Manuela Caniça

14:40 – 15:20 - Andrew Hemphil

Chairperson – Luís Tavares

Keynote Speakers

Session 1 Biomedical Sciences – One Medicine

Chairperson – Luís Lopes da Costa

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Hidden functions in the capsid protein of Dengue virus: biological relevance and drug development opportunities

Miguel A. R. B. Castanho, Ana Salomé Veiga

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal

macastanho@medicina.ulisboa.pt

The structure-function relationship of capsid protein of Dengue virus has been

extensively studied in our lab, in collaboration with partners having different expertise. A

multidisciplinary approach has revealed that the capsid protein of Dengue virus has the

ability to fuse lipid membranes and transport cargo across cell boundaries. We

hypothesize that this protein may participate in the mechanism of viral entry into the cells.

Moreover, we have identified domains of the peptide having antimicrobial activity.

Overall, these findings cast the discussion on the evolutionary meaning for such broad

multifunctionality.

Keywords

Multifunctional, virus, antimicrobial, membrane, transport.

Supporting references:

New potent membrane-targeting antibacterial peptides from viral capsid proteins, Frontiers in microbiology, 8, 775 [2017]

Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism, ACS Chemical Biology, 12, 1257-1268 [2017]

Receptors and routes of dengue virus entry into the host cells, FEMS microbiology reviews, 39, 155-170 [2015]

Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein: design and mechanism of action, The FEBS journal, 281191-215 [2014]

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Biomarkers and targets in metabolic liver disease

Cecília M P Rodrigues

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

Non-alcoholic fatty liver disease (NAFLD) is one of the most important causes of liver disease worldwide affecting both adults and children. With 20-30% prevalence, NAFLD will likely emerge as the leading cause of end-stage liver disease in the coming decades. The epidemiology and demographic characteristics of NAFLD vary worldwide, usually paralleling the prevalence of obesity, although a substantial proportion of patients are lean. Chronic liver injury results in inflammatory damage, matrix deposition, parenchymal cell death and angiogenesis leading to progressive fibrosis and later cirrhosis. Genetic polymorphisms, epigenetic marks and cofactors strongly modulate the risk of disease progression. Challenges in NAFLD include accurate identification of patients with simple steatosis and low risk of progression to advanced liver disease versus those with progressive features of the disease. In addition, there is an urgent unmet need for effective therapeutics given limitations of current strategies of life style modifications, such as weight loss and exercise. In this regard, miRNAs warrant full attention as epigenetic markers and targets.

Keywords: Diagnosis; miRNAs; non-alcoholic fatty liver disease; treatment

Keynote Speakers

Session 2 Animal Science and Production, Food Technology, Quality and Security – From Farm to Fork

Chairperson: Rui Caldeira

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New approaches to the enzymatic enhancement of ruminal fibre digestion

Tim McAllister

Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Canada

Sustainable conversion of forages and crop residues into ruminant protein presents

multiple benefits. Grasslands and permanent pastures store vast amounts of carbon,

reduce wind / water erosion and replenishment ground water reserves. Crop residues

and protein-rich legume forages can reduce the use of human edible grains and proteins

in ruminant diets. The fiber in plant cell walls is rich in polysaccharides including glucose,

xylose, arabinose, galactose and mannose, all of which can be fermented by rumen

microorganisms and provide energy for microbial protein synthesis and to the host.

However, only a fraction of the sugars in most forages are in a free form, with the majority

being contained within cellulose, hemicellulose and pectin. Furthermore, these

carbohydrate complexes, particularly hemicellulose, are decorated with a range of side

groups including acetyl, ferulic/coumaric acid and glucuranoic acids with covalent

linkages to lignin. These linkages with lignin must be oxidized to be hydrolysed and

therefore lignin is not degraded within the rumen. Even with this caveat, ruminants are

unique in their ability to convert lignocellulosic biomass into high quality meat and milk

protein. However, in spite of the rumen’s reputation as the most efficient biological

system at converting plant cell wall biomass into usable fermentation products, often less

than 50% of the energy in low quality forages is available for cattle. This apparent

inefficiency in what is deemed to be a highly efficient system arises primarily as a result

of lignin / phenolics restricting the access of rumen microbes and enzymes to the

carbohydrate stores within plant cell walls. This presentation will outline the latest

developments in technologies to enhance the conversion of low quality forage fibre into

energy within the rumen as a component of cost effective and sustainable dairy and beef

production.

17

Bioactive peptides from animal by-products

Fidel Toldrá, Marta Gallego, Milagro Reig, M-Concepción Aristoy and Leticia Mora

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Paterna (Valencia), Spain

Huge amounts of by-products like blood, bones, meat trimmings, skin, fatty tissues,

horns, hoofs, feet, skull and viscera among others are daily generated in the meat

industry. Collagen is another relevant by-product from the fish industry. Their treatment

and ecological disposal is very costly. However, these costs can be balanced through

innovative technologies to generate added-value products that increase its profitability.

Other by-products, especially those resulting from the production of dry-cured ham and

fermented sausages, have been traditionally used in Mediterranean household cooking

of broths and stews.

Recent valorisation strategies are based on the enzymatic hydrolysis of such by-products

to search for key active molecules with relevant nutritional properties like bioactive

peptides that can exert some important physiological effects such as antihypertensive,

antioxidant, antidiabetic, antimicrobial, etc. with promising applications in the food,

pharmaceutical and cosmetics industry. This lecture reports and discusses the latest

developments and trends in the production of added-value products like bioactive

peptides generated from meat and fish industry by-products.

Keynote Speakers

Session 3 Biotechnology and Genetics

Chairperson: Carlos Fontes

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The prebiotic potential of complex carbohydrates in mammalian gut bacterial communities.

Harry J. Gilbert

Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle

University, Newcastle upon Tyne, U.K.

Mammalian gut bacteria (MGB) make a significant contribution to the physiology and

health of its host. Glycans are major nutrients for MGB and in many of these communities

Bacteroides species are the major glycan degrading organisms. Understanding glycan

utilisation by MGB underpins prebiotic and probiotic strategies that combat dysbiosis,

and thus promote mammalian health and livestock production. A ubiquitous component

of the human diet are arabinogalactan proteins (AGPs). These proteoglycans occur in

plants from every taxonomic group, with high concentrations in processed foods. AGPs

comprise a E1,3-galactan backbone with E1,6-galactan side-chains, which contain

carbohydrate decorations. AGP utilisation is poorly understood, however, the prebiotic

potential of GA-AGPs was proposed through an increase in Lactobacillus,

Bifidobacterium and Bacteroides in some MGB communities. Maximising the health and

livestock production benefits of AGPs require development of robust models for the

metabolism of these proteoglycans by the HGM. Here we report a model for simple and

complex AGP utilisation by mammalian gut Bacteroides species. Microbial experiments

showed that only three Bacteroides organisms have the capacity to degrade AGPs.

These three species acted as keystone organisms supplying the other Bacteroides

species (recepients) with AGP-derived oligosaccharides that they could utilise as growth

substrates. We show that the presentation of a surface endo-glucanase on a recipient

Bacteroides promotes the bacterium to keystone status. We have also identified an

abundant sulphated AGP in red wine that it utilized by only a single Bacteroides species.

The mechanism by which this AGP is degarded and its prebiotic potential will be

discussed.

20

The Impact of Structural Biology in Deciphering Enzymatic Mechanisms

Maria João Romão

UCIBIO – Unidade de Ciências Biomoleculares Aplicadas- Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516

Caparica, Portugal

Email: mjr@fct.unl.pt; http://xtal.dq.fct.unl.pt/ Protein crystallography has had a major contribution in clarifying enzymatic reaction

mechanisms while correlating structural information with biochemical and kinetic data.

The field of metalloenzymes is very rich in examples where the knowledge of the crystal

structure unraveled for the first time complex metal cofactors and even allowed putting

forward novel reaction mechanisms [1-3]. Sometimes, the crystal structures enabled to

identify unexpected co-factors and to discover new metal ligands that challenged earlier

proposed functions of the individual active sites [3].

Molybdenum-dependent enzymes exist in all domains of life and their importance is

exemplified by their ubiquity, their roles in metabolic diversity and in global geochemical

cycles [1, 5]. In the talk different bacterial and mammalian molybdenum enzymes will be

considered to exemplify how the combination of crystallographic data with kinetic,

mutagenesis and molecular docking studies have made a decisive contribution to

understand the molecular basis of substrate and inhibition specificity for some of the

enzymes.

References:

1- M J Romão, DALTON TRANS., 2009, Jun 7;(21):4053-68. 2- HCA Raaijmakers and MJ Romão, J BIOL INORG CHEM. 2006 Oct;11(7):849-54. 3- C. Coelho, P.J. González, J.J.G. Moura, I. Moura, J. Trincão and M.J. Romão, (2011) JOURNAL OF

MOLECULAR BIOLOGY, 408, 932-948. 4- C Coelho, A Foti, T Santos-Silva, T Hartmann, S Leimkühler and MJ Romão, (2015) NATURE CHEMICAL

BIOLOGY 11, 779–783 (2015) 5- Mota C, Coelho C, Leimkuhler S, Garattin E, Terao M, Santos-Silva T, Romão MJ (2018)

COORDINATION CHEMISTRY REVIEWS 368:35-59.

Keynote Speakers

Session 4 Animal Health – One Health

Chairperson: Luís Tavares

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Antibiotic resistance and One Health perspective

Manuela Caniça

National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections (NRL-AMR/HAI), National Institute of Health Dr. Ricardo Jorge, Lisbon,

Portugal

Antibiotic resistance represents a global concern and it has manifest consequences to

humans, animals and environment. The decrease in resistance to antibiotics in all those

reservoirs is associated to the prevention of resistance mechanisms and respective

spread. Thus, antibiotic resistance should be tackled on all fronts in view to the essential

concept of One Health, where the transdisciplinary collaboration is obvious, with efforts

from science and society to have enough progress and impact. In addition, learning,

thinking, sharing, planning and working are the main drivers for the success of One

Health, namely in this context.

Definitely, joint efforts are needed to better understand the cross-species antibiotic

resistance transmission and emergence, as well as the constant adaptation of

microorganisms to the selective pressure exerted by antibiotics. Development and

evaluation of new diagnostic tools are also more and more needed, such as high

throughput methods (e.g. whole genome sequencing), namely for surveillance proposes.

Nowadays, one of the most worrying examples is the presence of carbapenemase-

encoding genes located on highly effective mobile genetic elements, either in human (in

pathogenic and commensal bacteria), and e.g. in the livestock environment, or even in

rivers. Their transmission is worrying and an important issue for public health. Indeed,

the One World - One Medicine - One Health need to be put in place in view to easily take

action against the antibiotic resistant bacteria.

Keywords

Antibiotic resistance, One Health, public health.

23

Novel drugs and drug targets for the treatment of toxoplasmosis and other diseases caused by cyst-forming apicomplexan parasites

Andrew Hemphill

Institute of Parasitology, University of Bern, Länggass-Strasse 122, 3012 Bern, Switzerland

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites, which can cause abortion and fetal defects in many animal species. While N. caninum is a veterinary problem and is most relevant in cattle, Toxoplasma induced abortion in sheep is a major problem, and T. gondii is also an important human pathogen, most notably when infection takes place during pregnancy, or in immune compromised patients. There is no approved treatment for neosporosis. The current treatments of toxoplasmosis are based on a combination therapy comprising sulfonamides and pyrimethamine or other antimicrobials including inhibitors of apicoplast division, and they cause serious adverse side effects. In many countries, there is no approved treatment for maternal and fetal T. gondii infections. Therefore, current therapies are not optimal and novel treatment options are required. Most drug efficacy studies for neosporosis and toxoplasmosis have been carried in non-pregnant models, although it is imperative for any drug to be considered for the treatment of these infections that no interference in pregnancy occurs. For neosporosis in cattle, the major mode of transmission is thought to occur via endogenous transplacental transmission, but there is no mouse model that can mimic this process; thus, pregnant neosporosis mouse models rely on inoculation of culture-derived tachyzoites during pregnancy. For T. gondii, a pregnant oocyst infection model has been recently established. The anti-coccidial toltrazuril has been earlier assessed in pregnant mice and cattle, with clear readout in the mouse model, but ambiguous results produced in bovines. More recently bumped kinase inhibitors (BKIs), which target calcium-dependent protein kinase 1, have emerged as novel treatment options in vivo. These drugs do not act parasiticidal, but are nevertheless highly efficient, and there are indications that immunity strongly contributes to the efficacy of BKIs. Studies on this phenomenon could shed light on novel potential treatments such as combined immune-chemotherapeutical options. The screening of open-source drug libraries such as the MMV Malaria Box and Pathogen Box has revealed several compounds that could be potentially repurposed for the treatment of neosporosis and toxoplasmosis in pregnant animals. One of these compound classes are endochin-like quinolones (ELQs), which target the cytochrome b Qi site on the cytochrome bc1 complex in the mitochondria of many apicomplexans. Interestingly, derivatives of decoquinate, an old anti-coccidal drug, have been recently synthesized, which target the cytochrome Qo site, and exhibit outstanding activities not only against T. gondii and N. caninum, but also Plasmodium falciparum and Mycobacterium tuberculosis.

16 November 11:20 - 12:20

Session 1 Oral Communications - CIISA Post-Docs and Junior Researchers

Chairpersons - António Duarte and Frederico Aires da Silva

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sdAb-immunoliposomes for brain targeting and drug delivery Aguiar SI1,3, Gaspar MM2, André A1, Martins D1, Ferreira M2, Carvalheiro M2, Neves V3, Coelho S3, Dias J1, Carrapiço B1, Gano L4, Galamba J4, Nobre R5, Almeida L5,6, Tavares L1, Gonçalves J6,7, Castanho M3, Aires-da-Silva F1

1. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisbon, Portugal. 2. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal. 3. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. 4. Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. 5. CNC-Center for Neuroscience and Cell Biology, Coimbra, Portugal. 6. Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Background: Brain disorders are one of the major causes of mortality. Despite the efforts, effective brain drug delivery has been systematically challenged by the impenetrability of the blood brain barrier (BBB). One promising approach is to target specific BBB receptors using monoclonal antibodies (mAbs). Some of these mAbs transmigrate the BBB and have been tested as vectors to deliver drugs into the brain. Yet, these rely on broadly expressed receptors bringing unwanted side effects and resulting in a low fraction of dose in the brain. Promising alternatives are single domain antibodies (sdAb). These show improved tissue penetration reaching targets inaccessible by conventional antibodies, low immunogenicity and lower manufacturing costs. Liposomes are vesicular concentric bilayer structures composed of lipid, separated by aqueous compartments. They present advantages over other delivery systems such as their ability to incorporate hydrophilic and hydrophobic drugs, low toxicity, low immunogenicity and targeted delivery. To construct an improved brain delivery system we are developing novel immunoliposomes, based of BBB targeted and transmigrating sdAbs conjugated to antibiotic containing liposomes for drug delivery validation in a meningitis model. Methods: A phage displayed-sdAbs library was constructed using 2 New Zealand White rabbits immunized with brain endothelial cells. In vitro and in vivo screens were performed for selection and identification of sdAbs towards new BBB receptors. In parallel different lipid compositions were prepared and tested for antibiotic encapsulation. Liposomes were characterized in terms of lipid and antibiotic contents and antimicrobial efficacy. Results: A specific immune response towards brain endothelial cells was obtained for both rabbits. Both serums showed BBB crossing properties in in vitro and in vivo models. sdAbs libraries were efficiently constructed with a diversity with ~107. Phage display selections and functional screenings were performed to identify potent BBB crossing sdAbs and novel endogenous receptors. A panel of 22 different liposomal formulations have been developed with DPPC: DPPG and SM:Chol:DcP being the most promising formulations and reaching up to 42% of encapsulation efficiency. Conclusions: We have selected brain specific sdAbs with BBB crossing properties and 2 promising vancomycin liposomal formulations for antibody linkage and drug delivery validation. Keywords: Blood Brain Barrier; Meningitis; sdAbs; Antibiotics; Immunoliposomes Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (BBB_BIO_0508_2014; IF_010100 and by UID/CVT/00276/2013).

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Dll4 and Jagged1 signaling are relevant players in mammalian spermatogenesis and sperm maturation Murta D1,2, Batista M1, Pinto M1, Silva E1, Trindade A1, Duarte A1, Lopes-da-Costa L1 1. Reproduction and Development Laboratory, CIISA, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, PORTUGAL; 2. CBIOS - Research Centre for Biosciences and Health Technologies, Faculty of Veterinary Medicine, Lusófona University of Humanities and Technologies, Campo Grande, Lisboa, PORTUGAL Spermatogenesis is the process by which male gametes (spermatozoa – SPZ) are produced in the seminiferous tubules of the testicles. These cells are then transported along the epididymis, experiencing maturational changes that will enable them to acquire fertilizing capacity. These complex finely tuned changes, comprising cell proliferation and differentiation events, are poorly understood. Deciphering these mechanisms will have a relevant impact in reproductive medicine through the development of new strategies for male fertility and contraception. Notch is an evolutionary well conserved cell signaling pathway involved in different cell fate decisions in several body systems. Recent work from our lab identified Notch signaling as a relevant regulator of spermatogenesis, and ligands Dll4 and Jagged1 as potential male fertility therapeutic targets. The aim of this study was to evaluate the role of Dll4 and Jag1 in spermatogenesis and sperm maturation. Dll4 and Jagged1 conditional inducible loss-of-function mutant male mice (n = 46) were treated with tamoxifen to induce loss-of-function. At days 11 (time required for epididymis sperm transport and maturation), 43 (time required for one spermatogenic cycle) and 70 after treatment mice were allowed to breed (2 superovulated female mice for each male). Following breeding, testicles and epididymis were collected for histology, immunohistochemistry and RT-PCR, and sperm samples were evaluated for motility, concentration, viability and morphology. Pregnancy, parturition and number of puppies were recorded for each female (fertility traits). Control (Genotype, n= C57BL/6) mice were included in the study. Dll4 and Jagged1 loss-of-function was confirmed by RT-PCR of these genes. Dll4 loss-of-function resulted both in spermatogenic and sperm maturation defects. Histologic analysis revealed seminiferous tubules without spermatogenic cells (Sertoli-cell only phenotype) and tubules with disrupted cell organization. Sperm samples showed a decrease in motility (31.4%±5.01 vs. 15.61%±3.29, p=0.011) , concentration (37.38±6.22 vs. 24.53±3.72, p=0.85) and viability (13.5±3.59 vs. 3.5±0.86, p=0.01). Females mated by Dll4 mutant males had decreased fertility traits (6.37±0.73 pups vs. 3.98±0.74 pups, p=0.025). Jagged1 loss-of-function also resulted in spermatogenic and sperm maturation defects, although at a less extent than Dll4 loss-of-function. Histologic analysis revealed disruption of seminiferous tubules organization. Intriguingly, affected seminiferous tubules showed an ectopic Dll4 expression pattern inside the citoplasmatic droplets, which is a Jagged1 specific location. Although sperm samples didn’t show any significant change, sperm mobility was decreased (43.57%±6.72 vs. 23.5%±7.89, p=0.066) and female fertility traits were significantly impaired (8.63±0.73 pups vs. 5.25±1 pups, p=0.023). In conclusion, results indicate that Dll4 and Jagged1 play a relevant role in both spermatogenesis and sperm maturation. Dll4 signaling is more crucial for male fertility than Jagged1 signaling. Absence of Jagged1 signaling may be compensated by ectopic Dll4 expression and signaling. Keywords: Notch, Dll4, Jagged1, Spermatogenesis, Sperm Maturation, Mouse Funding: CIISA-UID/CVT/00276/2013 and CIISA-9 from Fundação para a Ciência e a Tecnologia (FCT)

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The role of Sirt1 overexpression in vascular retinopathies Catita J1,2,3, Valença A1,4, Ramos D1,2, Bonet A1,2, Nacher V1,2,4, Navarro M1,2,4, Carretero A1,2,4, Mendes-Jorge L1,4, Ruberte J1,2,4 1. CBATEG - Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Spain; 2. Department of Animal Health and Anatomy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain; 3. Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal. 4. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon, Portugal Background: Sirt1, a NAD+-dependent protein deacetylase, is an important regulator in cellular stress response and cell survival. Sirt1 is considered a stress-induced protector in retinopathies with impaired vascularization and aberrant vessel growth such as age-related macular degeneration and diabetic retinopathy. In ischemic retinas, the expression of Sirt1 is induced to promote vessel regrowth by modulating the expression of angiogenic factors. While the list of Sirt1’s putative role in vascular retinopathies is growing, how the activity of Sirt1 affects the retinal blood vessels remains unclear. The aim of this study was to determine the effects of Sirt1 upregulation in the retinal blood vessels and to elucidate the mechanisms of its upregulation. Methods: To address the impact of Sirt1 upregulation, we used SIRT1 transgenic mice (Sirt1-tg). q-RT-PCR, western blot analysis and immunohistochemistry were used to detect VEGF-A, PDGF-B, PDGF-Rβ and NG2 in the retina of Sirt1-tg mice and wild-type littermates. Vascular density was analysed by immunostaining against collagen IV in whole-mount retinas and measured using AngioTool software. Results: Sirt1-tg mice showed a moderate increase of Sirt1 mRNA and protein levels in the retina. The analysis of retinal vasculature provided evidence that Sirt1 was overexpressed in both endothelial and mural cells of Sirt1-tg mice retinas. As a result of Sirt1 upregulation, we observed that mRNA and protein levels of the angiogenic factor VEGF-A were increased in Sirt1-tg retinas. However, no significant differences were found between the retinal vascular density of wild-type and Sirt1-tg mice, thus indicating the absence of neovascularization under VEGF-A overexpression. In this sense, we next investigated the ligand-receptor system PDGF-B/PDGFR-beta signalling, which plays a major role in vessel stabilization by promoting pericyte recruitment. The upregulation of PDFG-B/PDGF-Rβ pathway was confirmed by western-blot analysis. Accordingly, immunohistochemistry with pericyte markers NG2 and PDGFR-beta demonstrated that Sirt1-tg mice showed higher pericyte number and larger capillary surface covered by pericytes than wild-type. Conclusions: Our results indicated that although VEGF-A expression is increased in Sirt1 overexpressing retinas, neovascularization is absent due to an increase in pericyte coverage modulated by the upregulation of PDFG-B/PDGF-Rβ pathway, which is able to provide blood vessel stabilization. These results demonstrate a new role for Sirt1 in vascular stabilization, highlighting this protein as a therapeutic target in vascular retinopathies. Keywords: Sirt1, VEGF, pericyte recruitment, retina Funding Acknowledgements: Project PI16/00719 (CBATEG, Universitat Autònoma de Barcelona) – Instituto de Salud Carlos III, Spain.

16 November 12:20 - 13:00 Session 1 Poster Presentations - CIISA PhD students

Chairpersons - Antonio Duarte and Frederico Aires da Silva

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PhD Contest Poster P1 Characterization of HER2 Single Variants and Evaluation of the Anti-tumor Properties of Tyrosine Kinase Inhibitors in Feline Mammary Carcinoma Cell Lines Gameiro A, Almeida F, Correia J , Ferreira F

Center for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon Background: Feline mammary carcinoma (FMC) is the third most common type of neoplasia in cats (12-40%). HER2+ tumors represent 33-60% of all FMC and present a poor prognostic. Beyond monoclonal antibodies, a good alternative for breast cancer treatment is the use of tyrosine kinase (TK) inhibitors, such as Lapatinib and Neratinib, and also a combined therapy, for example, with Rapamycin, an mTOR pathway inhibitor. Resistance to HER2+ targeted therapy is common and can occur through mutations in the her2 gene. To improve chemotherapy options in FMC, the efficiency of TK inhibitors was evaluated in vitro, and the TK domain of her2 was sequenced to identify single variants (SV) in feline tumoral cell lines. Methods: The cytotoxicity of Lapatinib, Neratinib and Rapamycin was evaluated in four cell lines, three feline tumoral cells and one human breast cancer cell line. The different cell lines were incubated with increasing concentrations of each inhibitor for 72h and cell viability was determined by using WST-1 cell proliferation assay. HER2 expression in the four cell lines was evaluated by Western Blot, and genomic DNA from the feline cell lines was used as template to amplify her2 TK domain and submit to sequencing. Results: The cytotoxicity effect of Lapatinib, Neratinib and Rapamycin in the feline cell lines was similar to the one observed with the human cell line. Lapatinib enabled 100% cytotoxicity, while Rapamycin alone did not go above 60% cytotoxicity. The feline cell lines showed a lower level of HER2 expression in comparison with the human cell line. The her2 gene from the different cell lines showed a high percentage of identity among the four cell lines, and the majority of SVs appeared mostly in intron regions. The her2 from FMCp presented the higher number of SVs. Conclusions: The anti-tumor properties of the inhibitors used are comparable among human and feline cell lines, and with no indication of resistance to therapy. Our data demonstrates the possible use of these inhibitors in cats with FMC, particularly Lapatinib that showed promising results. Keywords: Feline mammary tumor, human epidermal growth factor receptor 2, tyrosine kinase inhibitors Funding Acknowledgements: This work was supported by Fundação para a Ciência e a Tecnologia (FCT/MCTES) through grant PTDC/CVT-EPI/3638/2014 and by the Centre for Interdisciplinary Research in Animal Health (CIISA), which is financed by national funds from FCT/MCTES (UID/CVT/00276/2013). AG is supported by a PhD fellowship (SFRH/BD/132260/2017) and FA by a post-doc fellowship (PTDC/CVT-EPI/3638/2014).

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PhD Contest Poster P2

TIM2: a new membrane receptor for H-ferritin in the mouse retina Valença A1,2, Ramos D1,2, Catita J2,3,4, Bonet A2,3, Nacher V1,2,3, Navarro M1,2,3, Carretero A1,2,3, Mendes-Jorge L1,2, Ruberte J1,2,3 1. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon, Portugal; 2. CBATEG - Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Spain; 3. Department of Animal Health and Anatomy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain; 4. Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal. The retina specifically needs iron, an essential component of the phototransduction cascade. However, imbalance of iron homeostasis is associated with oxidative damage and the progress of several retinopathies. Unfortunately, iron homeostasis and the mechanisms of iron dysregulation in the retina are still poorly understood. The BRB isolates the retina from the bloodstream, thus specific and tightly regulated mechanisms of iron uptake are required to cross this barrier and import the strictly necessary iron into the retina. Classically, transferrin endocytosis is known as the mechanism for iron uptake into the retina. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-carrier protein and can be regarded as a potential deliverer of a considerable large amount of iron to the retina compared to transferrin. Ferritin molecules are composed of L- and H- chain subunits that bind to Scara5 and TIM2 receptors, respectively. Although Scara5 was recently described in the retina for the first time, the presence of TIM2 remains unknown. This study aimed to unravel the presence of TIM2 in the mouse retina. We further intended to characterize the retinal localization of TIM2. Immunohistochemistry and western blot analysis were performed to detect retinal TIM2 expression. Spleen was used as a positive control tissue. The localization of TIM2 in the retina was analysed by means of double immunohistochemistry against TIM2 and specific markers for retinal cells. The presence of TIM2 in the retina was confirmed by western blot analysis. Immunolabeling revealed TIM2 expression throughout the entire retinal parenchyma, predominantly focused in the ganglion cell layer. Furthermore, TIM2 was strongly expressed in Müller cells with higher immunoreactivity in cell processes and endfeet along the internal limiting membrane, while weakly expressed in bipolar, amacrine, and horizontal cells and inner segments of rods. No expression of TIM2 was found in astrocytes, ganglion cells or cones. Additionally, although TIM2 was found surrounding blood vessels, no TIM2 expression was detected in endothelial cells or any other cellular component of the vessel wall. The present study revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly expressed in Müller cells, the principal retinal glial cells. Müller cells are responsible for maintaining retinal homeostasis and have been considered as important mediators of iron transport, distribution, and regulation in the retina. TIM2 expression in Müller cells, together with the involvement of these cells in iron homeostasis, unravels TIM2 as a new key player in retinal iron homeostasis, opening up to a new approach in iron metabolism regarding the putative role of TIM2 in this tissue. Keywords: TIM2 receptors, ferritin, iron homeostasis, retina Funding Acknowledgements: Doctoral scholarship SFRH/BD95330/2013 – FCT, Portugal. Project UID/CVT/00276/2013 (CIISA, FMV, ULisboa) – FCT, Portugal. Project PI16/00719 (CBATEG, Universitat Autònoma de Barcelona) – Instituto de Salud Carlos III, Spain.

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PhD Contest Poster P3 Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition, reduction of cancer stem cells and tumour cell intravasation Mendonça L1,2, Trindade A1, Carvalho C1,4, Badenes M1,3, Gigante J1,5, Duarte A1

1. Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA), Faculty of Veterinary Medicine, University of Lisbon (FMV-ULisboa), Lisbon, Portugal; Current addresses: 2. USF Vida +, ACES Cávado II Gerês/ Cabreira, ARS Norte, Braga, Portugal; 3. Instituto Gulbenkian de Ciência, Oeiras, Portugal; 4. Centro Champalimaud, Fundação Champalimaud, Lisbon, Portugal; 5. Boehringer Ingelheim, Lisbon, Portugal Systemic inhibition of Dll4 results in a vast reduction of cancer metastasis. Whether this effect is endothelial-mediated remains to be clarified. In particular, the connection between disrupting tumour vasculature and the metastatic process should be elucidated. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) frequency, and the intravasation of tumour cells. Lewis Lung Carcinoma (LLC) cells or LLC- green fluorescent protein (GFP)- marked were used to model mouse tumour metastasis, both in vitro, in cell culture and in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function (eDll4cKO) mice. We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumour vascular regression that leads to the reduction of tumour burden. Endothelial specific Dll4 inhibition induces an increase in tumoural blood vessel density, but these neovessels are poorly perfused, display increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumour, the number and burden of macro-metastases was significantly reduced. In fact, both EMT and CSC numbers were decreased by the endothelial-specific Dll4 loss-of-function. This is reinforced by an average two-fold decrease in tumoural intravasation and circulating LLC tumour cell count. Furthermore, our gene transcription analysis suggests that endothelial Dll4/Notch function intermediates tumour hypoxia-driven increase of EMT. We observed that the downregulation of Dll4/Notch1/Hey1 signalling caused a reduction in Snail-1 and Twist expression, known inducers of EMT, despite an observed increase in Hif-1alfa transcription. These findings show that the effect of Dll4 inhibition in suppressing cancer metastasis is at least partially endothelial-mediated. This is accomplished through the arrest of the EMT process and reduction of CSC clone selection in the primary tumour regulated by non-cell-autonomous endothelial signalling and also through reduction of tumour cell intravasation and subsequent circulation. Therefore, endothelial Dll4 may constitute a promising target to prevent metastasis. Keywords: Dll4/Notch, metastasis, epithelial-to-mesenchymal-transition, cancer-stem-cells, intravasation. Financial support: This work was supported by the Portuguese Foundation for Science and Technology, grants PTDC/SAU-ONC/116164/2009 and PTDC/SAU-ONC/121742/2010 to AT. CIISA has provided support through project UID/CVT/00276/2013, funded by FCT. LM is a PhD student supported by a studentship from FCT (SFRH/BD/74229/2010). AT is a Postdoctoral Researcher supported by FCT (SFRH/BPD/110174/2015).

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PhD Contest Poster P4 JAGGED1 and JAGGED2 participation in bovine sperm acrosome reaction Diniz P, Batista M, Silva MF, Mateus L, Lopes-da-Costa L, Silva E

CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal Mammalian spermatozoa (Spz) must undergo capacitation in order to acquire fertilization competence. One of the late capacitation-associated events consists in the Spz ability to undergo the acrosome reaction (AR), which is a pre-requisite for Spz-oocyte fusion. The Notch signalling pathway is an evolutionarily conserved mechanism involved in different cell fate decisions. In a previous study we detected Notch receptors NOTCH1, NOTCH2, NOTCH4 and ligands DLL1, DLL4, JAGGED1 (1) and JAGGED2 (unpublished results) in bovine Spz. The aim of this work was to evaluate the role of JAGGED1 and JAGGED2 in bovine Spz acrosome reaction. Viable frozen-thawed bovine Spz obtained following swim-up were incubated (2×107cells/ml) in TALP-Sperm with heparin and JAGGED1 (Abcam) or JAGGED2 (Abcam) protein (10, 50 and 100 ng/ml) for 3 hours. Samples were then treated with Calcium Ionophore A23187 (0.7mM) to induce the acrosome reaction or left untreated to evaluate the spontaneous acrosome reaction. Control samples without JAGGED1 and JAGGED2 supplementation were run in each assay. Spz were then processed for immunofluorescence using PNA, allowing for the differentiation of three acrosome status: non-reacted (NR), reacting (R) and acrosome reacted (AR). Each experiment was performed using Spz from 3 bulls and a total of 400-800 PNA-stained Spz cells where analysed for each treatment. Data were analysed using chi-squared tests. JAGGED1 supplementation had the highest effect at 10ng/ml, enhancing the spontaneous acrosome reaction (15% versus 8%, p<0.0001) and decreasing the proportion of NR Spz in induced acrosome reaction samples (6% versus 12%, p<0.001). JAGGED2 supplementation had the highest effect at 10ng/ml and 50ng/ml, decreasing the spontaneous acrosome reaction (16% versus 6%, p<0.0001), and at 100 ng/ml increasing the proportion of NR Spz (12% versus 7%; p<0.05). These results may indicate that JAGGED1 and JAGGED2 proteins are involved in the regulation of the acrosome reaction, showing opposite effects: JAGGED1 promoting the acrosome reaction and JAGGED2 preventing the acrosome reaction (acrosome stability). Keywords: Spermatozoa; Acrosome Reaction; Notch; JAGGED1; JAGGED2.

Funding: CIISA-UID/CVT/00276/2013, from Fundação para a Ciência e a Tecnologia (FCT); CIISA12/2016 and CIISA26/2017 projects, FCT scholarship SFRH/BD/130536/2017. 1 - Diniz et al.(2016) Reprod Domest Anim, Vol. 51, Suppl2. Pg 85.

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PhD Contest Poster P5 Sivelestat and noscapine modulate collagen transcription and prostaglandin secretion in mare endometrium Amaral A1, Fernandes C1, Lukasik K2, Szóstek-Mioduchowska A2, Rebordão MR1,3, Baclawska A2, Morazzo S1, Pinto-Bravo P1,3, Skarzynski DJ2, Ferreira-Dias G1

1. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2. Institute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, Poland;3. Coimbra College of Agriculture, Polytechnic Institute of Coimbra, Coimbra, Portugal Mare endometrosis is an important cause of subfertility/infertility in mares. Neutrophil extracellular traps (NETs), besides killing pathogens in the uterus, may also contribute to endometrial fibrosis. Elastase (ELA) is a NETs component that may stimulate in vitro endometrial collagen deposition. Thus, the aim of this study was to evaluate the effect of sivelestat (SIV) – an ELA inhibitor - and noscapine (Nosc) – an anti-fibrotic agent - on collagen transcription and prostaglandin secretion, in different estrous cycle phases. Follicular-FP (n=8) and mid luteal–MLP (n=7) phase explants were cultured for 24 or 48h with ELA (0.5µg/mL,1µg/mL), SIV (10µg/mL), ELA (0.5µg/mL,1µg/mL) + SIV (10µg/mL), Nosc (45µg/mL) or ELA (0.5µg/mL, 1µg/mL) + Nosc (45µg/mL). Collagen 1 (COL1) gene transcription was determined by qPCR and prostaglandins (PGE2; PGF2α) secretion in culture medium was assessed by EIA. In FP, at 24h, ELA 0.5 increased COL1 transcription (P<0.001), but its inhibition decreased it and PGF2α production (p<0.05). Also, ELA 0.5 + Nosc (P<0.01) and ELA 1 + Nosc (P<0.05) decreased COL1 transcripts and PGF2α production. PGE2 production increased with ELA 0.5 + SIV; ELA 1 + SIV; ELA 0.5 + Nosc and ELA 1 + Nosc incubation (P<0.05). At 48h, ELA 1 increased COL1 transcripts (P<0.01) and PGF2α production (P<0.001), but ELA 1 + SIV and ELA 1 + Nosc decreased them (P<0.05). Besides, ELA 1 + SIV and ELA 1 + Nosc incubation increased PGE2 (P<0.05). PGF2α also augmented with ELA 0.5 (P<0.001), but lowered with ELA 0.5 + SIV (P<0.01) and ELA 0.5 + Nosc (P<0.0001). In MLP, ELA 0.5 up-regulated COL1 mRNA levels (24h, P<0.01; 48h, P<0.001), but ELA 0.5 + SIV (24h, P<0.05; 48h, P<0.001) and ELA 0.5 + Nosc (24h, P<0.01; 48h, P<0.001) decreased it. At 24h, PGE2 production was upregulated in explants incubated with ELA 1 + Nosc (P<0.05). At 48h, ELA 1 also increased COL1 transcription and PGF2α production (P<0.05), but PGF2α production decreased with ELA 1 + SIV incubation (P<0.05). ELA 0.5 + Nosc and ELA 1 + Nosc treatments decreased PGF2α production (P<0.05). PGE2 release was higher in ELA 1 + SIV incubation (P<0.05). In conclusion, inhibition of ELA pro-fibrotic action by sivelestat, a specific elastase inhibitor, and the use of noscapine, which acts on myofibroblast differentiation, may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE2 and inhibiting pro-fibrotic PGF2α. Keywords: elastase, noscapine, sivelestat, fibrosis, mare. Grants: UID/CVT/00276/2013 (CIISA, Portugal); PTDC-CVT-REP-4202-2014 (Portugal); “Iuventus plus” Polish Ministry of Science and Higher Education (0463/IP1/2015/73); SFRH/BD/101058/2014 (PhD scholarship).

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Poster 6 Artificial tick-feeding system as a tool to investigate new approaches in tick control

Zúquete ST1, Santa C2,3, Santos D1, Pereira da Fonseca I1, Manadas B2, Alfaro-Cardoso L1 1. CIISA - Center for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal. 2. CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga Faculdade de Medicina, Pólo I, 1º andar, 3004-504 Coimbra, Portugal. 3. III - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal Background: Ticks are the most important arthropod vectors in Europe and in the United States. In order to evade host´s rejection during feeding, ticks upregulate salivary proteins expression. Salivary molecules play crucial roles in anti-haemostasis, inflammatory response suppression and immune evasion processes. Essential to feeding, ticks cement-plug is a possible target for the development of innovative tick control. Artificial tick feeding systems reduce hosts’ contamination on cement-cones posing as a model to study its composition and adhesion mechanisms. Methods: Experiments were conducted with the long hypostome tick Hyalomma lusitanicum Koch, 1844. Ticks were fed on defibrinated bovine blood in vitro. Handmade silicon membranes were glued to plastic feeding units and inserted in 6-well plates. Inside each feeding unit, ticks were secured in with cloth lids. Plates were put to float in an aquarium securing adequate temperature and humidity. Blood was changed twice daily (12h±3). Cement samples were collected and kept in acetone until solubilized in Laemmli buffer. Mechanical disruption was done using ultrasonic energy and protein extraction from pellet was performed twice. Main technique used included short GeLC-MS, consisting on a partially run SDS-PAGE followed by protein digestion and relative quantification by liquid chromatography coupled to tandem mass spectrometry. For identification, samples were run against a database assembled by transcriptomics analysis, against non-reviewed Ixodidae databases (Uniprot and GeneBank) and against bovine and rabbit databases. Results: Ticks were allowed to feed to repletion and originated viable larvae. A total of 125 proteins were identified but from these 109 relate to bovine, rabbit or other species and only 19 have correspondence to Ixodidae databases (3 ambiguous). Ixodidae matches were assembled accordingly to their gene ontology. Although, some correspond to putative cement proteins the majority of our findings relate to other sialome components: protease inhibitors; lipocalins; glycine-rich or proline-rich, collagen-like superfamily and enzymes (metalloproteases). Conclusions: An artificial feeding system is a tool to ticks’ cement composition disclosure and an alternative to reduce animal experimenting. The system was well adapted to H. lusitanicum biological needs. Such system is transferable to other areas of tick research, namely, pathogen-tick interactions and ivermectin resistance studies. Keywords: Cement – ticks – Artificial-feeding - control Funding Acknowledgements: This work is funded by National Funds through FCT - Portuguese Foundation for Science and Technology: Projects PTDC/CVT-EPI/3460/2012, CIISA – project UID/CVT/00276/2013 and POCI-01-0145-FEDER-007440, and CS is supported by a PhD fellowship SFRH/BD/88419/2012; and by the National Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125).

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Poster 7 Tubulin Cofactor B: a key player in Toxoplasma gondii host cell invasion Francisco S1, Delgado ILS1, Felix-Stortz J2, Jimenez-Ruis E2, Meissner M2, Zabala JC3, Leitão A1, Soares H4,5, Nolasco S1,4 1. Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Portugal. 2. Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. 3. Departamento de Biología Molecular, Facultad de Medicina, IDIVAL-Universidad de Cantabria, Santander 39011, Spain. 4. Escola Superior de Tecnologia da Saúde de Lisboa, Lisboa, Portugal. 5. Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Portugal Background: Tubulin cofactors participate in the folding, dimerization, and dissociation pathways of the tubulin dimer; being implicated in the control of tubulin proteostasis and consequently in the control of microtubule dynamics in vivo. We hypothesise that these proteins have a role in the microtubule cytoskeleton modifications observed during Toxoplasma gondii host cell invasion. In this context, we characterized the Tubulin cofactor B (TBCB) in T. gondii. TBCB is a CAP-Gly domain-containing protein that together with TBCE, interact with and dissociate the tubulin dimer. Methods: The TBCB sub-cellular localization in T. gondii was performed using an in-house anti-TBCB serum. TBCB overexpression T. gondii lines were obtained by random integration as well as TBCB conditional knockout lines by CRISPR/Cas9 system. TBCB transgenic clones were characterized by growing assays (plaque, invasion, replication and egress assays), western blot analysis and fluorescent microscopy (standard, confocal and super-resolution). Results: TBCB showed a polarized localization, at the anterior region of the parasite, under the conoid in close association with polar ring and subpellicular microtubules. It didn’t present a clear co-localization with the apical complex secretory vesicles, although the interaction with rhoptries and micronemes cannot be excluded. TBCB overexpression lines showed a significant decrease in the capacity to form plaques, attributable to a proportional reduction in the capacity to invade. No differences were observed in replication and egress. The TgTBCB knockout line, showed a complete depletion of the protein and a viability no longer than a week. These lines showed a strong reduction in their capacity to invade the host cell and in their replication rate. In the absence of TBCB, cells have an altered axis of division resulting in abnormal division. Some parasites show loss of the correct division axis and some parasites have four daughter cells forming inside instead of two. Conclusions: TBCB is a polarity marker in T. gondii and is involved in the invasion and replication processes. Its apical localization, together with TBCB mammalian partners already described (microtubule associated proteins), suggest that TBCB can be involved in the intracellular traffic of secretory vesicles depending on microtubules. Importantly, TBCB is an essential protein, constituting a good target for new control strategies. Keywords: Toxoplasma gondii, host cell invasion, tubulin, tubulin-binding cofactors (TBCs), Tubulin-Binding Cofactor B (TBCB), overexpression, knockout Funding Acknowledgements: SFRH/BD/101619/2014 (I.L.S.D) and SFRH/BD/79423/2011 (S.F.) and grants UID/CVT/00276/2013 and EXPL/CVT-EPI/1945/2013.

16 November 16:20 - 17:20 Session 2 Oral Communications - CIISA Post-Docs and Junior Researchers Chairpersons - Rui Bessa and José Santos Silva

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New insights on the ruminal biohydrogenation pathways of unsaturated fatty acids

Alves SP, Bessa RJB

CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa

The microbial processes that occur in the rumen, i.e. lipolysis of dietary esterified lipids and biohydrogenation of the non-esterified unsaturated fatty acids (UFA), affect the nutritional composition of ruminant derived foods. Biohydrogenation is the main process responsible for the extensive metabolization of the UFA into saturated end-products, limiting the escape of UFA from the rumen and deposited in tissues. At CIISA, we have been conducting several studies based on in vitro batch incubations with rumen inoculum to provide new information about the metabolization of UFA in the rumen. The final aim is develop the tools to increase the content of UFA in meat and milk. And so, we studied the ruminal biohydrogenation of ricinoleic acid (ROA, 12-OH,cis-9–18:1), linolenic acid (LNA, 18:3n-3) and eicosapentaenoic acid (EPA, 20:5n-3). The results of the in vitro experiments with ROA showed that it was metabolized in the rumen at a slow rate and the main products formed were 12-OH-18:0 and 12-oxo-18:0, by hydrogenation of the cis-9 double bond, followed by oxidation of the hydroxyl group, respectively. Our results also suggested that the 12-OH-18:0 and 12-oxo-18:0 escape rumen and are further metabolized through partial β-oxidation in ruminant tissues. In vitro experiments with 13C labelled and unlabelled LNA confirmed the origin of several fatty acids including the t10,c15-18:2 but surprisingly our results revealed that the t10-18:1 is not relevantly produced from the biohydrogenation of LNA in the rumen in contrast which have been proposed by some authors. Thus, we have proposed new biohydrogenation pathways of LNA. From the in vitro experiments with EPA from different sources, including deuterated-EPA, we demonstrated for the first time the kinetics of EPA biohydrogenation products in the rumen and the unequivocal formation of 20:0 from EPA. We also found that a marine microalgae, Nannochloropsis oceanica as a strong potential to be used as a natural source of rumen protected EPA to ruminants. As conclusion, we have identified novel biohydrogenation intermediates and refined the biohydrogenation pathways for several unsaturated fatty acids. This knowledge provides a solid base to future regulate meat and milk lipid composition by the host animal. Keywords Rumen, biohydrogenation, fatty acids, lipids, in vitro Funding Acknowledgements Fundação para a Ciência e a Tecnologia (FCT), Portugal, in the framework of the Project UID/CVT/00276/2013 (including CIISA projects: CIISA-10/2015 and CIISA-25/2017) and the project PTDC/CVT/120122/2010 are greatly acknowledge. S.P.A. also acknowledges the support of the FCT through the grant SFRH/BPD/76836/2011.

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Feeding strategies to improve fat partitioning and meat quality in pigs: from genetic background to dietary protein and amino acids Madeira MS, Alfaia CM, Pires VMR, Lopes PA, Prates JAM CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa

Commercial pig breeds produce very lean meat with poor eating quality. Thus, it is necessary to develop feeding strategies to increase intramuscular fat (IMF) content and consequently to improve meat quality. We hypothesized that the increased IMF content, pork eating quality and carcass fat partitioning in pigs induced by crude protein reduction in the growing–finishing phase of commercial crossbred pigs or Alentejano purebred could be modulated by dietary supplementation of amino acids or both without major undesirable effects on productive and carcass traits. In a first experiment, three diets differing in dietary protein (17.5 vs. 13.0%) and lysine levels were applied to two genetic divergent pig genotypes (Alentejano purebred and commercial crossbred pigs). In a second experiment, six diets differing in dietary protein (16 vs. 13%) and arginine and/or leucine supplementation were applied on commercial crossbred pigs. The third experiment was conducted with five diets with different protein levels (16 vs. 13%) supplemented with betaine and arginine on commercial crossbred pigs. The results from the first experiment showed that RPD increase IMF but decrease the productive performance in commercial crossbred pigs but not on Alentejano pigs, suggesting that lysine restriction mediates the effect of RPD on muscular lipid deposition. This result reflects a tendency for higher sensory scores in crossbred pigs. In the second experiment, reduced protein diet increased both IMF and backfat thickness. The increase of IMF was associate by increased SCD expression but did not improved meat sensory traits. Neither arginine nor leucine dietary supplementation increased IMF content. In the third experiment data confirmed that RPD increase IMF content and total fat content in SAT. Also, betaine and Arginine supplementation of Lys-deficient diets does not further increase IMF content but improves some pork sensory traits, including overall acceptability. Dietary betaine supplementation slightly affected fatty acid composition, although the arginine-supplemented diet did not influence fat content or fatty acid composition in any of these pig tissues. The results evidence that adipogenesis and lipogenesis are differently regulated in pig muscle and SAT and is specific of the genotype. These data contribute to understand the mechanisms of dietary regulation of fat partitioning in pigs and, therefore, could help improve pig feeding strategies to address industry needs and consumer demands. Keywords: pigs; reduced protein diets; amino acids; intramuscular fat; pork quality; lipid metabolism. Acknowledgements: This study was supported by Fundação para a Ciência e a Tecnologia (FCT) Grant (PTDC/CVT/2008/99210), CIISA project (UID/CVT/00276/2013) and an individual fellowship to M. S. M. (SFRH/BPD/97432/2013).

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Effect of two feeding levels on growth and development of the Lusitano foal Fradinho MJ1, Fernandes R2, Botelho M3, Francisco P4, Martin-Rosset W5, Santos AS6, Ferreira LMM6, Ferreira-Dias G1, Bessa RJB1, Caldeira RM1

1. Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, ULisboa, Av. Universidade Técnica, 1300-477 Lisboa, Portugal; 2. Faculdade de Medicina Veterinária, ULisboa, Portugal; 3.Coudelaria Henrique Abecasis,2050-041 Aveiras de Baixo, Portugal; 4.Coudelaria Ferraz da Costa, Vila Verde Ficalho, Portugal; 5.INRA, Centre de Recherche de Clermont-Ferrand/Theix, 63122 Saint Genes Champanelle, France; 6.CITAB-UTAD, Quinta de Prados, 5001-801 Vila Real, Portugal

Growth and development are particularly high during the first year of horse life, with body weight gain and skeletal growth closely related with feed intake level. Because growth rate has been associated with the occurrence of bone disorders, the knowledge of the most adequate growth model for each breed and purpose, as well as the factors that influence skeleton development, are fundamental for breeders and users. The present study was performed in order to evaluate the effect of two feeding levels on growth and development of Lusitano foals, on improved extensive systems. Eighty-nine Lusitano foals from two stud farms were monitored from birth to 3 years of age. During gestation and lactation periods their mothers were fed either 100 or 130% of their requirements according to INRA recommendations. Until weaning, foals were kept with their dams in the pasture and mares were fed accordingly to their group. From the 4th month onward, creep feed was distributed to all foals. During weaning and until the end of the study, the two groups of foals were respectively fed according to INRA recommended allowances for a moderate (M) or an optimized (O) growth level proposed for sport breeds. Foals were periodically weighed and measured, and a total of 7,008 records were obtained for body weight (BW), withers height (WH), girth (G) and cannon circumference (CC). The Richards growth function 𝑦 =𝐴(1 − 𝑏. 𝑒−𝑘𝑡)𝑀was fit to data using the NLIN procedure of SAS, and differences between growth models were analysed using the sum of squares reduction test. Growth models were different for BW, WH, G and CC (P<0.001), with higher values observed for O level. Estimated values at 36 months were 471 kg (M) vs. 508 kg (O) for BW, 157.1 cm (M) vs. 159.8 cm (O) for WH, 182.5 cm (M) vs. 187.9 cm (O) for G and 20.3 cm (M) vs. 20.6 cm (O) for CC. The results concerning body segments which have a bone base, in particular WH and CC, may support the option of breeders for optimized growth levels. However, the eventual effects of these growth levels on foals’ osteoarticular quality should be investigated. Keywords: Lusitano horse, growth and development, nonlinear models, feeding levels. Funding Acknowledgements: Projecto Proder Nº 23957 and Projecto CIISA UID/CVT/00276/2013.

16 November 17:20 - 18:00 Session 2 Poster Presentations - CIISA PhD students

Chairpersons - Rui Bessa and José Santos Silva

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PhD Contest Poster P8 Combined effect of nisin loaded pectin nanoparticles and high hydrostatic pressure on the extension of alheira’s shelf life Borges A1, Krivorotova T2, Sereikaite J2, Gama LT1, Fernandes MH1, Fernandes MJ1, Barreto A1, Fraqueza MJ1 1. CIISA. Faculty of Veterinary Medicine. University of Lisbon. Lisbon, Portugal; 2. Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius, Lithuania Alheira is a traditional Portuguese smoked and fermented meat sausage produced in Trás-os-Montes, a region in the north of Portugal. Its industry represents an important economic resource for the region. Bad practices can occur during alheira’s manufacture, storage and cooking, exposing consumers to microbial hazards. Considering these facts and with the aim of promoting its sustainable production, it is important to apply alternative technologies such as high hydrostatic pressure (HHP) and nisin loaded pectin nanoparticles (NLPN) to assure alheira’s safety and increase its shelf life while maintaining their organoleptic authenticity. Three batches of vacuum-packaged alheira were produced according to 4 conditions: without treatment, NLPN treatment, HHP processing (600MPa for 390 seconds at 10°C) and a combination of both treatments. The addition of NLPN was done during the mixing step of the process and the HHP processing was done after packaging. Microbiological analysis was carried out 1 day after treatments and then every month during a storage time of 5 months under 4ºC. C. perfringens and Listeria spp. were not detected in any of the samples analysed. The total aerobic microorganisms at 30ºC and lactic acid bacteria counts were over 7-8 log cfu/g before the application of technologies in study. The application of HHP significantly (P<0.05) reduced the levels of all microorganisms analysed and this effect was maintained over the time. The benefit of the NLPN addition was only verified in the coagulase-negative Staphylococci counts, maintaining this effect during the storage time. Moreover, the application of HHP on alheira produced with NLPN showed a synergistic effect, with better results when compared with those obtained when the technologies were applied alone (P<0.05). Overall, HHP combined with NLPN had a synergistic effect, and was considered the most suitable process for preserving and extending alheira’s shelf life. This study emphasizes the usefulness of using the synergism of two technologies to extend the shelf life of a fermented sausage and assure safety. Keywords: Emergent Technologies, Alheira, Fermented Meat Product, Shelf life, Synergism. Funding Acknowledgements: This work was conducted with the financial support of the project PRODER medida 4.1- Cooperação para a inovação no. 56345 “Novos produtos derivados da alheira” and CIISA Project UID/CVT/00276/2013.

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PhD Contest Poster P9 A putative new Campylobacter species potentially associated with Bovine Genital Campilobacteriosis Pereira G, Silva MF, Carneiro C, Duarte A, Mateus L, Lopes da Costa L, Silva E

Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal Bovine Genital Campilobacteriosis (BGC), a venereal disease caused by Campylobacter fetus subsp. venerealis (Cfv), is of worldwide concern in extensive beef herds, where natural mating is predominant. The bull, asymptomatic carrier, is the disseminator of the infection, which, in the case of pregnancy, induces embryo-fetal mortality (1). Recently it was suggested that the presence of several genomic components (virB/virD4 genes) of the Type IV Secretion system (T4SS) are associated with Cfv pathogenic potential (2). The objective of this study was to identify the etiologic agent of a clinical presentation compatible with BGC in farms at Alentejo. Preputial scrapings were collected from 16 bulls in 3 farms presenting BGC clinical evidence, for Cfv molecular identification. Isolation of Cfv was attempted in samples from one of those farms, according to standard techniques for Campylobacter species (3, 4), including transport of samples in selective enriched transport medium, but also transport in PBS and streaking into blood-agar plates. Cfv suspect colonies were submitted to biochemical identification (API Campy, Biomerieux), a conventional multiplex PCR directed to Campylobacter species identification (5) and the sequencing of a region of the 16S rRNA gene of the Campylobacter genus (6). Additionally, the isolates were screened by PCR for the presence of T4SS components and using qPCR assays based on two targets previously described for Cfv diagnostic purposes (parA gene and insertion element ISCfe1) (7, 8). These latter qPCR assays were also used directly on DNA extracted from the preputial samples. The isolation of Campylobacter-like colonies was only successful with the transport in PBS. However, in these colonies, both the biochemical identification and the multiplex PCR, although the later identified the Campylobacter genus, failed to identify the Campylobacter species. Also, the attempt to identify the species based on the sequencing of a fragment of the 16S rRNA gene was ineffective, since the best homology observed (96%) is far from the expected in isolates of the same species (over 98.7 %) (9). Surprisingly, the isolates were positive for the presence of several T4SS components (VirB4-6, VirB9, VirB11, VirD4) and for the parA and ISCfe1 qPCR assays. DNA extracted directly from the preputial samples was also positive for these qPCR assays and the T4SS components PCR. In conclusion, a Campylobacter species with Cfv molecular markers and T4SS virulence factor coding genes was isolated from the preputial fornix of bulls mating in farms presenting clinical evidence of BGC. Since the isolate’s 16S rRNA gene homology diverges over 1.3% with its phylogenetically closest species it’s very likely that we are in the presence of a new Campylobacter species, inhabitant of the preputial mucosa, with genomic virulence potential and putative clinical relevance. Keywords: Bovine Genital Campylobacteriosis; Campylobacter fetus subsp. venerealis species identification Funding: CIISA-UID/CVT/00276/2013; PhD scholarship-SFRH/BD/130923/2017 from Fundação para a Ciência e a Tecnologia (FCT); FCT-FEDER (Fundo Europeu de Desenvolvimento Regional) financed project BISCAMP (Projecto nº 30145). (1) Sahin O et al., 2017. Annu Rev Anim Biosci, 5: 21-42. (2) van der Graaf-van Bloois L et al., 2016. PLoS One. 11. (3) Chaban B et al., 2013. Am J Vet Res, 74(8):1066-9. (4) Monke HJ et al, 2002. J Vet Diagn Invest. 14(1):35-9. (5) Yamazaki-Matsune W et al, 2007. J Med Microbiol. 56(Pt 11):1467-73. (6) Iraola G et al, 2016. BMC Veterinary Research 12:286. (7) McMillen L et al., 2006. J Clin Microbiol, 44(3):938-945. (8) Abril C et al, 2007. Clin Microbiol Infect 13: 993–1000. (9) Stackebrandt E et al, 2006. Microbiol Today 33:152–5.

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PhD Contest Poster P10 Evaluation of molecular diagnostic methods for detection of Campylobacter fetus subsp. venerealis in bovine preputial samples Silva MF, Pereira G, Duarte A, Mateus L, Lopes da Costa L, Silva E Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal

Campylobacter fetus subsp. venerealis (Cfv) is the pathogen responsible for Bovine Genital Campylobacteriosis, a venereal disease associated with poor reproductive performance. Molecular-based diagnostic methods are considered to show higher sensitivity and specificity than culture-based methods. However, the specificity and sensitivity of molecular methods are often assessed with Campylobacter spp. strains rather than clinical samples, resulting in a lack of information on their diagnostic value in field samples (1, 2).The most studied targets for Cfv specific detection are the parA gene and the genomic insertion element ISCfe1, which have been used for diagnostic purposes. The objective of this study was to compare qPCR assays designed for the detection of Campylobacter fetus (Cf) species and subspecies (Cfv targets) in order to evaluate their performance on bull preputial samples. DNA samples extracted from 260 bulls’ preputial washings were screened for the presence of parA (2) and ISCfe1. A subset of the positive samples for Cfv targets were also tested for Cf species targets, including nahE (1) (n=79) and 16S rRNA (3) (n=73) genes, and a commercial diagnostic kit (VetMAX C. fetus kit, Life Technologies; n=31). Results were analysed using McNemar’s test. The qPCR for Cfv-specific subspecies targets ISCfe1 and parA detected 137 (52.7%) and 87 (33.5%) positive samples, respectively (p<0.05). Inconsistent results between these two targets were found in 80 samples (31%): ISCfe-1 positive and parA negative (n= 65; 25%), and ISCfe-1 negative and parA positive (n= 15; 6%). None of the field samples yielded a positive Cf species result. The higher number of positive ISCfe1 than parA positive samples may be associated to the existence of a higher number of genomic ISCfe1 than parA copies, allowing a higher sensitivity of the ISCfe1 assay, compared to the parA assay. However, the lack of agreement between these two Cfv qPCR detection methods (in particular the case of ISCfe1 negative and parA positive samples) is suggestive of horizontal gene transfer to other Campylobacter species, conferring a lower specificity to the parA assay, compared to the ISCfe1 assay. For the first time, both Cfv subspecies specific targets were simultaneously detected in the absence of positive Cf species specific targets. This intriguing finding may be associated either to a low sensitivity of the species targets assays or the horizontal transfer of both subspecies targets to other Campylobacter species. These results highlight the importance of re-evaluating current molecular diagnostic approaches. Keywords: Campylobacter fetus subsp. venerealis; Bovine Genital Campylobacteriosis; molecular diagnosis. Funding: CIISA-UID/CVT/00276/2013 from Fundação para a Ciência e a Tecnologia (FCT); BISCAMP (Project nº 30145 from FCT and Fundo Europeu de Desenvolvimento Regional (FEDER); FCT Scholarship: SFRH/BD/125657/2016. (1) van der Graaf-van Bloois L. et al., 2013. J Microbiol Methods, 95(1): 93-97. (2) McMillen L. et al., 2006. J Clin Microbiol, 44(3):938-945. (3) Iraola G. et al., 2016. BMC Vet Res, 12: 286.

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PhD Contest Poster P11 Portuguese nase (Iberochondrostoma lusitanicum) Aeromonas spp. biosurveillance program – filling a gap in endangered freshwater fish species conservation Grilo M1,2, Sousa-Santos C2, Tavares L1, Robalo JI2, Oliveira M1 1. Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2..MARE – Marine and Environmental Sciences Centre, ISPA Instituto Universitário, Portugal Background: Health assessments aiming at the direct evaluation of species’ fitness and ecosystem quality are lacking in Portuguese freshwater fish conservation programs. In Portugal, 6 cyprinid species are considered to be at risk of extinction. Among them, the Portuguese nase (Iberochondrostoma lusitanicum) presents a wider and more diverse habitat distribution, being an ideal model species for epidemiological comparisons of host-pathogen evolution in different anthropogenic and environmental scenarios. Bacterial infections are important causes of disease in fish, with Aeromonas spp. infections being the most frequent in freshwater species. Due to its impact and distribution, they represent suitable model species to unravel the impact of bacterial infections in fish populations. Methods: To evaluate the prevalence of Aeromonas spp. in I. lusitanicum during dry (September 2017) and wet seasons (February 2018), 64 individuals were collected at each season by electrofishing. Collection sites included 4 independent river basins presenting different associated human density and water quality levels: Jamor, Laje, Lizandro and Samarra (Lisbon district). Fish’s skin was swabbed for microbiological analysis and morphometric data of each individual were collected. Presence of hemorrhagic or ulcerative lesions was quantified. Bacteria isolation was performed using selective media, allowing the presumptive identification of Aeromonas spp. isolates. Chi-square analysis was performed to test independence between prevalence of lesions and sampling site (IBM SPSS Statistics®, version 24). Results: Aeromonas spp. prevalence in I. lusitanicum varied according to season (dry – 100%; wet – 83%), but was considered high at 4 sampling locations. Lesions’ prevalence was different among seasons (p≤0.05), ranging between 94.6% (Jamor) and 67.5% (Lizandro) in the dry season, and between 48.6% (Laje) and 22.5% (Samarra) in the wet season. Fish lesions’ rate was not independent from sampling site in the dry (p<0.001) and wet seasons (p<0.05). Standardized residuals analysis showed that dependency was influenced by the rate of fish not displaying lesions in Lizandro in the dry season and Samarra in the wet season. Conclusions: Results confirm the presence of a potential fish pathogen in populations of the critically endangered I. lusitanicum. Furthermore, seasonal and local differences observed may help to understand factors influencing Aeromonas spp. prevalence in I. lusitanicum. Keywords: Iberochondrostoma lusitanicum; Aeromonas spp.; biosurveillance; conservation medicine. Funding Acknowledgements: Work was supported by CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). Miguel Grilo holds a PhD fellowship (C10571K) from University of Lisbon. Authors would also like to thank MARE, Marine and Environmental Sciences Centre - ISPA (UID/MAR/04292/2013), Cristina Lima, Pedro Duarte Coelho and Sónia Fernandes (ISPA) and Sara Valente (FVM-ULisbon).

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Poster 12 Study of insect bite hypersensivity (IBH) in Lusitano horses in Portugal – preliminary contribution to the development of immunotherapy Pessoa VP1, Marti E2, Branco Ferreira M3, Tilley P1

1. CIISA (Centro de Investigação Interdisciplinar em Sanidade Animal / Centre for Interdisciplinary Research in Animal Health), Department of Clinics, Faculty of Veterinary Medicine, University of Lisbon, Portugal. 2. Department of Clinical Research and Veterinary Public Health, University of Bern, Bern, Switzerland; 3. Immunoalergology Unit, Internal Medicine Department, Faculty of Medicine, University of Lisbon, Portugal.

Background: Insect Bite Hypersensitivity (IBH) is a recurrent, seasonal pruritic dermatitis, and mainly a type I hypersensitivity reaction (IgE mediated) to salivary allergens from Culicoides spp, that affects a large number of horses worldwide. Though prevalence of HPI in Portugal is not yet known, the environmental characteristics are favourable to the activity of Culicoides spp, and common knowledge shows high occurrence in valuable Lusitano stud farms. This study describes the results of skin allergy tests, Intradermal (IDT) and Skin Prick Tests (SPT), performed in Lusitano horses with clinical diagnosis of IBH. Methods: 30 healthy controls and 30 IBH horses were tested. IBH horses were included based on medical history and physical examination supported by questionnaires and photos. They were all submitted to skin tests on the neck with 14 specific allergens, 13 different recombinant proteins from Culicoides spp. salivary glands (termed Cul1 to 11, Cul O1 and Cul O2) and C. nubeculosus (CN) whole salivary extract. Furthermore, 12 horses were also tested with another 6 allergens, 4 allergens from recombinant C. nubeculosus and 2 from recombinant C. obsoletus. The allergen concentrations used were 10µg/ml for IDT and 100µg/ml for SPT. Wheal diameters were assessed at 20 min (SPT/IDT), 6 hours (IDT) and 48 hours (IDT). SPT were considered positive when wheal diameter ≥0.9 cm, and IDT when wheal diameter was ≥50% of the histamine wheal. Results: Statistical differences were observed, between test and control groups, for CN, Cul 1, 7, 8, 9, CO23, CO110 (p<0.05); in the second allergen panel, statistical differences were observed for Cul 4 BAc, Cul 3 barley and COExt (p<0.05). Most animals studied seemed to be allergic to at least 4 of these allergens. CN was the allergen that all IBH horses were positive to, Cul9 being the second most frequent. Conclusions: Our results indicate that CN, Cul1, 7, 8 and 9 and CO110 and CO23 may potentially be the more relevant allergens in IBH in the population of horses studied. For the new allergen panel, although tested only in a reduced number of horses, we consider that CoExt, CulO3Bar and CulO4Bac, can potentially be relevant for IBH. SPT may be useful in IBH diagnosis, at a concentration of 100µg/ml, and readings assessed at 20 min. Key Words: Insect bite hypersensitivity, Allergens. Funding acknowledgements: This research was supported by CIISA Coordenação – Centre for Interdisciplinary Research in Animal Health hosted by the Faculty of Veterinary Medicine, University of Lisbon (CIISA-FMV-ULisboa).

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Poster 13 The use of Cistus ladanifer condensed tannin extract as additive to improve the soybean meal protein efficiency in lamb diets Dentinho MTP1,2, Paulos K1, Francisco A1,2, Belo AT1, Jerónimo E3,4, Almeida J1, Santos-Silva J1,2, Bessa RJB2,5

1. Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Fonte Boa, 2005-048 Santarém, Portugal; 2. Centro Investigação Interdisciplinar em Sanidade Animal (CIISA), Avenida Universidade Técnica, 1300-477 Lisboa, Portugal; 3.Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL), 7801-908 Beja, Portugal 4. Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, 7000 Évora, Portugal 5. Faculdade de Medicina Veterinária, Universidade Lisboa, Avenida Universidade Técnica, 1300-477 Lisboa, Portugal

Cistus ladanifer L. is a shrub distributed around the Mediterranean basin and very abundant on the central and southern regions of Portugal. In recent decades, due to the land abandonment and recurrent fire events, the area occupied by C.ladanifer has expanded considerably leading to loss of biodiversity and further increase in forest fire risk due to the high flammability of this resinous shrub. The use of this natural resource in animal nutrition may be a feasible solution that can contribute for the autonomy and sustainability of these agricultural and forestry areas considered critical by the low profitability and fire hazards. Cistus ladanifer is rich in phenolic compounds, mainly flavonoids and condensed tannins (CT) in leaves and stems. Condensed tannins complex with protein and this ability has been extensively studied for application in ruminant nutrition to improve the feed protein efficiency, namely in protecting the dietary protein from excessive microbial degradation in the rumen. Our aim was to study the use of C.ladanifer CT as additive to improve the soybean protein efficiency in lamb diets. A metabolic trial was performed with three rumen-cannulated rams in a 3 × 3 Latin square design to evaluate the effect of the C.ladanifer CT on apparent digestibility, N balance, rumen microbial N supply, ruminal fermentation characteristics, in sacco rumen degradability and in vitro intestinal digestibility of soybean meal. Soybean meal (SBM) was treated with 0, 15 and 30 g/kg of C.ladanifer CT. The treatment with 15 g/kg of CT was the most favorable reducing the protein rumen degradability and increasing the amount of protein absorbed in the small intestine, without compromising the digestibility of the diet. Thus, a productive trial was conducted to evaluate the effect of SBM treated with 15 g/kg on DM of C.ladanifer CT on lamb´s growth, carcass and meat quality. Twenty four Merino Branco lambs were assigned to three dietary treatments based on hay and concentrate (15/85 (w/w)) and fed at 4% live weight. Concentrates were formulated to contain: 16% of crude protein (CP) with untreated SBM (Control); 12% of CP with untreated SBM (Restricted protein (RP)); 12% of CP with SBM treated with 15 g/kg on DM of C.ladanifer CT (RPT). With CT inclusion, a positive response on lamb’s growth and feed conversion ratio were obtained and carcass traits and meat quality were not affected by all treatments. Results suggest that C.ladanifer CT extract has potential to be used as feed additive to increase the digestive efficiency of SBM protein in lamb diets. Thus, the protein content of diets can be reduced without compromising lamb performances and consequently decrease feed costs. Keywords: Cistus ladanifer, condensed tannins, protein, soybean meal, lambs. Funding Acknowledgements: This project was supported by European Fund for Regional Development (ERDF) (ALT20-03-0145-FEDER-000023 - CistusRumen - Utilização sustentável da Esteva (Cistus ladanifer L.) em pequenos ruminantes – Aumento da competitividade e redução do impacto ambiental).

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Poster 14

Chewing lice of wild birds from Portugal: neglected group of ectoparasites

Tomás A1, Palma R2, Rebelo MT1, Pereira da Fonseca I3

1. Center for Environmental and Marine Studies/Department of Animal Biology, Faculty of Sciences of University of Lisbon, Campo Grande 1749-016 Lisbon, Portugal; 2. Museum of New Zealand Te Papa Tongarewa, P.O. Box 467, Wellington 6011, New Zealand; 3..CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Av. Universidade Técnica, 1300-477 Lisbon, Portugal

Background: Chewing lice are permanent, obligate and host-specific ectoparasites commonly found on birds. Although chewing lice are relatively benign parasites, when present in large numbers they can cause changes in flight performance, thermoregulatory capacity, body mass and survival of the birds. According to BirdLife International approximately 310 species of birds occur in Portugal. However, the number of louse species documented is very limited. This study was carried out to determine chewing louse species of wild birds from Portugal. Methods: This study involves birds captured in mist-nets during the scientific ringing sessions and birds admitted at the Wildlife Rehabilitation and Investigation Centre of Ria Formosa. Chewing lice were collected using two methods: 1) visual search and collect for chewing lice in the plumage of each bird, during approximately 2 minutes; 2) fumigation chamber method, where birds are exposed to ethyl acetate for 15 minutes. Phthiraptera species were slide-mounted according to the Canada Balsam technique and identified. Results: Chewing lice were found on 43 (21.9%) of the 196 specimens of wild birds examined, independently of the method applied. One hundred and twenty-two birds were only visually examined, with 26 (21.3%) being parasitized. While for the fumigation method, 74 birds were examined, with 17 (22.9%) infested. Infested birds comprise 12 species and the chewing lice identified belong to suborders Amblycera (14 species) and Ischnocera (7 species). Conclusions: This is the more exhaustive contribution to knowledge of avian chewing lice associated with birds in Portugal. Even though all host-parasite associations have been previously reported in other studies, we record 20 species of chewing lice for the first time from wild birds in Portugal. These findings contribute to the knowledge of avian chewing lice from important bird areas in Portugal. Keywords: Chewing lice; birds; wildlife; Portugal. Funding Acknowledgements: Thanks are due for the financial support to CESAM (UID/AMB/50017 - POCI-01-0145-FEDER-007638), to CIISA (UID/CVT/00276/2013), to FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.

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Poster 15

Use of insect meal for total replacement of soybean cake in laying hens Fortunato R1,2, Saavedra, M1, Nunes R3, Nardozi B4, Moreira O4, Murta D1,3,5 1. CIISA, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, PORTUGAL; 2. Rações Zêzere, Ferreira do Zêzere, PORTUGAL; 3. EntoGreen - Ingredient Odyssey, Santarém, PORTUGAL; 4. EZN – INIAV, Vale de Santarém, PORTUGAL; 5. CBIOS, Faculty of Veterinary Medicine, Lusófona University of Humanities and Technologies, Campo Grande, Lisboa, PORTUGAL Population growth prompt the adoption of more efficient and sustainable production methods. Moreover, as the feed ingredient demand increase, its prices in the world market follow, and thus the industry is looking for alternative and more sustainable protein sources. However, soybean meal and fishmeal are still the main protein sources. On the other hand, insects may be used as a novel feed ingredient as they are very rich in protein and energy and can be produced with agriculture by-products. Black soldier fly (BSF) is one of the main insect species to be used as feed and there are already some producers in Europe. Seventy-Five Lhomann Brown classic hens, fed with the same diet till twenty five weeks of age, were divided into five groups (A, B, C, D and Control) and caged in individual cages for twelve weeks. During this period, each group had access to a specific diet. The control diet was a standard laying hens diet, formulated and produced by Rações Zêzere® with 20% soy. The experimental diets had an identical formula as the control diet, however diet A had a total replacement of soy, and thus 20% insect BSF meal, diet B 15% BSF meal and 5% soy, diet C 10% BSF meal and 10% soy and diet D 5% BSF meal and 15% soy. BSF larvae, provided by Entogreen®, were dried and sterilized, but not defeated. The replace of soybean cake by BSF meal didn’t resulted in a significant change in the animal’s life weight. However, it was possible to identify a significant difference between groups in egg weight mean as the control group achieved a superior egg weight mean of 64,83g throughout the trial, and groups A, B and C achieved 63,55g, 62,44g and 63,80g, respectively (p<0.05). Regarding weekly egg mass, the hens in the control group achieved a significantly higher average, 435,57g per hen, while groups A, B and C achieved 421,69g, 406,27g and 414,28g per hen, respectively (p<0.05). However, the weekly egg mass per hen, didn’t shown a statistically significant difference these two groups. It was not possible to identify statistically significant differences between groups in lay percentage, average daily feed intake and conversion ratio. This study shown that insect meal can completely replace the use of soybean cake in laying hens feed with the egg mass being the only negatively affected productive parameter. Nevertheless, we believe that a lower replacement of soybean cake can be more effective, as it can contribute to better performances. Keywords: Laying hens, eggs, insect meal, black soldier fly, novel proteins Funding Acknowledgements: This study was conducted in the scope of the EntoValor project (POCI-01-0247-FEDER-017675).

17 November 10:50 - 11:50 h

Session 3 Oral Communications - CIISA Post-Docs and Junior Researchers

Chairpersons - José Prates and Alexandre Leitão

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Assembly of Ruminococcus flavefaciens cellulosome involves novel single binding mode cohesin-dockerin interactions Bule P1, Alves VD1, Israeli-Ruimy V2, Smith SP3, Gilbert HJ4, Najmudin S1, Bayer EA2, Fontes CMGA1

1. CIISA – Faculdade de Medicina Veterinária, ULisboa, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot 76100 Israel; 3. Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada; 4. Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom. Cellulosomes are highly specialized macromolecular nanomachines that centralize anaerobic bacteria’s efforts to hydrolyse plant cell wall polysaccharides, a major untapped source of carbon and energy. The assembly of the cellulosomal components occurs via highly ordered protein: protein interactions between cohesins, located in the scaffoldins, and dockerins, typically found in the enzymes. The genome of the ruminal bacterium Ruminococcus flavefaciens FD-1 revealed a particularly elaborate cellulosome system that is assembled from a library of more than 200 different components. In this study, a medium-throughput approach was used to investigate the molecular basis for the organization of R. flavefaciens cellulosomes. The genes encoding 48 representative dockerins and 10 cohesins were cloned and transformed into E. coli competent cells so that each strain contained one cohesin and one dockerin expressing plasmid, generating 480 different recombinant strains. The formation of recombinant cohesin-dockerin complexes was evaluated through SDS-PAGE, leading to the establishment of the various cohesin-dockerin specificities required to organize R. flavefaciens cellulosomes. To complement these studies and to elucidate the structural basis of cellulosome assembly in R. flavefaciens, we have solved the structures of four novel cohesin-dockerin complexes. Typically, the Coh-Doc complexes that incorporate enzymes into the cellulosome possess a dual binding mode of interaction. This means that the dockerin can bind the cohesin in two different orientations due to its highly symmetrical nature. R. flavefaciens cellulosomal enzymes, were shown to be incorporated directly into primary scaffoldins, or indirectly via an adaptor scaffoldin termed ScaC. Structural data of complexes containing enzyme-born dockerins bound to either primary scaffoldin cohesins or the cohesin of ScaC, have shown that the dockerins lack the structural symmetry required to support a dual binding mode. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structure of the Coh-Doc complex responsible for the interaction of adaptor scaffoldin ScaB with primary scaffoldin ScaA was solved. The data showed that this interaction also involves a single-binding mode revealing a novel mechanism of cellulosome assembly that challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility. Keywords: celulossome, cohesin, dockerin, protein-protein complexes, Ruminococcus flavefaciens Funding Acknowledgements: This work was supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008, EXPL/BIA-MIC/1176/2012 and PTDC/BIA-MIC/5947/2014

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Broadening the miRNA catalogue in livestock species: a contribution to the functional annotation of animal genomes consortium Amaral AJ1, Marthey S2, Hoffman A3, Lagnel J4, Haack F5, Pokharel K6, Palasca O7, Seemann S7, Arya A8, Gama LT1, Groenen MAM8, Kantanen J9, Crooijmans RPMA8, Giuffra E9, Stadler PF3, Anton C7, Madsen O8, Gorodkin J7 1-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisboa, Portugal; 2-INRA, UMR GABI, Jouy-en-Josas, France; 3- Bioinformatics Group, Department of Computer Science University of Leipzig, Leipzig, Germany; 4-INRA PACA, Montfavet Cedex, France; 5-Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany; 6-Natural Resources Institute Finland, Jokioinen, Finland; 7-Center for noncoding RNA in Technology and Health, Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark; 8-Wageningen University, WAGENINGEN, Netherlands; 9-INRA, UMR de Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France MicroRNAs play a crucial role in the regulation of gene expression. Their action is crucial in many biological processes and functions, such as cell development and differentiation, and in response to disease. Moreover, it has been shown that polymorphisms in miRNAs can be linked to diseases and complex traits. An improved annotation of miRNAs in domestic animals is therefore required in order to acquire a comprehensive understanding of their impact on livestock traits. For this purpose a working group was established for the development of analysis pipelines including for the automated processing of small-RNA-seq data in the framework of COST-Action FAANG-Europe. A total of 328 quality approved small-RNA-seq datasets available from public repositories for five livestock species (Gallus gallus, Sus scrofa, Equus caballus, Ovis aries and Bos taurus) was used to quantify miRNA expression in different tissues as well as to identify putative novel miRNA candidates. For Bos taurus, our analysis identified a total of 434 novel miRNAs, allowing an increase of 50% of the total miRNA (769) available in miRBase 21. Interestingly, for Ovis aries, we have identified a large number of putative novel miRNAs (261) that share the seed with pre-existing miRNAs in other species. We have further identified many putative novel miRNAs in pig (252), in chicken (172) and in horse (122). Currently we are studying the miRNA syntheny across species at functional and genomic level, in order to infer their involvement in functional pathways and how these are conserved in the different species. Furthermore, we will also present the occurrence of other types of ncRNAs, e.g. tRNAs and snoRNAs in the analyzed data, some of which are known to be processed into miRNA. We have created a comprehensive ncRNA annotation for the studied species as well as interspecies pairwise and multiple genome-wide alignments. We believe these findings will further contribute to the understanding of the functional genome of the studied species. Keywords: FAANG, miRNAs, livestock, regulation of gene expression, bioinformatics Funding Acknowledgements: AJA is supported by a Post-doctoral fellowship within IMAGE H2020 project (REF 677353-2). This abstract is based upon the work from COST Action FAANG-Europe (CA15112) supported by COST (European Cooperation in Science and technology) and co-funded by FCT grant UID/CVT/00276/2013.

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Gene silencing (siRNA) of Bos taurus prion-like genes, and its relation to progesterone (P4) and 17E-estradiol (E2) concentrations Pimenta J1,2, Pires VM2, Nolasco S2,3, Castelo-Branco P5, Apolónio JD5, Marques CC1, Lopes-da-Costa L2,4, Prates JA2,4, Azevedo ARA1, Fonseca EA1, Pereira RMLN1,2 1INIAV-Instituto Nacional de Investigação Agrária, I.P., Fonte Boa, 2005-048 Vale de Santarém, Portugal; 2CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal. Faculdade de Medicina Veterinária, Universidade de Lisboa. Av. Universidade Técnica, 1300-477, Portugal; 3ESTeSL, Escola Superior de Tecnologia da Saúde de Lisboa; 4Faculdade de Medicina Veterinária, Universidade de Lisboa. Av. Universidade Técnica, 1300-477, Portugal; 5Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro Background: In order to clarify and evaluate key questions related to the biological characteristics and physiological functions of sprn and prnp prion family genes in cattle female reproduction, with particular emphasis on their participation in the granulosa follicular cells steroidogenesis, we established a bovine granulosa cell (GC) culture system where we conducted post-transcriptional gene silencing (siRNA) of sprn and prnp prion family genes. Methods: We evaluate (by RT-PCR) a possible relationship between the referred gene silencing and the expression of two genes involved in the steroidogenesis pathway (CYP19A1 and CYP11A), as well as in the protein translation, by quantifying progesterone (P4) and 17E-estradiol (E2) concentration in GC culture medium by radioimmunoassay (RIA). Estradiol concentrations were determined by solid phase Radioimmunoassay method, using the ESTR-US-CT cisbio kit and the Wallac 1470 Gamma reader; and Progesterone concentrations where determined by using the Progesterone Kit in the Immulite 2000 Immunoassay System, both from Siemens Healthcare. Results: The group where we conducted the sprn silencing (that presented the lowest value for CYP11A1), also exhibited the lowest value for Progesterone (182.9 µg/L). However, the difference between this group and the control group (average=247,3 µg/L), was not statistically significant, which might be explained in part by a possible relatively shorter half-life of CYP11A1 compared to a more stable half-life of Pregnenolone (P5), and thus Progesterone (P4). Conclusions: In conclusion, we established a structured approach to consistently determine the relationship between these prion-family genes and steroid hormone biosynthesis, within the scope of bovine folliculogenesis pathways. Keywords: Gene silencing, prion-like genes, bovine granulosa cells, steroidogenesis Funding acknowledgements: Projects UID/CVT/276/2013 (CIISA) and ALT20-03-0246-FEDER-000021.

17 November 11:50 - 12:30 Session 3 Poster Presentations - CIISA PhD students

Chairpersons - José Prates and Alexandre Leitão

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PhD Contest Poster P16

African swine fever virus (ASFV) encodes for QP509L and Q706L, two RNA helicases, essential for viral replication

Freitas FB, Frouco G, Martins C, Ferreira F CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, 1300-477 Lisbon, Portugal

Background: ASFV is a double-stranded DNA virus that causes a fatal disease in domestic pigs and wild boar. It is solely controlled by the implementation of strict sanitary measures due to the fact that neither vaccine nor treatment are available to control the infection, leading to devastating social and economic impact in affected countries. The deeper knowledge on ASFV genes involved in viral replication and transcription open new insights towards the identification of candidates to design efficient vaccines and/or as antiviral drug targets. With this end we have characterized two putative ASFV Superfamily 2 RNA helicases (QP509L and Q706L). These type of enzymes are known to be essential for RNA mechanisms being involved in RNA elongation, termination and release among other roles in different viral infections and other biological systems. Methods: Phylogenetic analyses were performed to better clarify the relation between QP509L and Q706L RNA helicases; the homology pattern, among virulent and non-virulent isolates and also with other ASFV and non ASFV RNA helicases. The transcript levels for QP509L and Q706L were quantified by qPCR along the infection. Protein levels were also quantified by immunoblot in parallel with protein localization during the infection. siRNA assays were developed in order to uncover the role of these proteins during ASFV replication cycle in particular for transcription. Results: Phylogenetic analyses showed that QP509L and Q706L are conserved between isolates, although, some variation was found for different genotypes. Our analyses also showed that each of these enzymes (QP509L or Q706L) cluster individually with other proteins belonging to the NCLDV clade. Further experiments showed that QP509L and Q706L are actively transcribed from 4 hours post infection reaching a maximum peak of accumulation at 12 and 10 hours respectively. Immunoblot analysis revealed that both proteins co-localized in the viral factories at 12 hours post infection, however, QP509L was also detected in the cell nucleus. Moreover, Vero cells infected and transfected with siRNA against QP509L or Q706L presented a reduction of the late viral transcripts up to 46.2% and 77,7% respectively. siRNA effects were also extended to genome replication, with reductions up to 53.6% (QP509L) and 71.43% (Q706L). Finally, downregulation of QP509L and Q706L, drastically reduced the viral progeny up to 99.4% and 98.4% respectively. Conclusions: Our results suggest that QP509L and Q706L have non-redundant roles and are essential during viral infection, emphasising the potential use of these enzymes as target for drug and vaccine development against ASF. Keywords: ASFV, RNA helicases, vaccine design, siRNA. Acknowledgments: This work was supported by Fundação para a Ciência e a Tecnologia (CIISA-UID/CVT/00276/2013) and by the European Union's Seventh Framework Programme (FP7/2007-2013, Grant 311931, ASFORCE). FBF and GF were supported by doctoral scholarships from Fundação para a Ciência e a Tecnologia (SFRH/BD/104261/2014, SFRH/BD/89426/2012).

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PhD Contest Poster P17

A High-Throughput approach to assess microalgae cell wall disruption by enzymatic digestion

Coelho D1, Lopes PA1, Cardoso V2, Ponte P2, Madeira MS1, Alfaia CM1, Bandarra N3, Fontes CMGA1,2, Prates JAM1,2.

1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa; 2. NZYtech, Estrada do Paço do Lumiar, Campus do Lumiar, Ed. E, 1649-038 Lisboa, 3. DivAV, Instituto Português do Mar e da Atmosfera, Rua Alfredo Magalhães Ramalho, 1495-006 Lisboa

The microalgae Chlorella vulgaris and Arthrospira platensis exhibit high lipid and protein content, richness in carotenoids, vitamins and minerals, which make its use in monogastric animal feed quite appealing. However, microalgae display a recalcitrant cell wall that hinders the access to bioactive compounds, being therefore necessary to develop mechanical and non-mechanical cell wall disruption methods. A rational combination of 200 pre-selected Carbohydrate-Active enZymes (CAZymes), produced by High-Throughput (HTP) technologies, was tested according to its ability to degrade C. vulgaris and A. platensis cell wall. After the individual screening of the most functional enzymes, their characterization was assessed, in particular pH, temperature stability, resistance to proteolysis and ability to function in the animal gastrointestinal tract. These enzymes were combined into a Mix of 4 and 2 enzymes and incubated with C. vulgaris and A. platensis suspension, respectively. The disruption of microalgae cell wall was evaluated through an increase up to 1.21 mg/mL and 2.42 mg/mL of reducing sugars released in C. vulgaris and A. platensis treated with the Mix relative to no treatment (P<0.05) and (P<0.001). To further complement those findings, a new protocol to quantify oligosaccharides by HPLC was implemented, and found an increase by 8 and 7 fold in C. vulgaris and A. platensis treated with the Mix relative to no treatment (P<0.001), respectively. With resort of fluorescence microscopy, 47% reduction of fluorescence intensity was achieved in C. vulgaris and 36% in A. platensis by using Calcofluor White staining, a fluorochrome that evaluates cell wall integrity (P<0.001). Overall, these results indicate that: 1) both enzyme Mix lead to a partial degradation of recalcitrant cell wall, which may be advantageous in maximising the nutrients bioavailability of microalgae for monogastric diets, in particular and in facilitating the cost-effective use of microalgae for animal feed industry, in general; 2) the methodological approach herein presented has been proven as successful to assess microalgae cell wall digestion. Keywords: Chlorella vulgaris, Arthospira platensis, cell wall disruption, CAZYmes, methodological approach Funding Acknowledgements: This study was supported by Fundação para a Ciência e a Tecnologia (FCT, Lisbon, Portugal) through PTDC/CVT-NUT/5931/2014 grant, CIISA project (UID/CVT/00276/2013), and individual fellowships to DC (SFRH/BD/126198/2016) and PAL (Post-Doc, Portugal2020 08/SI/3399/2015).

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PhD Contest Poster P18

A novel functional strategy for the development of highly specific antibody drug conjugates for non-Hodgkin’s lymphoma André A1, Dias J1, Aguiar S1, Oliveira S2, Ministro J2, Gano L3, Correia J3, Tavares L1, Aires-da-Silva F1 1. Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal; 2. Technophage, Lisboa, Portugal; 3. Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Portugal. Background: Cancer is a leading cause of death worldwide, remaining one of the most challenging diseases to treat. As such, novel therapies are urgently needed to limit tumor relapse or progression and to promote better patient outcomes. Immunotherapies in the form of monoclonal antibodies (mAbs) have emerged as a clinically validated and effective treatment strategy. Due to their high specificity, activity, valuable pharmacokinetics and standardized manufacturing processes, mAbs have been considered a promising alternative to conventional chemotherapy, often associated with severe side effects and the development of mechanisms of resistance. Among mAb-based therapies, antibody-drug conjugates (ADCs) are considered one of the most promising strategies, combining the tumor selectivity with the cytotoxic potency of small molecules. Using the potential of single domain antibody fragments (sdAbs), we aim to develop a highly selective and potent new anti-cancer ADC therapeutic molecule. Methods: A panel of highly specific sdAbs against non-Hodgkin’s lymphomas (NHL) were selected using a novel functional approach of in vitro selections followed by an in vivo biopanning on a xenograft murine model. For that purpose, bioluminescence canine and human B-cell lymphoma xenograft models were established. For in vivo imaging, a stable RajiGFP+Luc and CLBL-1GFP+Luc cell lines were generated using a lentiviral system encoding firefly luciferase and green fluorescent protein (GFP) reporters. After GFP and luciferase expression confirmation by FACS and ELISA assays, 5x106 cells were injected subcutaneously in SCID mice to establish the tumor. When tumors reached 1000 mm3, a round of selection was performed. To identify the best sdAbs clones, expression and binding to Raji and CLBL-1 cells extracts were evaluated by ELISA and next generation sequencing (NGS). Results: A stable RajiGFP+Luc and CLBL-1GFP+Luc cell lines were generated. All xenograft mice presented tumor development. All tumors were efficiently monitored and quantified by bioluminescence imaging. The final screening on these in vivo models allowed the selection of a panel of highly specific sdAbs against NHL receptors. Expression and binding properties were assessed, confirming that sdAbs recognized both human and canine NHL targets. Conclusion: These results validate a novel functional strategy for in vivo antibody selection and contribute for the development of a promising sdAb-based therapy for NHL. Keywords: ADC, non-Hodgkin’s lymphoma, antibodies, animal model, phage display Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (BBB_BIO_0508_2014; IF_010100, SFRH/BD/131468/2017, and by UID/CVT/00276/2013).

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PhD Contest Poster P19

Development of a hyaluronic acid and chitosan nanoparticulated system for topical ocular delivery of erythropoietin Silva B1,3, Marto J1,4, São Braz B3, Delgado E3, Gonçalves LMD1,2

1. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa; 2. Cardiovascular Autonomic Function Laboratory, Faculty of Medicine, Universidade de Lisboa; 3. CIISA - Centro de Investigação Interdisciplinar em Saúde Animal, Faculty of Veterinary Medicine, Universidade de Lisboa; 4. Laboratório Edol, Produtos Farmacêuticos S.A., 2795-225 Linda-a-Velha, Portugal. Background: Recent studies in animal models of ophthalmic neurodegenerative diseases has shown that erythropoietin presents neuroprotective and neuroregenerative properties. Delivery to retina of recombinant human erythropoietin beta (EPOβ) through the subconjunctival route was effective. However, the topical route for EPO administration has not yet been studied. Drug delivery systems based on chitosan nanoparticles can improve drug topical ocular delivery by increasing contact time and mucoadhesion. These properties can be further improved by using hyaluronic acid, a natural polymer. Therefore, this study aimed to develop and evaluate the behaviour of a hyaluronic acid and chitosan nanoparticulate system for topical ocular delivery of EPOβ. Methods: The nanoparticles (NP) were prepared using an ionic gelation technique, with a low molecular weight chitosan and hyaluronic acids of three different molecular weights (50 kDa - AH1, 300 kDa - AH2, 3000 kDa – AH3). The nanoparticles were characterized in terms of size, zeta potential (ZP), polydispersity index (PI) and mucoadhesion properties. EPOβ covalently labelled with Oregon Green fluorophore was loaded into the NPs. In vitro release and permeation assays and ex vivo permeation assays were performed. The percentage of encapsulation efficiency (EE) and drug loading (DL) were determined. Ex vivo permeation assays were performed in Franz-type diffusion cells, using corneas, scleras and conjunctivas dissected from fresh pig eyes obtained from a slaughterhouse. Results: The NPs presented a size on the range of 260-300nm, a positive ZP (+29 - +30mV) and low PI (0.136-0.219). These parameters did not differ significantly for the three HA. The EPOβ EE and DL were on the range of 35-39 % and 17-18%, respectively. The kinetic Korsmeyer–Peppas model is describing the in vitro release, with n<0.5 corresponding to a Fickian diffusion mechanism, for all NPs. The results of the ex vivo assays with AH2 NPs showed that the conjunctiva is the most permeable membrane to EPOβ followed by sclera and cornea. Conclusions: The molecular weight of the HA used in this study does not interfere in the nanoparticles characteristics and release assays. The ex vivo assay showed that porcine ocular membranes were permeable to the nanoparticles with EPO. These promising results could support the use of NPs here developed as a system for topical ocular delivery of erythropoietin. Keywords: Erythropoietin, chitosan, hyaluronic, nanoparticles, ophthalmology. Support: Fundação para a Ciência e Tecnologia (FCT) (Pest-UID/DTP/04138/2014); Project UID/CVT/276/2013 (CIISA) and FCT scholarship: SFRH/BD/130476/2017.

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PhD Contest Poster P20

A potential role of the dll4 / notch signaling pathway in the stabilization of atherosclerotic plaques

Carinhas JG1, Djokovic D1*, Fernandes AC1, Trindade A1, Carmona D2, Duarte A1, Tavares AT3

1. Centro Interdisciplinar de Investigacao em Sanidade Animal (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Lisbon, Portugal; 3. Centro de Estudos de Doencas Cronicas (CEDOC), Nova Medical School, Lisbon, Portugal. *Current address: Departmento de Obstetricia & Ginecologia, Hospital S. Francisco Xavier - CHLO, Lisbon, Portugal

Background: Atherosclerosis is a degenerative disease of large arteries characterized by endothelial dysfunction, vascular inflammation, and the progressive development of intima plaques. As the atherosclerotic plaque develops, new vessels arising from the adventitial vasa vasorum grow into the media and intima layers, modulating plaque growth and stability. The new microvessels are fragile and leaky and may lead to intraplaque hemorrhages, thrombosis and vessel obstruction. Inhibition of atheroma neovascularization has therefore been proposed as an approach to restrict lesion growth and promote its stabilization. Methods: For this study, ApoE-/- mutant mice were used as an animal model of atherosclerosis induced by hyperlipidemia. When fed with a high-fat cholesterol-rich diet, these mice develop severe hypercholesterolemia and atherosclerotic lesions similar to those observed in human arteries. Functional analysis was performed on ApoE-/- mutant lines inducible for endothelial-specific Dll4 overexpression and subexpression (ApoE-/- Dll4lox/lox VE-Caderin-CreERT2 and ApoE-/- Tie2-rtTA TetO7-Dll4, respectively), and in cultures of aortic rings in three-dimensional collagen gels. Human atherosclerotic plaques from patients undergoing carotid endarterectomy were also analysed. Results: Dll4 is expressed in vasa vasorum of atherosclerotic plaques from ApoE-/- mice, from early to advanced stages of plaque development. With the progression of the lesion, vasa vasorum become more leaky and immature, and mRNA levels show an inactivation of Dll4/Notch signalling. Dll4 endothelial loss-of-function mice had a greater area of atherosclerotic lesion with higher microvascular density compared to controls (unstable and immature microvessels), with the opposite being observed in the mice with endothelial gain-of-function of this ligand. Also in aortic rings culture, treatment with Dll4Fc (Dll4/Notch pathway inhibitor) induced greater vessel growth, as opposed to the effect of this pathway enhancer (Dll4 protein immobilized). In human atherosclerotic plaques, analysis of DLL4 expression at different stages of lesion progression corroborates the hypothesis of the important role of this ligand in microvascular stability. Conclusions: The results suggest that Dll4 endothelial overexpression may promote the stabilization of microvessels and restrict the development of atherosclerotic plaques, evidencing the Dll4/Notch signaling pathway as a potential therapeutic target for the development of anti-angiogenic strategies in this pathology. Keywords: Apolipoprotein E, vasa vasorum, Dll4/Notch signalling, vascular maturation, plaque stabilization Funding acknowledgements: Project UID/CVT/276/2013 (CIISA); FCT SFRH/BD/73264/2010; PTDC/SAU-OSM/102468/2008

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PhD Contest Poster P21

Mutant prevention concentration of nisin towards canine oral enterococci Cunha E1, Janela R1, São Braz B1, Moreira da Silva J2, Tavares L1, Veiga AS3, Oliveira M1 1. CIISA - Centro de Investigação em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica de Lisboa, 1300-477, Lisboa, Portugal; 2. Virbac de Portugal Laboratórios, Lda., Rua do Centro Empresarial, Quinta da Beloura 2710-693, Sintra, Portugal; 3. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal. Background: New antibacterial strategies are urgent, as worldwide mortality rate associated with infections promoted by multi-drug-resistant (MDR) bacteria dissemination is increasing. Antimicrobial peptides (AMP), such as nisin, are promising alternatives for the treatment of MDR infections, since resistance and cross-resistance to these compounds has rarely been observed, as they directly disturb cell membranes’ integrity. However, AMP-based therapeutic protocols must not promote the selection and amplification of resistant mutants. The mutant prevention concentration (MPC) is an important parameter that allows to determine the antimicrobial concentration that impairs the formation of mutant strains, defined as the drug concentration at which no colony is recovered when a high inoculum is applied onto drug-containing agar plates. Methods: The aim of this study was to determine the MPC value of nisin regarding a collection of oral enterococci (n=20), obtained from dogs with periodontal disease, previously characterised. Nisin minimum inhibitory concentrations (MIC) ranging from 6.25 µg/mL to 600 µg/mL were applied onto Muller-Hinton agar plates, for MPC determination using a diffusion method. MPC was defined as the lowest nisin concentration that prevented the growth of any resistant mutant subpopulations after a 72-hour incubation period. Results: Nisin MPC values ranged from 400 µg/mL to > 600 µg/mL, with an average value of 437.5 µg/mL. Considering the nisin MIC average value previously determined for the same isolates (14.90 µg/mL), the MPC values obtained were 27 to 40-fold higher than the MIC ones, revealing the importance of MPC determination in order to establish accurate treatment protocols, based on the appropriate doses of antimicrobial compounds. Conclusions: To our knowledge, this was the first report of MPC determination for nisin towards against canine enterococci, being a useful method to antimutant dosing strategies. Keywords: Dogs; enterococci; mutant prevention concentration; nisin; periodontal disease. Funding Acknowledgements: Work was supported by CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) (Project UID/CVT/00276/2013 and internal project CIISA 5/2018).

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PhD Contest Poster P22

Characterization of gut microbiome of healthy companion animals from households and shelters Belas A1, Aboim C1, Leal R1, Mendes CI2,3, Marques C1, Carrico JA2, Pomba C1

1. Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2. Microbiology Institute, Molecular Medicine Institute, Faculty of Medicine, University of Lisbon, Portugal; 3. University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, Netherlands.

Background: Studies about gut microbiome of companion animals are still limited, especially in animals from shelters. The goal of this study was to characterize the gut microbiome diversity in healthy companion animals (dogs and cats) living in human households (contact with humans) or in shelters. Methods: DNA from fresh fecal samples obtained from cats [(N=22; N=11 from shelters and N=11 from households)] and dogs [(N=73; N= 50 from shelters and N= 23 from households] were extracted using DNA isolation Kit. The V4 region of the 16S rRNA gene was amplified and the resulting amplicons sequenced on an Illumina MiSeq platform. Sequences were clustered into operational taxonomic units picked at 99% sequence similarity and assigned taxonomy using Greengenes reference database. Analyses included the QIIME2 and SAS statistical package. A p < 0.05 was used. Results: The Principal Coordinate Analysis plot demonstrated that the gut microbiome from household and from shelter dogs were fairly well separated. Regarding cats with and without human contact, there was no evident separation of bacterial communities. The phylum Firmicutes was predominant in both species. Concerning the gut microbiome from both dog groups, there was also a dominance of Bacteroidetes, Fusobacteria and Proteobacteria phyla. There was no significant difference in Firmicutes and Bacteroidetes phyla frequency between househould and shelter dogs (p>0.05). However, in shelter dogs the phyla Fusobacteria and Proteobacteria were identified in higher percentages (94%, n= 47/50; 96%, n=48/50, respectively) than in the household dogs (65.2%, n=15/23, 30.4%, n=7/23, respectively) (p< 0.05). A clear dominance of the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria was identified in the core microbiota from household and shelter cats (p> 0.05). Conclusions: To our knowledge, this is the first study that compares the gut microbiome composition between household and shelter companion animals. In this study, we found a significantly higher frequency of Proteobacteria phylum in shelter dogs. This phylum is a microbial group of concern to veterinary medicine, as it includes several clinically important pathogens, which can contribute to dysbiosis. Despite the fact that shelter dogs can be clinically asymptomatic, this study highlights that they can be reservoirs of potential pathogenic bacteria and can contribute for its dissemination through fecal contamination. Keywords: Microbiome, gut, companion animals, household, shelter. Funding Acknowledgements: This work was supported by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the Programa Operacional Factores de Competitividade (COMPETE) and by National funds through the Fundacao para a Ciencia e a Tecnologia (FCT) [Interdisciplinary Centre of Research in Animal Health Project UID/CVT/00276/2013 and PET-Risk Consortium Project JPIAMR/0002/2016]. Adriana Belas (SFRH/BD/113142/2015) and Catia Marques (SFRH/BD/77886/2011) hold FCT PhD grants.

17 November 15:50 - 16:50 Session 4

Oral Communications - CIISA Post-Docs and Junior Researchers

Chairpersons - Luísa Mateus and Miguel Fevereiro

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Developmental versus oncogenic Notch function in the prostate Trindade A1, Pedrosa AR1,2, Graça JL1,3, Carvalho S1, Peleteiro MC1, Duarte A1 1. Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal. Current addresses: 2. Centre for Tumour Biology, Barts Cancer Institute, London, United Kingdom; 3. Instituto de Investigação em Ciências da Vida e Saúde, University of Minho, Braga, Portugal Tumour development commonly involves the re-activation of developmental molecular mechanisms. The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis. Current data on this subject are incomplete and disperse. We proposed to analyse Notch components transcription and expression, in developmental, adult normal and tumour bearing mouse prostate. Transcriptional analysis by RT-PCR and protein localization studies by immunohistochemistry/immunofluorescence analysis in histologic sections of prostatic tissues were carried out. The formation of the prostatic buds starts at E16.5 and is divided in 3 phases: 1-Specification; 2-Initiation and; 3-Elongation. During prostatic bud differentiation, Dll1, Notch2, 3 and 4 appear to be the Notch pathway components involved with the specification phase. For the initiation phase Dll1 remains unspecific and so seems to be less important. Notch3 also seems to be more important for the specification phase, loosing epithelial expression during the initiation phase. Jagged1 is detected at the onset of the Initiation stage and decreases transcription throughout the remaining stages. Dll4, Jagged2, Notch2 and Notch4 seem to be most involved with the Elongation phase of prostatic development. In the adult healthy mouse prostate, we observed that Dll1 and Dll4 are expressed in the luminal compartment, whereas Jagged2 expression is restricted to the basal and stromal compartments. Additionally, Notch2 and Notch4 are normally expressed in the luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer. As prostate tumour development takes place, there is up-regulation of Notch components, particularly, increased expression of Jagged1 and 2, Notch3 and Hey1. We have also detected the presence of activated Notch3 in prostatic tumor lesions that co-express Jagged1 and ultimately the Hey1 effector. In conclusion, our data indicates that the Notch axis Jagged1/Notch3/Hey1 is highly relevant for prostate cancer development. Interestingly, after the first stage of prostatic epithelial bud determination, a stage when the specific identity of what is to become a prostatic epithelial cell (luminal and basal) is first determined and the selected cells are still not terminally differentiated, Jagged1 and Notch3 expression is switched off during the following phase of prostate development and is kept off during the postnatal prostate maturation phase and in homeostasis only being detected again at the onset of neoplasic transformation. We believe that the current findings identify Notch3 and Jagged1 as prostatic oncogenes. Keywords: Notch, Prostate, Cancer, Embryology, Oncogenes Funding: Portuguese Foundation for Science and Technology; Grant numbers: PTDC/SAU-ONC/116164/2009; PTDC/SAU-OSM/102468/2008; PTDC/CVT/115703/2009; PTDC/SAU-ONC/ 121742/2010; UID/CVT/00276/2013; SFRH/BD/44964/2008; SFRH/BPD/110174/2015

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Besnoitia besnoiti protein disulfide isomerase: inhibition of its catalytic activity and of tachyzoite host cell invasion

Marcelino E1, 2, Morais J2, Lopes A 3, Santos D2, Novo C3, Leitão A1

1. CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa - Lisbon, Portugal; 2. Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias - Lisbon, Portugal; 3. Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa - Lisbon, Portugal Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and emerging in Europe. Despite the great economic losses associated with besnoitiosis, the disease has been underestimated and poorly studied having no effective therapy or a safe vaccine available. Protein disulfide isomerase (PDI), an essential enzyme for the acquisition of the correct three-dimensional structure of proteins, plays an important role in host cell invasion in the closely related Neospora caninum and Toxoplasma gondii and may represent a promising drug target. We have previously determined the B. besnoiti PDI gene sequence (accession number DQ490130) and produced a recombinant B. besnoiti PDI (recBbPDI) and truncated versions corresponding to the PDI domains a, b, b’ and a’c. By hybridoma technology we developed a panel of monoclonal antibodies (mAb). Four best performing mAb were selected and tested, by ELISA and western blot, for cross reactivity with N. caninum and T. gondii PDI, as well as for domain recognition of the truncated versions. The results obtained were overlapped with the in silico prediction of epitopes using BepiPred and Ellipro and the probable epitope recognition site determined. PDI activity was measured by a turbidimetric assay based on the polymerization of reduced insulin. Both recBbPDI and truncated version a´c (recBb-a´c) showed a dose dependent activity, that was sensitive to the PDI inhibitors bacitracin, tocinoic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4-chloromercuribenzoic acid (pCMBA). The activity of recBbPDI was also inhibited by three mAb (exception was mAb T8a), while recBb-a´c activity was not influenced by mAb presence. We showed that B. besnoiti PDI is part of the secretory-excretory compartment in tachyzoites. Using the above mentioned membrane impermeant inhibitors and anti-BbPDI mAb during the invasion of Vero cells by B. besnoiti tachyzoites we were able to decrease the invasion rate, which shows that this protein is probably involved in the host cell adhesion/invasion process. The inhibitions obtained, both in the host cell invasion ability and in the enzyme catalytic activity, confirm that PDI represents a potential target for the treatment and/or prevention of besnoitiosis. Keywords: Besnoitia besnoiti, protein disulfide isomerase, PDI, bovine besnoitiosis Funding Acknowledgements: Fundação para a Ciência e Tecnologia (FCT), through the fellowship SFRH/BD/ 31445/2006 and the project grant PTDC/CVT/65674/2006. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa.

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High-throughput expression of marine animal venom toxins in Escherichia coli to generate libraries of recombinant venom peptides for drug discovery

Brás, JLA1,2, Sequeira AF1,2, Fernandes VO1,2, Abecassis DMA3, Vincentelli R4, Fontes CMGA1,2

1.NZYTech, Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, 1649-038 Lisbon, Portugal; 2. CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal; 3. Centre of Marine Sciences (CCMAR), Universidade do Algarve, 8005-139 Faro, Portugal; 4.Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France

Background: Animal venoms are composed by a wide variety of reticulated peptides, called venom peptides. These molecules are biologically active disulphide-reticulated peptides, which show high specificity, low immunogenicity and higher resistance to degradation than linear peptides, thus representing a new class of organic products with great pharmacological and biomedical potential. These properties make venom peptides noble candidates for drug discovery. It is estimated that the set of all animal venoms represents over 40 million bioactive molecules with potential drug application, which remain almost completely uncharacterized. In this context, marine animals are considered one of the most important reservoirs of this diversity, currently unknown. However, recombinant production of reticulated peptides containing disulphide bonds in Escherichia coli (E. coli) is challenging, especially when associated with the production of bioactive molecules for drug screening.

Methods/Results: Here, we report the development of a high-throughput automated platform to generate large libraries of recombinant venom peptides from transcriptomic data. Globally, transcriptomic data collected from marine species were used to design 96 optimized genes for efficient recombinant production in E. coli. From 96 synthetic genes produced, 53 peptides (55%) were efficiently produced in E. coli using DsbC as a fusion tag, which allowed the efficient formation of folded disulphide bridges in bacteria periplasm. These animal venom peptides were cleaved using Tobacco etch virus (TEV) protease to remove fusion tag and recover the venom peptides in the native state.

Conclusions: Overall, this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides that could be representatives of diversity prevailing on venomous marine animals. These results open up new possibilities to explore these new molecules in drug discovery programs.

Keywords: Venom peptides, High-throughput expression, drug discovery library

Funding Acknowledgements:

This work was supported by the PORTUGAL 2020 – Programa Operacional Regional de Lisboa, Projecto 011199.

17 November 16:50 - 17:50 Session 4 Poster Presentations - CIISA PhD students

Chairpersons - Luísa Mateus and Miguel Fevereiro

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PhD Contest Poster P23 Antimicrobial activity of the antimicrobial peptide pexiganan against a diabetic foot infection Pseudomonas aeruginosa strain. Santos R1,2, Gomes D1, Tavares L1, Veiga AS2, Oliveira M1 1. Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal; 2. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Background: Diabetes mellitus affects 422 million people worldwide and its prevalence is expected to double by 2030. Up to 25% of diabetic patients develop diabetic foot ulcers in their lifetime and around half of these ulcers become clinically infected. Diabetic foot infections (DFIs) are caused by a polymicrobial community of pathogens, being Pseudomonas aeruginosa the predominant Gram-negative species. A major obstacle in the treatment of DFIs is the increasing incidence of bacterial strains resistant to different antibiotic classes. Thus, it is of vital importance to develop new treatment strategies to effectively eradicate these infections. Antimicrobial peptides (AMPs) provide a potential alternative to conventional antibiotics. Pexiganan is included in this group, being a synthetic AMP that presents a broad-spectrum of action against both Gram-negative and some Gram-positive bacteria. Methods: This work evaluated the antimicrobial potential of pexiganan against a P. aeruginosa strain isolated from a patient with DFI, as well as the potential of guar gum gel as a topical delivery system for this AMP. Characterization of P. aeruginosa in vitro susceptibility profile was performed by determining the minimum inhibitory (MIC) and the minimum bactericidal (MBC) concentrations, using standard broth microdilution assays. Results: Pexiganan’s MIC value was 10.67±4.62 µg/mL for the P. aeruginosa clinical isolate under study. Regarding its bactericidal activity, the AMP MBC value was 10.67±4.62 µg/mL. When incorporated in the guar-gum gel, pexiganan’s MIC value was 32.00 ± 0 µg/mL, approximately 3-fold higher, and its MBC value was 53.33 ± 18.48 µg/mL, approximately 5-fold higher. Conclusions: Results show that pexiganan is able to inhibit and eradicate P. aeruginosa clinical strains and that the guar-gum gel is a potential candidate to be used as a topical delivery system for this AMP. The inclusion of antimicrobial peptides in DFI therapeutics may in the future contribute to decrease the administration of conventional antibiotics to these patients. Keywords: Antimicrobial peptides; Diabetic Foot Infections; Diabetes mellitus; Pexiganan; Pseudomonas aeruginosa Funding Acknowledgements: Work was supported by CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa (Project UID/CVT/276/2013) and by FCT (Project PTDC/SAU-INF/28466/2017 - Polyphasic validation of antimicrobial peptides as alternative treatment for diabetic foot infections). Raquel Santos acknowledges FCT – Fundação para a Ciência e a Tecnologia, Portugal (fellowship SFRH/BD/100571/2014).

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PhD Contest Poster P24 Miltefosine and meglumine antimoniate treatments combined with allopurinol restore lymphokine normal levels in canine leishmaniosis

Santos M1, Alexandre-Pires G1, Pereira MA2, Marques CS1, Gomes J1, Correia J1, Gomes L1, Rodrigues AV2, Basso A1, Reisinho A1, Meireles J1, Santos-Gomes GM2, Pereira da Fonseca I1.

1. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. GHTM - Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugal

Background: Canine leishmaniosis (CanL) caused by Leishmania infantum is an endemic zoonotic disease of worldwide concern. Leishmaniostatic or leishmanicidal drugs or a combination of both are usually used to treat sick dogs. Such treatments improve dogs’ clinical condition, although when therapy is discontinued relapses can occur. Hence, the objective of the present study was to evaluate the effect of the two most commonly used treatments on cell-mediated immune response to CanL. Methods: Five and six symptomatic dogs, naturally infected with L. infantum, were treated respectively with meglumine antimoniate (MG) or miltefosine (MT) combined with allopurinol (A). Peripheral blood, lymph node and bone marrow samples collected before treatment (Tp0) and one (Tp1), two (Tp2) and three (Tp3) months after were used to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, samples obtained from five non-infected dogs were also evaluated. Results: Peripheral blood of non-treated sick dogs showed reduced gene expression of all evaluated cytokines, except for IFN-γ (p > 0.05). Bone marrow presented increased gene expression of IFN-γ (p > 0.01), IL-12 (p > 0.05) and TNF-α (p > 0.05). Lymph node exhibited increased accumulation of IFN-γ (p > 0.01) and IL-10 mRNA (p > 0.01). These results indicate that changes in cytokine gene expression caused by Leishmania parasites seem to be tissue specific. By Tp3, both treatment protocols restored the gene expression of most lymphokines. However, with MG+A a reduction of IL-4, IL-5, IL-10 and TGF-β in blood and of IL-4 and TGF-β in bone marrow was recorded. The MT+A protocol was able to recover most lymphokine levels in blood except IFN-γ, as well as in lymph node and bone marrow except for TNF-α and IFN-γ. Conclusions: These results point out a drug-induced reduction of Th2 and Treg lymphokines with the MG+A protocol, while the MT+A maintained an initial Th1 profile against the parasite. Nevertheless, both treatments were able to normalize the cellular immune response and improve the clinical conditions in all dogs. Keywords: Canine leishmaniosis; Immune response; Mononuclear cells; Lymphokines; Treatment. Funding Acknowledgements: This work was funded by FCT’s (Foundation for Science and Technology) project (PTDC/CVT/118566/2010), the Centre for Interdisciplinary Research in Animal Health through project UID/CVT/00276/2013 and Global Health and Tropical Medicine through project UID/Multi/04413/2013. Marcos Santos holds a PhD scholarship (SFRH/BD/101467/2014) from FCT.

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PhD Contest Poster P25 Species composition of the mosquito fauna in the Lisbon zoological garden

Madeira S1, Osório H2, Gaspar N3, Boinas F1

1. Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon; 2. Centre for Vectors and Infectious Diseases Research, National Institute of Health Doutor Ricardo Jorge; 3. Lisbon Zoological Garden Background: Zoological gardens are a unique collection of microhabitats in which exotic species, native fauna, humans and arthropods can coexist. In these ecosystems, mosquitoes can find ideal conditions for survival: large range of vertebrate hosts, breeding habitats and shelter. In this way, zoos can have an important role as surveillance and monitoring areas, and in the investigation of mosquito ecology. The aim of this study was to examine the species composition of mosquito populations in different habitats inside the Lisbon zoological garden. Methods: Mosquito captures were performed in three different habitats inside the Lisbon zoological garden: near zebras, birds and giraffes. CDC miniature light traps baited with CO2 were operated during one night, fortnightly, from May to September. Adult mosquito species were morphologically identified according to identification keys. Culex pipiens were subject to molecular identification using the CQ11 polymorphic microsatellite marker. Results: A total of 99 mosquitoes were collected in the eight trapping nights. Of the 40 species reported in Portugal, only four were detected in the zoo: Culex pipiens s.l. (80.8%), Culiseta longiareolata (12.1%), Aedes caspius (3%) and Culex theileri (3%). No exotic species were detected. The trap located closest to the giraffes had the largest number of mosquitoes as well as highest species diversity. Female mosquitoes accounted for 71.7% of the total mosquitoes captured, and 45% were gravid females. Molecular analysis revealed the presence of two biotypes, Cx. pipiens pipiens and Cx. pipiens molestus, as well as hybrids of these two forms. Conclusions: Although only four different species were captured during this study, three of them are recognized vectors of disease agents of medical and veterinary importance. The most common species identified, Culex pipiens s.l. is one of the most common and widespread species in Portugal, and have been implicated as vectors of West Nile virus (WNV) in Portugal. The two biotypes and their hybrids, all identified at the zoo, have different physiology and behavior. Hybrids can feed both in avian and mammalian hosts, which can make them bridge vectors of WNV between birds and humans. The presence of gravid females indicates that mosquito populations have found in the zoo the ideal habitats for their establishment. This highlights the importance of zoos for the assessment of the risk of mosquito-borne diseases. Keywords: Mosquitoes; Vector; Zoological garden; Culex pipiens biotypes; hybrids Funding Acknowledgements: Fundação para a Ciência e Tecnologia – SFRH/BD/117431/2016. Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon - UID/CVT/276/2013.

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PhD Contest Poster P26

The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma

Dias J1, Aguiar S1, Pereira D2, Andre A1, Gano L3, Correia J3, Carrapico B1, Rutgen B4, Malho R5, Peleteiro MC1, Gonçalves J2, Rodrigues C2, Gil S1, Tavares L1, Aires-da-Silva F1 1. Centro de Investigacao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Universidade de Lisboa, Lisboa, Portugal; 2. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal; 3. Centro de Ciencias e Tecnologias Nucleares, Instituto Superior Tecnico, Universidade de Lisboa, Estrada Nacional, Bobadela LRS, Portugal; 4. Department of Pathobiology, Clinical Pathology Unit, University of Veterinary Medicine, Vienna, Austria; 5. Biosystems and Integrative Sciences Institute, Faculdade de Ciencias, Universidade de Lisboa, Lisboa, Portugal Background: Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. Methods: To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, the cytotoxicity of a panel of HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. To elucidate mechanisms of action, histone acetylation status was assessed using immunoblotting studies. Furthermore, levels of caspase 3/7 activation and Annexin V/7-AAD analysis were performed to clarify the nature of cell death induced. Finally, the anti-tumor effect of panobinostat was further tested in a murine tumor xenograft model. CLBL-1 cells were injected subcutaneously in SCID mice to establish tumors. After tumor induction, mice were randomly assigned to one of 3 groups (n=5): vehicle, 10 mg/kg/dose and 20 mg/kg/dose. Results: Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. Conclusions: In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology. Keywords: HDAC inhibitors; panobinostat; LBH589; non-Hodgkin Lymphoma; canine lymphoma Funding Acknowledgements: The authors thank the Portuguese Funding Agency, Fundacao para a Ciencia e a Tecnologia, FCT IP, for financial support: IF/01010/2013 to FAS, SFRH/BPD/100522/2014 to SA and PhD fellowships SFRH/BD/90514/2012, SFRH/BD/131468/2017 and SFRH/BD/96517/2013 to JD, AA and DP, respectively. CIISA has provided support through Project UID/CVT/00276/2013, funded by FCT. LG and JDGC gratefully acknowledge FCT support through the UID/Multi/04349/2013 project, RM through UID/MULTI/00612/2013. Part of this work has received support from European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT under the Programme grant SAICTPAC/0019/2015.

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PhD Contest Poster P27 Companion animals as reservoirs of Klebsiella pneumoniae Marques C1, Belas A1, Menezes J1, Aboim C1, Cavaco-Silva P2,3, Trigueiro G4, Telo da Gama L1, Pomba C1 1. Interdisciplinary Centre of Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Centro de Investigacao Interdisciplinar Egas Moniz, Instituto Universitario Egas Moniz, Caparica, Portugal; 3. Technophage, Lisboa, Portugal; 4. Laboratorio de Analises Clinicas Dr. Joaquim Chaves, Lisboa, Portugal Background: Klebsiella pneumoniae is an important nosocomial pathogen that is frequently multidrug-resistant (MDR) and associated with urinary tract infections (UTI). The role of dogs and cat as reservoirs of K. pneumoniae is still poorly studied. This study aimed: 1) to characterize the fecal colonization and sharing of K. pneumoniae between healthy companion animals and humans living in close contact; 2) to compare the population structure of faecal K. pneumoniae with uropathogenic strains. Methods: Fecal samples from healthy humans (n=44), dogs (n=18) and cats (n=8) belonging to 18 households were screened for the presence of K. pneumoniae using MacConkey agar plates with and without cefotaxime. Up to five K. pneumoniae colonies per participant were isolated and compared with uropathogenic K. pneumoniae strains (n=25 from dogs and cats; n=76 from humans) by PFGE after XbaI macro-restriction (Dice/UPGMA, 1.5% tolerance). All unique K. pneumoniae pulse-types from companion animals and third-generation cephalosporin (3GC) resistant K. pneumoniae from humans were typed by MLST. The underlying 3GC resistance mechanisms were characterized by PCR and sequencing. Results: Twenty-one K. pneumoniae pulse-type/STs were detected in faecal samples from 7 dogs and 9 humans. Notably, in household (H) 15 two colonized dogs shared each strain with one co-living human (ST252 and ST1241). Furthermore, one dog from H16 shared one undistinguishable K. pneumoniae ST17 strain with two humans from different households. Dogs were colonized by K. pneumoniae clonal lineages previously associated with human infections (ST17, ST188, ST281, ST252, ST423, ST1093, ST1241). Most uropathogenic K. pneumoniae from dogs and cats belonged to ST15 (60%, n=15/25) which were frequently MDR CTX-M-15 producers (73.3%, n=11/15). The hospital-adapted ST11 and ST147 harbouring CTX-M-15 were also detected in animals. Some faecal K. pneumoniae strains from dogs were >80% similar to uropathogenic strains from humans. Of note, H10 dog was colonized by a closely related K. pneumoniae ST423 (92.3% similarity) to one strain from a hospitalized patient with UTI. Conclusions: This study shows that dogs and cats may be colonized and infected with high-risk clonal lineages to humans. By detecting humans and dogs sharing undistinguishable K. pneumoniae strains, these results highlight the potential role of dogs as reservoir of K. pneumoniae to humans and vice-versa. Good hygiene practices are advised. Keywords: Klebsiella pneumoniae, UTI, Colonization, Companion Animals, High-risk clonal lineages

Funding acknowledgements: This work was supported by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the Programa Operacional Factores de Competitividade (COMPETE) and by National funds through the Fundacao para a Ciencia e a Tecnologia (FCT) [Interdisciplinary Centre of Research in Animal Health Project UID/CVT/00276/2013 and PET-Risk Consortium Project JPIAMR/0002/2016]. Adriana Belas (SFRH/BD/113142/2015) and Catia Marques (SFRH/BD/77886/2011) hold FCT PhD grants.

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PhD Contest Poster P28

Contribution to the staging of severe equine asthma syndrome (EAS) in the field using lung function testing Simões J, Sales Luís J, Tilley P

Clinical Research Lab, CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. Universidade Técnica, 1300-477 Lisboa, Portugal Background: Severe EAS remains the most common respiratory disease in adult horses. Affected animals exhibit increased respiratory effort at rest, coughing and even poor performance1, and although severe EAS phenotype can be easily identified when disease exacerbation occurs, mild clinical cases prove harder to diagnose2. In 2012, our team3 developed a clinical staging method for severe EAS. In the present study we further analysed information provided by lung function tests. As such, we aimed to evaluate the contribution of data obtained from arterial blood gas analysis, pleural pressure measurement, flowmetric plesthysmography and histamine bronchoprovocation for severe EAS staging. Methods: Twenty one horses with severe EAS phenotype and 21 healthy controls, aged between 8 and 23 years (mean age 14 ± 4 years) were examined by two independent clinicians for disease staging and lung function evaluation. Clinical staging3, arterial blood gas analysis, pleural pressure measurement, flowmetric plethysmography and histamine bronchoprovocation were performed. Discriminant analysis was performed considering an α-level of P<0.05. Results: The discriminant model included the variables which displayed significant differences between group means, namely clinical score, BALF neutrophil percentage, ΔPpl and histamine concentration (P<0.05) and also PO2 (P=0.062), which tended towards significance. The first two canonical linear functions obtained were statistically significant, with the first function explaining 97.3% of data variability and the second function 2%. Together both functions explain 99.3% of data variability. The validation of our staging method resulted in 94.7% of original cases grouped correctly and 86.8% of cross-validated cases correctly classified, confirming the major contribution of the independent variables used. These results validate the discriminant analysis performed, indicating its ability to discriminate between groups using the linear functions. Conclusions: In the present study we developed a clinical staging method using discriminant analysis to determine the contribution of several variables provided by a clinical score and ancillary diagnostic tests. The staging model proposed showed very good results in this population, and as such the discriminant linear functions may be used to identify and stage severe EAS, having the great advantage of being used in the field, as well as in diagnostic and research centers. Keywords: severe equine asthma syndrome; disease staging; discriminant power; lung function tests; diagnosis. Funding acknowledgements: This research had financial support by CIISA – Centre for Interdisciplinary Research in Animal Health hosted by the Faculty of Veterinary Medicine, University of Lisbon (CIISA-FMV-ULisboa) through the Project UID/CVT/00276/2013.

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PhD Contest Poster P29

Canine Behavioural Assessment and Research Questionaire (C-BARQ) study of its factorial structure in European Portuguese

Canejo-Teixeira R1, Almiro PA2, Serpell JA3, Baptista LV4, Niza NMRE1

1. CIISA, Faculdade de Medicina Veterinária/ULisboa; 2. CINEICC, PsyAssesmentLab, Faculdade de Psicologia e de Ciências da Educação da Universidade de Coimbra; 3. Center for the Interaction of Animals and Society, University of Pennsylvania School of Veterinary Medicine; 4. Faculdade de Ciências Sociais e Humanas, Universidade Nova de Lisboa.

Introduction: The human-dog relationship is the oldest domestic animal partnership1, serving the needs of both the human and the dog2, however these relationships can become problematic. The most common sign of dysfunctional human-dog partnerships is problem canine behaviour and their identification requires evaluation of the dog. The Canine Behavioural Assessment and Research Questionnaire(C-BARQ) uses owner knowledge to do this3,4. It has been validated for use in various languageseg;5,6,7, showing consistency and validity. In a clinical setting, the use of the C-BARQ could help veterinarians to identify problems. This study establishes the psychometric properties of a European Portuguese version of the C-BARQ and validates it. Materials & Methods: This C-BARQ was based on the 100-items 14-factors version used in the study by Duffy and Serpell4, but excluding 22 ‘Miscellaneous’ items. Questionnaires where completed online, using Google FormsTM, or in person throughout the Greater Lisbon Metropolitan Area during an 8-month period. Construct validity was accessed by exploratory factor analysis using IBM SPSS™ Statistics 20.0. Results: In total 344 questionnaires where competed and 13 factors were extracted, explaining 58.42% of the total variance. Most of the items loaded onto the expected factors4 excepting Dog-directed Aggression(DDA), Dog-directed Fear(DDF), item 8(“will fetch or attempt to fetch sticks, balls or objects”) which loaded into factor “Energy”(EL) and 43(“when examined/treated by veterinarian”) which loaded into factor “non-social fear”(NSF). Discussion: The results obtained for the European Portuguese version of the C-BARQ suggest good validity and reliability indices with a robust 13-item factor structure. It followed the original4, excepting DDA and DDF subscales, with all items loading strongly onto one factor, renamed Dog Associated Fear/Aggression(DAF). Owners can be unreliable in distinguishing between signs of fear and aggression in their dogs8, and lack of experience could explain this grouping. Item loaded onto the subscale EL instead of the original Trainability(TR)4. Fetching may not be considered an act of training but of playing, dogs that owners identify scoring high on the EL factor may be more extroverted9

displaying fetch-like behaviours but not basic obedience. The item 43 loaded onto the NSF instead of the original Touch Sensitivity(TS) subscale4. The reaction of a dog when examined by a veterinarian may not be an accurate measurement of TS, but of fear5. This study has demonstrated the validity of the European Portuguese version of the C-BARQ which could be of vital importance to help resolve behavioural problems in owned dogs. Keywords: C-BARQ, psychometric properties, dog, behaviour 1Wang, G.-D. et al. Out of southern East Asia: the natural history of domestic dogs across the world. Cell Res. 26, 1–13 (2015).2Houpt, K. A. et al. Proceedings of a workshop to identify dog welfare issues in the US, Japan, Czech Republic, Spain and the UK. Appl. Anim. Behav. Sci. 106, 221–233 (2007).3Hsu, Y. & Serpell, J. A. Development and validation of a questionnaire for measuring behavior and temperament traits in pet dogs. J. Am. Vet. Med. Assoc. 223, 1293–1300 (2003).4Duffy, D. L. & Serpell, J. A. Predictive validity of a method for evaluating temperament in young guide and service dogs. Appl. Anim. Behav. Sci. 138, 99–109 (2012).5Hsu, Y. & Sun, L. Factors associated with aggressive responses in pet dogs. Appl. Anim. Behav. Sci. 123, 108–123 (2010). 6Svartberg, K. A comparison of behaviour in test and in everyday life: Evidence of three consistent boldness-related personality traits in dogs. Appl. Anim. Behav. Sci. 91, 103–128 (2005).

7González-Ramírez, M. T., Quezada-Berumen, L. & Landero-Hernández, R. Assessment of canine behaviors using C-BARQ in a sample from Northern Mexico. J. Vet. Behav. Clin. Appl. Res. 20, 52–58 (2017). 8Mariti, C. et al. Perception of dogs’ stress by their owners. J. Vet. Behav. Clin. Appl. Res. 7, 213–219 (2012).9Ley, J., Bennett, P. & Coleman, G. Personality dimensions that emerge in companion canines. Appl. Anim. Behav. Sci. 110, 305–317 (2008).

Posters

Master Students Contest

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Master students contest Poster M30 Assessing the capacity of lysozyme into improve the nutritive value of Spirulina-containing diets for broilers Santos H1, Puerta B1, Pestana J1, Alfaia CM1, Lordelo M2, Prates JAM1, Fontes CMGA1 1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa, Portugal; 2. LEAF - Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisboa, Portugal Microalgae, mainly Spirulina, may be incorporated in diets for monogastric animals, in particular poultry. This abundant biomass resouces may be a viable alternative of energy and protein as well as other bioactive compounds that may improve meat quality. However, microalgal cell walls are largely indigestible by monogastric animals due to the complex matrix of cross-linked insoluble carbohydrates. In the case of Spirulina, the cell wall is rich in peptidoglycan. Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan. In addition, lysozyme can serve as a natural alternative to antibiotics used to improve feed efficiency. Here we have tested the capacity of lysozyme to improve the nutritive value of Spirulina containing diets for broilers One hundred and twenty old male Ross 308 birds were housed used 40 battery brooders with three birds per cage. The experimental period lasted from day 21 to 35. Chickens were given 4 different treatments, as follows:1)- corn based diet (control); 2)- diet+15% corn/soybean ; 3)Spirulina diet (15%)+Rovabio commercial enzyme mix (0,005%); and 4)-Spirulina diet (15%)+LIsozyme commercial enzyme(0,01%). At 35 days, birds were slaughtered, and breast and thigh collected. Overall the data suggest that incorporation of microalgae did not affect the performance of broiler chicks. Enzymes used to supplement diets had no effect in performance although meat quality was significantly altered. The data confirm that microalgae are a viable feed for monogastric animal diets and may contribute with a significant proportion of bioactive compounds to modulate quality of broiler meat. Keywords: Lysozyme, Spirulina, broilers, meat quality Funding Acknowledgements: This study was supported by Fundação para a Ciência e a Tecnologia (FCT, Lisbon, Portugal) through PTDC/CVT-NUT/5931/2014 grant and CIISA project (UID/CVT/00276/2013).

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Master students contest Poster M31

Genetic relatedness of Listeria monocytogenes isolates from a ready-to-eat meat-based food producing industry with a history of persistent contamination Andrade JC1, Henriques AR1

1. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal. Contamination of ready-to-eat foods with L.monocytogenes can be traced back to post-processing contamination from environmental sources. Also, certain L.monocytogenes subtypes are known to persist in the food-producing environment for months, or even years. This work aimed to investigate the genetic relation among L.monocytogenes isolates collected during a 6-year period in a ready-to-eat meat-based food (RTEMP) producing industry located in Évora with a history of persistent contamination. A total of 90 samples were collected, of which 19 were raw materials, 11 were intermediate products, 23 were final products, and 37 were equipment swabs samples. L.monocytogenes detection was performed according to ISO11290-1. Presumptive isolates were confirmed and serogrouped by multiplex PCR, according to Kérouanton et al. (2010). Genetic characterization of the isolates was performed using the PulseNet standard procedure (Graves & Swaminathan, 2001) for L.monocytogenes typing with ApaI and AscI enzymes. A dendrogram was built in BioNumerics software package version 6.10. PFGE patterns were analyzed with an optimization setting of 1.5% and a band-position tolerance of 1.5%. Cluster analysis was performed using the unweighted pair group method with arithmetic averages (UPGMA) and band-based Dice correlation coefficient. Of the twenty presumptive isolates obtained by conventional microbiological methods, only 17 revealed to be L.monocytogenes using PCR. Four different serogroups were found in food products and food-related environment: IIa (35.3%), IIb (5.9%), IIc (52.9%) and IVb (5.9%). The 17 L.monocytogenes isolates from different food and environment samples presented 6 PFGE types. Pulsotypes were considered clones when they had at least 90% of similarity. Isolates with identical restriction patterns were isolated from raw materials, intermediate and final products and equipments at different times. The presence of genetically related L.monocytogenes strains throughout the studied time-frame emphasizes the possibility of a common source. Suppliers should be addressed to understand the origin of some strains, although results underline cross-contamination as a possible way of disseminating L.monocytogenes in the assessed food industry. Likewise, equipment’s sanitizing method should also be considered and validated, because it might select isolates that are able to survive and adapt to the food processing environment. Keywords: Listeria monocytogenes, persistent contamination, meat-based food products, molecular subtyping. Funding acknowledgements: Centre for Interdisciplinary Research in Animal Health, Project UID/CVT/00276/2013.

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Master students contest Poster M32

Assessing Listeria monocytogenes growth in ready-to-eat chicken salads as a function of temperature

Bernardo R, Henriques AR

CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal

Currently, there is a growing preference for convenience foods, such as ready-to-eat (RTE) foods that are associated to long refrigerated shelf-lives and do not require a heat treatment prior to consumption.Unlike the majority of foodborne pathogens, Listeria monocytogenes is able to grow at refrigeration temperatures. Inconsistent temperatures during commercial production and distribution, as well as at consumer’s household, may gather the conditions for the pathogen to thrive, reaching unsafe limits. L.monocytogenes causes a rare but severe human illness. Listeriosis is almost exclusively transmitted through the consumption of contaminated foods, predominantly RTE foods. In developed countries with epidemiological surveillance networks, listeriosis incidence is low, but with high fatality rates, peaking at 20% in risk groups, the highest for any foodborne pathogen, even with antibiotic therapy. Therefore, it is of upmost importance to understand the behavior of L.monocytogenes in RTE foods during storage under abusive conditions. L.monocytogenes growth behaviour in RTE chicken salads as a function of temperature was studied in order to develop L.monocytogenes’ predictive growth models, taking into account the processing and storage conditions, and the foreseen shelf-life. RTE chicken salads were inoculated with a three-strain mixture of cold-adapted L.monocytogenes (≈4 log cfu/g), and stored at 4, 12 and 16°C, for 8 days. At appropriate time intervals, a series of decimal dilutions were prepared from each sample and plated onto ALOA agar, after which the plates were incubated at 37oC for 48 h, under aerobic conditions, and L.monocytogenes were enumerated. The four-factor modified logistic growth model described by Baranyi & Roberts (1994) was fitted to the derived microbiological data for the estimation of the pathogen's growth kinetic parameters: lag time (λ), maximum growth rate (μmax), and maximum population density (Nmax). The predicted μmax varied from 0.021±0.008, to 0.052±0.024 and to 0.066±0.009 log cfu/g/h at 4oC, 12oC, and 16oC, respectively. The considered storage temperatures significantly influenced L.monocytogenes μmax (p<0.05). Finally, the square-root-type model proposed by Ratkowsky et al. (1982) was used to describe L.monocytogenes growth rate as a function of temperature. The present study provides predictive growth models for L.monocytogenes in RTE chicken salads which can be used in subsequent quantitative microbial risk assessments. Keywords: Listeria monocytogenes, RTE chicken salads, storage temperatures, predictive growth models, growth kinetic parameters. Funding acknowledgements: Centre for Interdisciplinary Research in Animal Health, Project UID/CVT/00276/2013.

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Master students contest Poster M33

Production of bivalve molluscs and associated risk factors Pires D1, Grade A2, Ruano F2 Afonso F1

1. CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon. Av. da Universidade Técnica, 1300-477 Lisbon fafonso@fmv.ulisboa.pt; 2. IPMA - Instituto Português do Mar e da Atmosfera, 1495-165 Lisbon

Background: The culture of bivalve molluscs (molusciculture) is an activity with high expression in Portuguese aquaculture, representing 55% of the total production. The production of clams represents about 24% of the total produced. European clam, Ruditapes decussatus, is economically the most important species. However, in recent years its production has decreased associated with several factors such as overfishing and diseases like Perkinsiosis. To face the decrease of R. decussatus, it was replaced by the Japanese clams, Ruditapes philippinarum, which appears to have greater resistance to diseases and growing rates. Despite the rapid expansion of this species in Portuguese waters, the native clam remains commercially more important and appreciated than the Japanese clam because of its quality. Methods: The objective of this study was to evaluate methods of diagnosing Perkinsiosis in two species of clams from different sites. The RFTM test (reference test) and histopathology (complementary method) were used as methods. Samples were collected in October 2017 and April 2018 from 6 different sites, with an n=50 for each site (except in the Tejo Estuary (2017) with n=47) corresponding to a total of 297 specimens examined. Results: The results showed that the European clam had a higher prevalence of the disease, mainly those produced in Olhão. The Japanese clams also had positive cases, although, with a lower level of infection. This study suggested that to diagnose Perkinsiosis, the RFTM test is effective, less expensive and faster. Nonetheless, it should be complemented by histopathological analyses especially in animals with low infection. Conclusion: Taking in account the environmental conditions in different sites, it is clear that the environmental stress caused by high salinities, water temperatures, low oxygen concentration in the sediments, high densities and physiological stress due to the spawning season, contribute to an increase in the prevalence and spread of the disease. Keywords: Bivalve molluscs, Clams, Aquaculture, Diseases. Funding Acknowledgements: This work was supported by SNMB-MONIT,” Sistema Nacional de Monitorizacao de Moluscos Bivalves”, 16.02.01-FMP-0043

78

Master students contest Poster M34

Occurrence and characterization of White Striping in broilers breast meat in a Portuguese slaughterhouse Rocha A, Alves S, Bessa RJB, Fraqueza MJ CIISA - Centro de Investigacao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Universidade de Lisboa, 1300-477 Lisboa. Broiler meat consumption increased in the last years, causing a high pressure on the production to meet the increased demand. Due to this pressure, the broiler industry intensified the genetic selection in order to improve the broilers average daily gain, feed conversion, and yield in breast muscles. The success of the selection was high but some unexpected problems arose like the increased incidence of a quality defect on broiler breast called white striping. This defect was characterized by white strips along the breast muscle, with different intensity levels causing impact of consumer preferences during purchase moment. The present study aims to collect information about the occurrence of “white striping” on broilers breast meat at slaughterhouse level. The screening study was performed in a slaughterhouse under commercial conditions, in Lisbon, Portugal. A total of 5791 birds from 150 different flocks were classified after slaughtering process for the presence and severity of white striping on their breast muscle. Sex, strain and other production factors such as broilers live weight and age at slaughter day, were also studied for possible association with white stripping defect. A total of 36 samples of chicken breast muscle from 10 different flocks were sampled for further analysis. Meat quality parameters, such as pH and colour (Lab system) were assessed. Lipid extraction and solid phase extraction separation (SPE) of lipid fractions were done on meat samples. The fatty acid composition was determined by flame ionizing detection gas chromatography (GC-FID). From the total 5791 birds classified, 67.5% presented white stripping defect. For the studied factors when ordered by groups was observed a significant difference in classification of white stripping according to age, live weight and sex (P<0.01). No differences on defect occurrence were observed for the two commercial strains used in the study. Colour and pH showed no differences for different white stripping quality levels. Regarding breast lipid composition was noticed differences related to the classification of white stripping found in total lipid contents (P<0.007), and also neutral lipids and polar lipids (P<0.001). Finally, in the fatty acid composition, only for 18:0 (P<0.001), 20:0 (P<0.01) and 203n-3 (P<0.05) were observed differences regarding the classification of white stripping. The occurrence of white striping quality defect on broiler meat was high at slaughter level, similarly to the frequency observed in other recent studies. Age, live weight and sex have been found to be factors implicated on white striping classification. Higher contents of neutral lipids were present in meat with white stripping defect. The samples classified as presenting severe white stripping lesions presented the highest content of 18:0 and 20:0 fatty acids. Keywords: White Stripping, Broiler, Meat quality, lipids

79

Master students contest Poster M35

Mineral and amino acid profiling of muscle and liver of Damara, Dorper and Australian Merino lambs under restricted feeding

Ribeiro DM1, Madeira MS2, Martins CF1, Kilminster T3, Scanlon T3, Oldham C3, Greeff J3, Freire JPB1, Prates JAM2, Mourato MP1, Almeida AM1

1. LEAF - Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, University of Lisbon, Lisboa, Portugal; 2. CIISA - Centro Interdisciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veterinária, University of Lisbon, Lisboa, Portugal; 3. Department of Agriculture and Food Westerna Australia, Perth, WA, Australia

Seasonal weight loss (SWL) is a major constraint in extensive animal production systems in the tropics and in the Mediterranean, namely for the Australian sheep production that is based on merino breed, which is sensitive to SWL. SWL-tolerant alternative breeds, such as the fat-tailed Damara and the Dorper, have been increasingly used in Australia, due to their hardiness. Previous studies have demonstrated the impact that feed restriction has on the muscle and liver mineral and amino acid profile. The aim of this work was to better understand the profiles of these organs of Australian Merino (AM), Damara and Dorper, when subjected to Seasonal Weight Loss and by comparison to control animals. Twenty-four lambs per breed were divided into growing (control) and restricted groups. The trial lasted 42 days in which bi-weekly weightings were registered to evaluate growth performance. At the end of the trial, animals were slaughtered. Liver and muscle samples were collected immediately after and snap frozen in liquid nitrogen. Mineral assessment as carried out using Inductively Coupled Plasma – Optic Emission Spectrometry. Amino acid profiling was achieved through High Performance Liquid Chromatography with fluorescence detection. Three-factor variance analysis was carried out using the SAS. Overall, mineral concentration was lowered in the muscle of restricted animals, as a consequence of fat tissue mobilization. Increase in certain mineral concentrations, such as zinc and iron, were recorded indicating a putative increase of enzymatic activity. Outstanding concentrations of Cu in the liver of Dorper indicate greater ability to accumulate this element, even under SWL: with 192.08 mg/kg DM, compared to restricted groups of Damara (97.05 mg/kg DM) and Australian Merino (42.90 mg/kg DM). Restricted animals accumulated more ferritin in the liver, causing comparative increase of iron between subgroups of 334.92 mg/kg DM (AM), 197.44 mg/kg DM (Damara) and 331.01 mg/kg DM (Dorper). Weight loss also induced generalized muscle protein breakdown in all breeds, with varying intensity. Dorper breed seems to mobilize less muscle amino acids when under these conditions, with the Damara having frequent significant differences. This may indicate yet another mechanism that provides the Damara the ability to endure harsh conditions. Amino acid concentration in the liver confirmed gluconeogenesis triggered by poor nutrition. Overall Damara seems to be less susceptible to nutritional stress, Dorper having intermediate classification and Merino the highest susceptibility. Keywords: Seasonal Weight Loss, Minerals, Amino acids.

80

Master students contest Poster M36

Development and optimization of liposomes encapsulated with potent cytotoxic compounds for immunotherapy

Rico I1; Dias J2; Andre A2, Aguiar SI2, Gaspar MM3, Gil S2, Aires-da-Silva F2

1. Integrated Master Student of Faculty of Veterinary Medicine, University of Lisbon, Portugal. 2. CIISA – Center for Interdisciplinary Research for Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal. 3. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

Background: Cancer is one of the leading causes of death in both humans and dogs. There is a rising owners’ concern about their dogs’ health and therefore the search and development of new methods to improve cancer therapy, dog’s welfare and to overcome cancer resistance is growing up. Furthermore, integration of dogs in clinical trials reveals itself as a benefit for both dogs and humans since it allows the access to other therapy protocols inaccessible to Veterinary Medicine, providing results on an animal model much more similar to humans, before starting human clinical trials. Liposomes are being investigated as a drug delivery system of chemotherapeutic agents in order to improve their pharmacokinetics, to reduce the negative side effects and to target cancerous cells. The aim of this study is to access the cytotoxic effects of a potent anti-tumor agent in canine large B-cell lymphoma – Panobinostat HDAC inhibitor – using targeted liposomes as a drug delivery system and comparison with cytotoxic effects of Doxorubicin and liposomal Doxorubicin. Material and Methods: The effects of Panobinostat, liposomal Panobinostat and lipossomal Panobinostat targeted with folates on CLBL-1 cell viability were measured using the alamarBlue® reagent, 24h after the treatment. Two replicate wells were used for each drug concentration and three independent experiments for each drug were carried out in different days. Best-fit IC50 values were calculated using the log (inhibitor) vs response (variable slope) function. The data was statically analyzed using ANOVA and Tukey test in R version 3.5.0. (2018). Results and Conclusion: Results showed a lower IC50 value for Panobinostat statistically different of Doxorubicin’s IC50. There was no statistic difference between Panobinostat’s IC50 value and its liposomal formulation IC50. However, liposomal formulation of Panobinostat with folates showed a slightly lower IC50 comparing to Panobinostat, with a pvalue<0,05. This results indicate that Panobinostat has a higher cytotoxicity activity comparing to Doxorubicin, in vitro, in agreement with previous studies(1). The use of liposomal formulation could be advantageous in reducing the cytotoxic effects, as shown in previous studies for liposomal Doxorubicin(2), since its efficacy is similar to the free drug. Keywords: Panobinostat, HDAC inhibitors, canine lymphoma, liposome Funding: This work was supported by Fundacao para a Ciencia e Tecnologia (BBB_BIO_0508_2014; IF_010100, SFRH/BD/90514/2012, SFRH/BD/131468/2017 and by UID/CVT/00276/2013).

(1) Ishida, T., Kirchmeier, M. J., Moase, E. H., Zalipsky, S., & Allen, T. M. (2001). Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1515(2), 144–158. (2) Kanter, P. M., Bullard, G. A., Ginsberg, R. A., Pilkiewicz, F. G., Mayer, L. D., Cullis, P. R., & Pavelic, Z. P. (1993). Comparison of the cardiotoxic effects of liposomal doxorubicin (TLC D-99) versus free doxorubicin in beagle dogs. In Vivo, 7(1), 17–26.

81

Master students contest Poster M37

IsomiR Window: A platform for analysing small-RNA-seq data of mammalian species in an integrative and user-friendly manner Vasconcelos AM1, Carmo B2, Ferreira B2, Viegas I2, Carvalho MG2, Ferreira A1, Amaral AJ3 1. Lasige - Laboratório de Sistemas Informáticos de Grande Escala, Faculty of Sciences, University of Lisbon, Lisbon, Portugal; 2. BioISI - Biosystems and Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal; 3. CIISA- Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal Since the development of Next-Generation Sequencing (NGS) methods, several variants of microRNAs (miRNA), isomiRs, have been discovered. These variants vary in length and/or sequence when compared to their canonical forms and can display additions or deletions of one or more nucleotides at the 5’ or 3’ ends or both, internal editings or 3’ end tailings. Although the existence of isomiRs is widely known, there are still questions regarding the biological function of these molecules. With the aim of creating an analysis pipeline that would allow to contribute to the identification and understanding of IsomiR function, we have developed IsomiR Window, a platform for integrated analysis of small-RNA-seq data that enables the discovery of IsomiRs and characterization of small non-coding RNAs. This platform was build with two main components, the IsomiR Window pipeline for data processing and the IsomiR Window web-interface. The pipeline is written in Perl language and is integrated in a Graphical User Interface (GUI) developed using the Laravel framework for web-interface development. It integrates over ten third-party softwares for the analysis of small-RNA-seq data and holding a new algorithm that is the first to allow the detection of all possible types of isomiRs. It further includes all required databases (genome and non-coding RNAs). IsomiR Window is free and is distributed as a virtual machine prepared in a Linux environment including all softwares and it was designed to run in ordinary workstations. This tool allows users to process several datasets in parallel without the restriction of input file size, using well established tools for several steps of the analysis (alignment, miRNA quantification and prediction, differential expression and functional analysis) in an automated manner. The versions of the IsomiR Window pipeline was created, one for a basic user profile, executed through the web-interface for users without any bioinformatics skills, and other for bioinformatics and advanced users, enabling users to invoke the Perl scripts directly, allowing a broad diversity of experimental designs. The web-interface establishes the analysis settings and executes the Perl pipeline automatically, allowing the users to retrieve results in charts and tables in different file formats. The pipeline provides two modules, Annotation and Functional Analysis, which can be used independently, enabling a high degree of flexibility when analysing small RNA-seq data. It also allows users to review results from previous analyses. This tool, compared with other currently available tools, provides new functionalities essential to perform the analysis of isomiRs in an accurate and user-friendly way, being capable of producing results in an efficient time manner, minimizing human technical errors while ensuring reproducibility, which may be used for investigation or in clinical purposes. Keywords: IsomiR; miRNA; Annotation Analysis; Functional Analysis; Web Application Funding acknowledgements: Bolsas de Investigação para Licenciado, no âmbito do Programa Estratégico da Unidade de I&D “LASIGE – Laboratório de Sistemas Informáticos de Grande-Escala”, com a referencia “UID/CEC/00408/2013”, financiado por fundos nacionais atraves da FCT/MCTES (PIDDAC) no âmbito do programa de financiamento dos Programas Estratégicos das Unidades de I&D.

82

Master students contest Poster M38

Molecular survey of vector Borne Diseases in two groups of domestic dogs from Southern Portugal Dordio M1, Nunes, T1, Beck R2, Pereira da Fonseca I1, Gomes J1,3

1. Faculdade de Medicina Veterinária, Universidade de Lisboa; 2. Croatia Veterinary Institute, Zagreb; 3. Instituto Nacional de Investigação Agrária e Veterinária Background: Canine vector-borne diseases (CVBD) are caused by a wide range of pathogens transmitted by arthropods, and they had been an issue of growing importance in recent years. Since there is a lack of information about the vector-borne pathogens circulating in Portugal, the aim of the present study was to detect canine vector-borne bacteria and protozoa with veterinary and zoonotic importance using molecular methods and to do a correlation with the clinical signs and changes in the Complete Blood count (CBC). Methods: One hundred and forty-two dogs from southern Portugal were tested, including 48 dogs clinically suspected of vector-borne diseases (Group 1), collected from a veterinary hospital and 94 apparently healthy dogs from shelters (Group 2). All the clinical data was register and CBC was performed for the Group 1 and microhematocrit for the Group 2. Anaplasma spp./Ehrlichia spp., Babesia spp., Hepatozoon spp. and Mycoplasma spp. infections were evaluated by PCR in blood samples. Results: Single infections were detected by PCR in thirty-five dogs (24.64%) and co-infections were found in thirteen (9.15%) dogs. B. canis was detected in two dogs, both belonging to Group 1, with the owners reporting anorexia and lethargy during 2-4 days and clinical manifestations were compatible with an acute disease. Concordance between PCR and peripheral blood smears observation results was substantial for Babesia spp. diagnosis. Hepatozoon canis single infections were found in group 1 of sick animals, with clinical signs compatible with hepatozoonosis and CBC changes such as anemia, lymphocytosis and neutrophilia. Musculoskeletal changes were found only with H. canis infection. There was evidence of the lower sensitivity of the microscopic observation of blood smears for the detection of intracytoplasmatic gamonts compared with molecular diagnosis. Two co-infections with A. platys suggest this can potentiate disease pathogenesis altering clinical manifestations. Conclusions: This is the first molecular identification of B. canis and M. haematoparvum in dogs from southern Portugal. The identification of CVBD agents helps to guide the clinical approach of veterinarians at the practice and reinforces the importance of a One Health approach, to prevent the risk of the transmission to humans. Keywords: CVBD; Dogs; Molecular methods; Southern Portugal; Ticks Funding Acknowledgements This work was supported and funded by National Institute for Agrarian and Veterinary Research, INIAV, Lisbon; Croatia Veterinary Institute, Zagreb and Centre for Interdisciplinary Research in Animal Health Project UID/CVT/00276/2013.

83

Master students contest Poster M39

Renal injury in bitches diagnosed with pyometra Martins A1, Basso MA2, Mateus L3 1. Integrated Master Student of Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2. Laboratory of Clinical Pathology M. Braco Forte, Faculty of Veterinary Medicine, University of Lisbon, Portugal; 3. Reproduction and Development Laboratory, Center for Interdisciplinary Research for Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal Renal dysfunction is a common finding in bitches with pyometra, leading in some cases to elevated serum urea and creatinine. These high values are assigned to dehydration, reduced renal perfusion and/or toxemia. However, observation of increased serum concentrations of urea and creatinine are not a constant and it has been questioned if renal histological changes are attributed to pyometra or just age related (1). The aim of this study was to evaluate the degree of renal damage in patients diagnosed with pyometra using different markers of renal damage: renal proteinuria (urine protein/ creatinine ratio (UPC) and dipstick protein), serum concentrations of urea, creatinine and symmetric dimethylarginine measurements (SDMA). A total of twenty-eight bitches with confirmed diagnose of pyometra were included in this study [mean age: 8.9 years; range 4-13 years]. Blood and urine samples were collected and assayed in the day of ovariohysterectomy. Data were statistically analyzed using R Commander (Fox, J., and Bouchet-Valat, M. (2018). Rcmdr: R Commander. R package version 3.5-1). In the dipstick test, protein was increased in 82% of the patients. However, this protein may be due to other sources as urinary tract inflammation or haemorrhage and not only due to renal loss (2). UPC evaluation considering microscopic sediment analysis allowed to exclude other sources of protein then kidney injury and only 10.7% animals had reliable UPC alterations. SDMA was increased in 35.7% bitches. There was no correlation between age and increased SDMA (p > 0.05) which supports the idea that renal injury is associated with pyometra and not only age related. Furthermore, it was shown a correlation between polyuria and polydipsia and SDMA concentration (p < 0.05). On the other hand, UPC, creatinine and urea values showed no correlation with SDMA which is in accordance with previous studies showing that SDMA can detect renal injury in earlier stages when compared to creatinine (3). Of the complications associated with pyometra, renal injury is one that veterinarians must recognize. The clinicians awareness of this complication could prevent a deterioration of renal lesions by adapting their therapy protocols and by having a closer follow-up in long term, regarding bitches’ renal function. Keywords: Canine pyometra; Renal function; Symmetric dimethylarginine; ratio. Funding: UID/CVT/00276/2013 from Fundacao para a Ciencia e Tecnologia (FCT); MIMV 4.2017 from CIISA. (1) Heiene, R. et al. (2007). Renal histomorphology in dogs with pyometra and control dogs, and long term clinical outcome with respect to signs of kidney disease. Acta Veterinaria Scandinavica, 49(1), 13. (2) Vaden, S. L. et al. (2004). Effects of urinary tract inflammation and sample blood contamination on urine albumin and total protein concentrations in canine urine samples. Veterinary Clinical Pathology, 33(1), 14–19. Dimethylarginine Assay Validation, Stability, and Evaluation as a Marker for the Early Detection of Chronic Kidney Disease in Dogs. Journal of Veterinary Internal Medicine, 29(4), (3) Nabity, M. B. et al. (2015). Symmetric 1036–1044.

84

Master students contest Poster M40

Equine piroplasmosis: hematological and inflammatory biomarkers changes in horses with clinical and subclinical disease Barros C1,2, Duarte A1, Brito T1, Rosa T2, Magalhães M2, Tavares L1, Lamas L1,2 1. CIISA FMV-UL. Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. SCUE FMV-UL. Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal

Background: Currently, real-time PCR (qPCR) is considered the best technique to diagnose Theileria equi and Babesia caballi infections. Furthermore, the role of inflammatory biomarkers in equine piroplasmosis (EP) is still not clear, not only for diagnostic and monitoring purposes but also for its use as disease predictive indicators. Considering the obligate intracellular nature of EP agents, the following hypothesis was set: the inflammatory response in EP differs from other inflammatory diseases. Moreover, the following questions were asked: 1) What is the usefulness of hemogram and acute inflammation markers in the early detection of EP?; 2) What are the advantages of using qPCR to diagnose EP?

Material and methods: In this study, inclusion criteria were: horses with unknown origin fever (UOF) in the last 72 hours and asymptomatic horses with EP suspicion. Each blood sample was subjected to a qPCR for T. equi and B. caballi detection. Positive samples were subjected to nested PCR and direct sequencing of the amplicons to validate the qPCR result. We also performed a hematological analysis and determination of total proteins, SAA, plasma fibrinogen and plasma iron. According to qPCR results, the following groups were created: horses with clinical piroplasmosis (PC), horses with subclinical piroplasmosis (SC) and horses with UOF but negative to EP (NS).

Results: All positive samples by qPCR were confirmed by amplification and sequencing of a larger fragment of the 18S rRNA gene for both agents. In 17 positive samples, 15 were positive to T. equi and 2 to B. caballi. There were significant differences (p <0,05) in monocytes absolute value between PC-NS and PC-SC groups, in SAA between PC-SC groups and in plasma iron between PC-NS and PC-SC groups. 75% of SC group horses showed neutrophilia without left shift. We just found T. equi in blood smears of horses that had a parasitemia equal or higher than 7,2x102 parasites/µL.

Conclusion: This study highlights the usefulness of qPCR in EP diagnosis and that this disease is characterized by an inflammatory response associated with hemogram and inflammatory markers changes, in which monocytes absolute value, SAA and plasmatic iron demonstrate higher sensitivity in the detection of EP.

Keywords: Equine piroplasmosis; PCR; Inflammatory response; Biomarkers Funding acknowledgments: Funded by Project UID/CVT/276/2013 CIISA (Centro de Investigação Interdisciplinar em Sanidade Animal da Faculdade de Medicina Veterinária da Universidade de Lisboa).

85

Master students contest Poster M41

Expression of pro-inflammatory and Th1 cytokines in canine allergic conjunctivitis Varandas C, Cartaxeiro C, Delgado E, Gil S Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal Background: Canine allergic conjunctivitis (cAC) is often included in the symptoms of canine atopic dermatitis (cAD). Although an allergic reaction is the most common cause of conjunctivitis in dogs, cAC is still poorly understood. Thus, the aim of the present study was to determine the gene expression of the pro-inflammatory (IL-6 and TNF-D) and Th1 (IL-12) cytokines in cAC. Methods: Twenty cAD patients were evaluated for ocular signs of allergic conjunctivitis (conjunctival hyperemia, chemosis, epiphora, ocular discharge, pruritus and concurrent keratitis), graded 0 to 3 according to severity, and cAD Extent and Severity Index (CADESI)-4. Furthermore, conjunctival samples were collected from atopic dogs and 21 control dogs. mRNA was isolated from this samples and a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantify cytokines expression. Results: Cytokines expression was higher in the atopic group, IL-6 and IL-12 mRNA expression were respectively 291,5 (p=1,31e-09) and 4,9 (p=0,0003) times more present in atopic dogs when compared with the control samples. The average expression of TNF-D also seemed to be higher in atopic group, however statistically there were no significant differences between atopic and healthy groups (p=0,18). The mRNA levels of all cytokines in the atopic group exhibited a negative correlation (RhoIL-6=-0,28; RhoTNF-D=-0,57; RhoIL-12=-0,50) with allergic conjunctivitis clinical score, yet for IL-6 this correlation wasn’t statistically relevant (pIL-6=0,24; pTNF-D=0,02; pIL-12=0,049). Statistically there was no significant correlation between cytokines gene expression levels in the atopic group with (CADESI)-4 values (p>0,5). Conclusions: Results obtained indicate that IL-6 and TNF-D contribute to the development of the allergic response, as well as the IL-12, which is a critical factor in Th1 differentiation. Thus, our results suggest an increase of pro-inflammatory cytokines which are strongly involved on the innate immune response, and a Th1 subset activation. This raises the possibility of new therapeutic modalities involving immunotherapy to help in the clinical control of allergic conjunctivitis. Keywords: allergic conjunctivitis, canine atopic dermatitis, interleukin 6, TNF-D, interleukin 12 Funding Acknowledgements: This work was supported by CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) (Project UID/CVT/276/2013), and by “Study of the immune response in canine allergic conjunctivitis.” (CIISA MIMV 5).

86

Master students contest Poster M42

Biocide and antibiotic cross-resistance in equine medicine: is there a link? Trigo da Roza F , Cunha E , Tavares L, Oliveira M, Lamas LP

CIISA/Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal Background: The dissemination of multidrug-resistant (MDR) bacteria is a recognized worldwide problem for both human and veterinary medicine. Among MDR strains, extended spectrum ß-lactamase (ESBL) producing bacteria are frequently implicated in nosocomial infections1. In veterinary medicine, the use of biocides is considered a key element to control infections promoted by such strains3, including surgical site and wound infections3,4. In fact, the dissemination of resistant bacteria has led to an increased use of these compounds in veterinary hospitals, including in equine clinics5,6. Could this disseminated and uncontrolled use of biocides be potentiating some of the resistance mechanisms of bacteria? Methods: To address this question, an adaptation of the MEGA-plate methodology described by Baym et al.7 was used. Two isolates collected from wounds of hospitalized horses were used as bacterial models: a commensal E. coli fecal isolate and a K. pneumoniae isolate obtained from a surgical site infection, both with known antimicrobial resistance. Isolates were inoculated in a MEGA-plate and submitted to increasing concentrations of two surgical preparation biocides: chlorhexidine (from 0% to 0,04%) and povidone-iodine (from 0% to 3%). By the end of each assay, new isolates were collected and identified and their antimicrobial resistance profile determined using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute. Results: The evolution of bacterial growth observed in the 4 assays suggests that these biocides are not capable of inhibiting bacterial growth over time and space. The difference between growth evolution of the E. coli and K. pneumoniae isolates seemed not to be significant, as both isolates were able to reach the MEGA-plate region with maximum biocide concentrations within the same time period. Also, differences were observed between the antimicrobial resistance profiles of the initial isolates and the ones collected at the end of the assays, with the last presenting increased resistance to aminopenicilins and 1st generation cephalosporins. Conclusions: This study suggests that use of biocides in veterinary medicine, in particular in equine clinics, should be reviewed, and that their use can be linked to cross-resistance to antibiotics. Keywords: Escherichia coli; Klebsiella pneumoniae; Biocides; Antibiotic; Resistance. Funding Acknowledgments: This work was supported by the Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). References [1] Abuzaid et al.(2012). Journal of Hospital Infection, 81(2), 87–91. [2] Weber et al. (2007 Antimicrobial Agents and Chemotherapy, 51(12), 4217–4224. [3] Southwood, L. (2009). In E. Robinson & K. Sprayberry (Eds.), Current Therapy in Equine Medicine (6th ed., pp. 394–401). St. Louis, Missouri: Elsevier Inc. [4] Wilkins & Unverdorben (2013). Advances in Skin & Wound Care, 26(4), 160–163. [5] Maddox et al. (2015). Equine Veterinary Journal, 47(6), 756–765.[6] White & McDermott (2001). Current Opinion in Microbiology, 4, 313–317. [7] Baym et al. (2016). Science, 353(6304), 1147–1151.

87

Master students contest Poster M43

New administration formula of parasiticide fungi spores to prevent infection by gastrointestinal nematodes in pasturing horses. Evangelista F1, Pena MV2, Meissner M2, Luís J1, Palomero A2, Arias MS2, Sánchez-Andrade R2, Torres MI2, Gomes L1, Madeira de Carvalho LM1, Paz-Silva A2

1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. Universidade Técnica, 1300-477 Lisboa, Portugal; 2. COPAR - Grupo para el Control Parasitario, Departamento de Patologia Animal, Faculdade de Veterinaria, Universidade de Santiago de Compostela, 27002-Lugo, Espanha Background: Anthelmintic resistance in horses has increased in recent years and the continuous search for alternative control methods has led to the development of complementary approaches such as biological control. Following this line of research, a new formula has been developed using a lyophilized product that contained both Duddingtonia flagrans and Mucor circinelloides spores for the control of gastrointestinal nematodes in horses. Method: Following product manufacture, normal spore morphology and viability were assessed. Petri dishes were assembled with mashed product to verify in vitro development of new spores and a sealed plastic box containing product was stored for three months to verify product stability. From September 2017 to March 2018, to observe product effect in horse faecal egg count (FEC) of eggs per gram (EPG), one group of 5 horses received 10 g of product per os (with M. circinelloides spores and around 105 D. flagrans chlamydospores per horse) 3 days a week. Another group of 7 horses served as control. After treatment with Ivermectin pour-on in September, a faecal sample was collected from each horse monthly and FEC was assessed with a Modified McMaster technique. Faecal culture method was also used in November and January to identify L3 genus and species in both groups. Results: Spores present in the product were able to produce new spores in all Petri dishes after the 10 days and the product showed no degradation or alteration during the three months storage period. Only gastrointestinal nematode eggs, namely strongyle eggs, were observed with Modified McMaster technique. EPG average from each group was compared for each individual month and overall to see the reduction effect achieved with the fungi treatment. Statistically significant differences were found between the two groups in February (72% reduction), March (64% reduction), and overall, 66% reduction. The horses in the test group only reached a cut-off value of 300 EPG two months after the horses in the control group. Faecal culture method showed only the presence of cyathostomin larvae. Conclusions: This study allowed the successful development of a new formula for the administration of parasiticide fungi to horses, based on lyophilized product, which increases the possibilities for future product development and application. New and improved ways of biological control should be developed and implemented to increase parasite control and reduce anthelmintic resistance cases. Keywords: Parasites, biological control, Duddingtonia flagrans, Mucor circinelloides, horses. Funding ackowledgements: The experimental design has been approved by the Ethical committee of the University of Santiago de Compostela, complies with the Directive 2010/63/EU and was partly supported by the Research Project protocol number CTM2015-65954-R (Spanish Ministry of Economy and Competitiveness; FEDER) and Project UID/CVT/276/2013 (CIISA)

88

Master students contest Poster M44

Splenic hemangiosarcoma in dog: association between erythrocyte parameters and the prognosis Santos I1, Vicente G2, Nunes T3, São Braz B3

1. Faculdade de Medicina Veterinária, Lisboa, 2. Hospital Escolar/Faculdade de Medicina Veterinária, Oncology Department, Lisboa, 3. CIISA/Faculdade de Medicina Veterinária, Lisboa Background: Hemangiosarcoma is the most common splenic tumor in dogs, most of which are anemic due to internal hemorrhage, after splenic rupture. The primary goal of this study was to evaluate the association between erythrocyte parameters markers of anemia and the prognosis of dogs with splenic hemangiosarcoma. The initial hypothesis formulated is that the survival time will be lower in animals with lower hematocrit values, erythrocyte counts and hemoglobin concentration; with presence of splenic rupture; and in those who underwent splenectomy only. Methods: Dogs with histopathological confirmed splenic hemangiosarcoma, presented at FMV Scholar Hospital, between January of 2011 and February 2018, were included in this retrospective study (n=35). A Spearman correlation was calculated between the erythrocyte parameters and the survival time, and a Kaplan Meier survival analysis was made between the amount of dogs with values greater than or equal to, and below a cut-off established for each one of the erythrocyte parameters (26.2% for the hematocrit, 8.97 g/dl for the hemoglobin concentration and 3.78x106/μl for the erythrocyte count). Additionally, a Kaplan-Meier survival analysis was made according to the presence of splenic rupture (n=28) or its absence (n=5); whether the dog was stage I (n=5), II (n=13) or III (n=15); and depending on the treatment to which they were subjected: surgery only (n=26) or surgery followed by chemotherapy (n=7). Results: The median survival time was significantly lower in dogs with splenic rupture (59.5 days) compared with those without splenic rupture (240 days). Despite the statistical significance, stage III was associated with a lower median survival time (45 days), compared to stage II (83 days) and stage I (240 days) dogs. The animals submitted to surgery only had a median survival time lower (50 days) than those submitted to a chemotherapy treatment after splenectomy (121 days). A positive correlation was calculated between all the erythrocyte parameters and the survival time. The median survival time obtained for the dogs characterized by erythrocyte parameters greater than or equal to the cut-off values established (120 days) was significantly higher than those with a hematocrit, erythrocyte count and hemoglobin concentration below the cut-off values (45, 13 and 13.5 days, respectively). Conclusions: A more severe anemia is associated with a worse prognosis, as well as the presence of splenic rupture and surgical treatment only. Key words: Splenic hemangiosarcoma, dog, erythrocyte parameters, prognosis

89

Master students contest Poster M45

ESBL resistance genes in fecal E. coli of calves fed waste milk with antimicrobial residues Cardoso M1, Prata I3, Rebelo I2, Nunes T1, Carneiro C1, Bexiga R1

1. Faculdade de Medicina Veterinária, Universidade de Lisboa - Centre for Interdisciplinary Research in Animal Health; 2. Proleite – Cooperativa Agrícola de Produtores de Leite C.R.L.; 3. CEVA Saúde Animal

Extended-spectrum β–lactamases are enzymes capable of hydrolysing β-lactam antimicrobials, providing resistance to many, including 3rd and 4th generation cephalosporins. Infections caused by E. coli harbouring ESBLs have increased in incidence in humans and dairy cattle. Feeding waste milk to calves, which may include milk with antimicrobial residues, has been observed to increase antimicrobial resistance in faecal isolates of calves. This study aimed to evaluate the association between feeding waste milk to calves and occurrence of antimicrobial resistance of calf fecal isolates by ESBLs. In each of 32 farms, fecal samples were collected from 5 healthy calves in the 1st month of life and pooled. Five isolates from each sample were selected and confirmed as E. coli by 16S gene amplification. ESBL production was determined phenotypically on 148 isolates from 31 farms by antimicrobial susceptibility testing to cefotaxime, ceftazidine both alone and after media impregnation with clavulanate. Genotypic confirmation was performed by PCR for the blaCTX-M-1, 2, 8, 9 and blaCMY-2 genes. A questionnaire was performed regarding potential risk factors for the emergence of antimicrobial resistance. A univariate analysis was performed to evaluate risk factors to include in a multivariable logistic regression. Phenotypically 40 E. coli isolates from 15 farms were found, whereas genotypically 55 isolates from 20 farms were confirmed. The questionnaire revealed that 28/31 farms fed waste milk to calves. On 7/31 farms an antimicrobial was added to milk, to prevent calf diarrhoea. The number of different intramammary tubes used as treatment options for mastitis varied from 1 to 7. The multivariate analysis identified the use of a higher number of antimicrobial options for mastitis treatment as a significant risk factor for calves shedding ESBL-producing E. coli. For each additional option used for mastitis treatment, there was a 2.2 times higher risk of calves shedding ESBL-producing E. coli. Reducing the use of antimicrobials for mastitis treatment should be encouraged to reduce antimicrobial resistance by ESBL. Keywords: ESBL, waste milk, calves, E. coli Acknowledgments: Magnomics is acknowledged for providing financial support for reagents and Dr. Lina Cavaco, National Food Institute, Technical University of Denmark is acknowledged for providing positive control isolates. Participating farmers and veterinarians are acknowledged for providing information and access to farms.

90

Master students contest Poster M46

Effects of methadone on intraocular pressure in dogs and cats Nunes M, Delgado E CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal Background: The purpose of this study was to determine the effects of methadone as a solo-agent of anaesthetic premedication, on intraocular pressure (IOP) in dogs and cats undergoing both elective surgeries or diagnostic procedures. Methods: The study group was composed of 32 dogs and 5 cats with 8,22 ± 3,73 years. Ophthalmic exam was normal. The baseline IOP (T0) of the subjects were registered before IV methadone at the 0.2 mg kg-1 dosage. IOP variations were registered ten (T10) and twenty (T20) minutes after the drug administration. IOP values were measured with rebound tonometry (Icare®, Helsinki, Finland) each animal being positioned in sternal recumbency, without e-collars and with the head maintained relaxed at the level of the thorax. All variables were compared at each specific time point using a repeated-measures analysis of variance (ANOVA) with R® 3.3.3 software and the R-Commander extension. The differences were considered significant when P < 0.05. Results: The mean ± SD baseline (T0) and post-treatment (T10, T20) IOP values were respectively: 17,05 ± 3,32 mm Hg, 16,92 ± 3,37 mm Hg and 16,25 ± 3,33 mm Hg. IOP increased transiently at T10 in the majority of the animals but the mean values at T20 were lower than in T0. There were no statistically significant differences between baseline values and post-treatment values (p=0.296). Conclusions: There were no significant variations in IOP values in dogs and cats after the administration of methadone as a solo-agent of anaesthetic premedication. Methadone may be a good alternative as anaesthetic premedication in intraocular surgery since it apparently does not interfere with IOP. Keywords: Glaucoma, opioids, methadone Funding Acknowledgements: Funded by Project UID/CVT/276/2013 (CIISA - Centre for Interdisciplinary Research in Animal Health).

91

Master students contest Poster M47 Hemogram of pregnant Labrador Retriever bitches Dourado R1; Soares B1; Alves I2; Mateus L3 1. Integrated Master Student of Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2. School of Agriculture, University of Lisbon, Portugal; 3. Center for Interdisciplinary Research for Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal. Pregnancy is a critical period in the reproductive life of a female dog. It is characterized by many physiological changes, which may appear to be pathological in the non-pregnant bitch. Therefore, Veterinarians should be aware of the normal alterations in the pregnant bitch. The aim of this study was to compare haematological parameters between pregnant and non-pregnant bitches. A total of nineteen Labrador Retriever bitches belonging to a single kennel were included in this study [11 pregnant (P = 11) and 8 non-pregnant (NP = 8); mean age: 3.2 years; range 1-6 years]. Blood sampling for complete hemogram were collected and assayed in 4 time periods (in estrus (T1), between 25-35 days (T2), 40-50 days (T3) and 61-63 days (T4) after the LH peak day). Data were statistically analyzed using MANOVA in Statistica for Windows (Statistica 6.0, StatSoft Inc., Tulsa, OK, EUA, 1995). In the pregnant group, near parturition (T4), a normocytic, normochromic mild anemia was observed in all females and leukocytosis due to neutrophilia were observed in 55 % of the females. In both groups, there was a decrease in the number of leukocytes (mainly neutrophils) from estrus to the first half of the diestrus (T2). This decrease remained until two thirds of diestrus (T3) in the non-pregnant group. The results obtained in the eritrogram may be explained by the hemodilution caused by the increase in total plasma volume (1). The decreased erythrocyte half-life may also account to the decreased erythrocyte number (2). The results obtained in leukogram are probably associated with the physiological stress induced by pregnancy leading to increased cortisol concentrations (3). The decrease in the leukocyte numbers observed in diestrus (T2 and T3) compared to estrus (T1) may be justified by the high concentrations of progesterone found in the first half of diestrus (4). Of the physiological changes characteristic of the gestational period, the normocytic, normochromic mild anaemia and in some cases leukocytosis due to neutrophilia are hallmarks of late pregnancy that Veterinarians must be aware. Keywords: Labrador Retriever; Pregnancy; Hemogram; Anemia; Leukocytosis. Funding: UID/CVT/00276/2013 (1) Concannon, P. W., Powers, M. E., Holder, W., & Hansel, W. (1977). Pregnancy and parturition in the bitch. Biology of Reproduction, 16(4), 517–526. (2) Chandra, S., Tripathi, A. K., Mishra, S., Amzarul, M., & Vaish, A. K. (2012). Physiological changes in hematological parameters during pregnancy. Indian Journal of Hematology and Blood Transfusion, 28(3), 144–146. (3) Awodu, O. A, Enosolease, M. E., Ubaru, A G., & Famodu, A A. (2002). Leucocyte counts in pregnant Nigerian women with sickle cell trait. African Journal of Reproductive Health, 6(3), 112–116. (4) Bartoskova, A., Ondrackova, P., Leva, L., Vitasek, R., Novotny, R., Janosovska, M., & Faldyna, M. (2014). The effects of in vitro exposure to progesterone and estradiol-17ß on the activity of canine neutrophils. Veterinarni Medicina, 59(4), 202–209.

92

Master students contest Poster M48

The role of alpha-1 acid glycoprotein in post operatory monitoring of chronic gingivostomatitis in the cat: an exploratory study Rosa R1, Branco V2, Iglésias L1, Pissarra H3, Niza M3, Mestrinho L3

1. Faculdade de Medicina Veterinaria, Universidade de Lisboa, Avenida da Universidade Tecnica, 1300-477 Lisboa, Portugal; 2. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; 3.

Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinaria, Universidade de Lisboa, Avenida da Universidade Tecnica, 1300-477 Lisboa, Portugal Background: Inflammatory biomarkers such as alpha-1 acid glycoprotein (AGP) are valuable tools in monitoring inflammatory disease progression in response to an established therapy.1 AGP serum levels were evaluated in cats with chronic gingivostomatitis (FCGS) compared with a control group and at 2 post-extraction moments in order to explore the role of this acute phase inflammatory protein in the clinical monitoring of this disease. Methods: Twenty cats were recruited for the study, 10 controls and 10 cases. Blood samples were collected at day 0 for both groups and for the case group, 30 and 60 days after surgical procedure with dental extractions. Serum AGP was determined using an AGP-8 enzyme linked immunosorbent assay (ELISA) kit. Results: In this study all diseased cats presented severe clinical lesions of caudal stomatitis and alveolar stomatitis, histological findings of severe inflammation, and positive isolation feline calicivirus antigen, reinforcing the homogeneity of the group. The serum concentration of AGP was significantly superior in the FCGS group compared with the control group, confirming that these cats are in a systemic inflammatory state with a positive statistically significant association between AGP values and caudal stomatitis lesions at day 0 and alveolar stomatitis at day 30 and 60 respectively. Higher AGP levels were found in cats with unfavorable response to treatment although such levels at presentation were not indicative of unsuccessful outcome. However, the absence of AGP decrease from 0 to 60 days was significantly related with an unsuccessful outcome. Indicating that cats not responding to surgical extractions at 60 days showed no decrease of AGP. Conclusions: This preliminary study suggests that this biomarker may be useful in monitoring this disease. Keywords: Feline chronic gingivostomatitis, alpha-1 acid glycoprotein, dental extractions, biomarker, feline calicivirus. Funding: The present work was funded by the project UID/CVT/276/2013 from the Center for Interdisciplinary Animal Health Research (CIISA), Lisbon, Portugal, internal grant Ref. 012/2018. Vasco Branco is funded by a Post-doc Fellowship, SFRH/BPD/85219/2012, from Fundação para a Ciência e Tecnologia (www.fct.pt).

93

Master students contest Poster M49 Evaluation of adverse effects – increase in serum creatinin and/or urea, in cats undergoing doxorubicin administration Fernandes M1, Vicente G2, Nunes T3, São Braz B3

1. Integrated Master Student of Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2.

School Hospital, Faculty of Veterinary Medicine, University of Lisbon, Portugal; 3. CIISA, Faculty of Veterinary Medicine, University of Lisbon, Portugal. Doxorubicin is an anthracycline with anti-neoplastic action and nephrotoxic potential. The characterization of this toxicity in cats is still ongoing1. One of the kidney responses to injury is the development of chronic renal disease (CKD). The renal dysfunction may result in a decreased glomerular filtration rate with a consequent increase in serum creatinine and/or urea among other alterations2. The aim of this study was to evaluate a sample of cats submitted to doxorubicin administration regarding the presence of azotemia. The clinical history of the feline population sample that was presented at the Oncology Service at the Veterinary School Hospital between 2013 and 2018 was analyzed. The study included 28 tumor bearing-cats (median age: 11 years, range: 6- 17kg, range: 3-6.8kg) submitted to single protocols with doxorubicin and with blood samples taken prior to each session for serum creatinine and urea measurements. Doxorubicin was administered at a dose of 25 mg/m2 by intravenous infusion (20-30 minutes) after diluting the 2 mg/mL solution in 50mL of 0.9% NaCl with a 21 day interval. The animal was considered to have azotemia when serum creatinine and/or urea values were higher than the upper limit value. In order to standardize the results the consensus of the Veterinary Cooperative Oncology Group (VCOG-CTCAE) was used. Statistical analysis of data was performed with R Commander® software. The incidence of azotemia was 61% and a statistically significant difference was found between the initial and final values of creatinine and urea (p=0.002; 0.018) with a median cumulative dose of 100 mg/m2. Survival analysis showed a median time of 155 days until the development of azotemia (TA) and a significant difference (p=0,000682) in TA between age. The persistence of the azotemia with subsequent development of CKD was observed in 39% of the cats. The incidence of azotemia is higher than the verified by Reiman et al. and Poirier et al. while the TA observed is lower3. Although a possible cause-effect relationship is intuitive to observe it is difficult to assess whether progressive renal dysfunction in geriatric tumor bearing-cats with possible subclinical renal disease is directly due to the use of doxorubicin1. Thus, careful evaluation of renal function prior to administration of the chemotherapeutic agent and monitoring during treatment is advisable. Prospective studies about the nephrotoxic mechanisms of doxorubicin in cats are needed. Keywords: Doxorubicin; cats; creatinin; urea; nephrotoxicity.

1Reiman, R. A., Mauldin, G. E., & Mauldin, G. N. (2008). A comparison of toxicity of two dosing schemes for doxorubicin in the cat. Journal of Feline Medicine and Surgery, 10(4), 324–331. https://doi.org/10.1016/j.jfms.2007.12.009 2Schnellmann, R. G. (2013). Chapter 14: Toxic Responses of the Kidney. Em C. Klaassen, L. J. Casarett, & J. Doull, Casarett & Doull’s Toxicology. (8a). Blacklick: McGraw-Hill Publishing. 3Poirier, V. J., Thamm, D. H., Kurzman, I. D., Jeglum, K. A., Chun, R., Obradovich, J. E., … Vail, D. M. (2002). Liposome-Encapsulated Doxorubicin (Doxil) and Doxorubicin in the Treatment of Vaccine-Associated Sarcoma in Cats. Journal of Veterinary Internal Medicine, 16(6), 726–731. https://doi.org/10.1111/j.1939-1676.2002.tb02415.x

Posters

95

Poster 50 Diffusion of a nisin-biogel on a Diabetic Foot Ulcer collagen model Gomes D1, Rego P2, Reis S1, Carvalho S1, Dias S3, Santos R1,3, Mendes JJ4,5, Peleteiro MC1, Tavares L1, Veiga AS3, Oliveira M1

1. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa; 2. Centro de Química Estrutural, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa; 3. Instituto de Medicina Molecular (IMM), Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa; 4. Institute of Microbiology, Faculty of Medicine, University of Lisbon, Avenida Professor Egas Moniz, 1649-028 Lisboa; 5. Hospital Professor Doutor Fernando Fonseca, IC19, 2720-276 Amadora, Portugal Background: There is a crucial need to develop new strategies to treat Diabetic Foot Infections (DFI), a major complication of Diabetes mellitus (DM), considering its increasing prevalence worldwide and its association with biofilm-producing and antimicrobial resistant strains. Antimicrobial peptides (AMP) are potential substitutes for conventional antibiotics. One of the best characterized AMP, nisin, is broadly used as a food preservative to control Gram-positive bacteria. Our team already demonstrated its antimicrobial activity against Staphylococcus aureus DFI isolates collected in Lisbon Medical Centers, including biofilm-producing and Methicillin-Resistant strains. We also evaluated the potential of a guar-gum biogel as a topical delivery system for nisin, showing that this AMP can rapidly diffuse from the biogel and eradicate planktonic cells and biofilms. However, the DFI microenvironment is complex, and antimicrobials diffusion and efficacy may be impaired by biofilms and host biomolecules. Methods: The diffusion ability of the nisin-biogel was evaluated using an in vitro model mimicking the DFI microenvironment. A collagen wound model was established and exposed to one dose of the nisin-biogel for 24h or to 3 consecutive doses at every 8h. After incubation, wound model was removed and sectioned in 3 areas for AMP distribution evaluation, by performing nisin quantification in the different collagens layers using an improved agar diffusion assay. Results: When a single dose of the nisin-biogel was applied, nisin concentrations at the different areas were 1.858 (area 1), 1.104 (area 2) and 0 mg/mL (area 3). When 3 doses of the nisin-biogel were applied during a 24h period, nisin concentrations in the 3 areas increased to 6.754 (area 1), 4.400 (area 2) and 1.575 mg/mL (area 3). Conclusions: The application of the nisin-biogel to the wound model every 8h allows nisin diffusion, reaching area 3 in concentrations higher than its MIC value (0.022 mg/mL), but lower that the Daily Dose Intake of 2 mg/Kg established by FAO (2013) for the oral consumption of this AMP. In the future, this new biogel may be applied to the effective treatment of DFI, ultimately contributing to decrease the frequency of lower limb amputation, improving health care and life quality of DM patients and to decrease antibiotic administration in the clinical setting, impairing resistance dissemination. Keywords: Antimicrobial peptides; Collagen wound model; Diabetic Foot Infections; Nisin-biogel. Funding Acknowledgements: Work was supported by the Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa (FMV/UL), (Project UID/CVT/00276/2013) and by FCT (Project PTDC/SAU-INF/28466/2017 - Polyphasic validation of antimicrobial peptides as alternative treatment for diabetic foot infections).

96

Poster 51

Wnt-Prostaglandin Pathways interaction in Murine Embryo Development Pinto M, Batista M, Diniz P, Murta D, Silva E, Lopes-da-Costa L, Torres A Reproduction and Development, CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon Early pregnancy loss in humans, which is often due to defects that occur around implantation time, is a worldwide social and economic concern. Prostaglandins (PG) influence embryo implantation and embryo spacing (1,2). Canonical Wnt pathway plays an important role in embryo development and uterine decidualization (3). The interaction between the above pathways in mammalian embryo development is unknown, although already established for other biological systems (4,5). This work aimed to evaluate the interaction of Wnt and PG pathways in embryo development. Embryos (2.5 days post coitum) from CD1 superovulated female mice (20 embryos/group/session; n= 5 sessions) were collected and cultured in vitro in the presence of DKK1 (canonical Wnt inhibitor) or vehicle until blastocyst stage. Following culture, embryos were either processed for RNA extraction (n= 26 embryos/group) and qRT-PCR of PG pathway genes (COX-1, COX-2, cPGES, mPGES-1, mPGES-2, PGFS, PGIS), or transferred into pseudopregnant females (14 embryos/female, n= 10 females, 5 sessions). After 48 hours, evaluation of the implantation sites was performed using the blue dye method, and endometrial tissue was collected and processed for RNA extraction and qRT-PCR of PG pathway genes. Transcription of COX-1 was present in both expanded and hatched blastocysts, but transcription of COX-2 was not detected. There was an increased transcription (p<0.05) of mPGES-1 and PGFS in expanded blastocysts and of cPGES and mPGES-2 in hatched blastocysts. The blockade of canonical Wnt pathway had no significant effect on transcription of PG pathway genes in pre-implantation embryos and decidual tissue. In conclusion, this is the first report on the transcriptional pattern of PG pathway genes in murine pre-implantation embryo development. Genes coding for PGE2 synthesis are transcribed during blastocyst expansion, hatching and implantation. The intriguing absence of COX-2 transcription may suggest a preference for the COX-1/PGES pathway in PGE2 synthesis. Finally, there was no evidence of an interaction between the Wnt and PG signaling pathways in murine embryo development. Keywords: Wnt; Prostaglandins; Embryo development; Implantation Funding: CIISA UID/CVT/00276/2013 – CIISA3/2016 1 Kennedy, T.G. (1977). Biology of Reproduction, 16, 286-291; 2 Wellstead, J. et al. (1989). The Anatomical Record, 225,101-105; 3 Chen, Q. et al. (2009). Molecular Human Reproduction, 15 (4), 215-221; 4 Goessling, W. et al (2009). Cell, 136 (6), 1136-1147; 5 Oshima, H. & Oshima, M. (2010). Pathology International, 60, 599-607

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Poster 52

Portuguese traditional PDO cheeses: characterization of the autochthonous microbiome Ruivo M1, Crespo MTB2,3, Barreto AS1 and Semedo-Lemsaddek T1

1. Centro de Investigação Interdisciplinar em Sanidade Animal -CIISA, Faculdade de Medicina Veterinária da Universidade de Lisboa, Portugal; 2. iBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 3. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal In Mediterranean countries, such as Portugal, many dairy foodstuffs are manufactured carrying out the artisanal ways, without the use of starter cultures. Hence, fermentation processes rely on in house microorganisms, known as the autochthonous microbiome, whose technological skills are responsible for the organoleptic specificities of each product/region. The present study analysed two Portuguese traditional PDO cheeses (Azeitão and Nisa), samples being collect over a three years period (2016, 2017 and 2018) at seven dairies (five from Azeitão and two from Nisa). In order to characterize the autochthonous microbial community present in the cheese samples, conventional and molecular microbiology, as well as a metagenomic approach (sequencing of the V1-V3 region of the 16S RNA), were applied. Data gathered led to the identification of Lactococcus and Leuconostoc genus as the most abundant among cheese samples, despite region of origin. Although results showed significant variations on the microbial diversity between dairies, regions and over time, several lactic acid bacteria could be identified as being characteristic of specific dairies/regions. These eco-specific bacteria belong mainly to the genus Enterococcus and remained constant over-time, suggesting their importance for the manufacture of the products under study. Overall, results obtained demonstrated the complexity of the microbial ecology of traditional food products and proved the relevance of the autochthonous microbiome, leading towards the need for a further characterization of these in house microorganisms, especially regarding pathogenicity potential and technological features. Keywords: Portuguese artisanal cheese; Protected Denomination of Origin; autochthonous microbiome; lactic acid bacteria; microbial diversity Funding Acknowledgements: Experimental work was supported by Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). Teresa Semedo-Lemsaddek is financially supported by FCT (SFRH/BPD/108123/2015). Authors also thank PDO cheese producers for supplying the samples.

98

Poster 53

Pulmonary pathological processes and animal welfare in pigs Dias S1, Rodrigues C2, Veloso MG1, Nunes TP1, Cota JB1 1. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. Direção Geral de Alimentação e Veterinária, Divisão de Alimentação e Veterinária de Setúbal, Quinta da Várzea - Estrada dos Ciprestes, 2910-315 Setúbal, Portugal Background: Respiratory tract diseases are a major problem in pig health, negatively affecting pig intensive production systems. Pneumonias and pleuritis are frequently detected on the slaughter line, being directly responsible for marked losses in production indexes and the profitability of swine production sector. In order to achieve a greater control and enhance the sanitary status of pig farms several countries have implemented lesion monitoring programs performed during post mortem inspection. The main objective of this study was to assess the incidence of pathological processes affecting the lungs of fattening pigs, reporting the data on a monthly basis to the veterinarians responsible for the pig farms. These reports aimed to create awareness amongst the veterinarians for the diseases affecting the animals and to aid potential changes on prophylactic or therapeutic plans or even in rearing conditions. Methods: The presented study focused on a total of 15894 fattening pigs, from 118 different lots slaughtered on a Portuguese abattoir. Each carcass was evaluated regarding the presence of lesions indicative of enzootic pneumonia, viral pneumonia, pleuropneumonia and pleuritis. Lung samples from 33 different carcasses with suggestive pneumonia lesions were collected for histopathological diagnosis, in order to verify if there was a correspondence between the macroscopical diagnosis performed on the slaughter line and the microscopical patterns of the diseases under evaluation. Results: During the post mortem inspection, about half (53.6%) of all of the carcasses evaluated presented at least one of the lesions under study. From these, the most prevalent finding was enzootic pneumonia (56.6%), followed by pleuritis (37.7%) and finally pleuropneumonia (3.2%) and viral pneumonia (2.5%). The histopathologic analysis carried out validated all the macroscopical diagnosis performed by the veterinary inspectors. Conclusions: The high prevalence of lesions revealing ongoing respiratory disease has a significant impact both on an economical level as well as on animal welfare. Thus, the implementation of lesion monitoring programs, followed by the report of the post mortem inspection findings to the herd’s veterinarians, should promote greater animal health, welfare and productive performance. Keywords: Respiratory disease; Fattening pigs; Monitoring program; Slaughterhouse; Funding Acknowledgements: The authors would like to acknowledge the staff of the Pathology Laboratory of the Faculdade de Medicina Veterinária – ULisboa for the technical support.

99

Poster 54

Antimicrobial activity of essential oils against bacteria of the oral microbiota Aires A, Barreto AS, Semedo-Lemsaddek T

CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal Oral microbiota is composed by Enterococcus, Streptococcus and Actinomyces, among other microrganisms. These bacteria are known for forming dynamic communities of sessile cells, interact and adhere irreversibly to solid surfaces and each other. Concurrently, the construction of extracellular polymeric substances (EPS) will cover and protect the producing strains, conferring protection against exterior agents. These biofilms are a major public health concern due to their contribution to infection and therapeutical failure, as well as for facilitating genetic exchanges. Nowadays, natural compounds, such as essential oils -EOs-, are being analysed as alternatives to penetrate the biofilm and eliminate the bacteria present, contributing to the eradication of these structures. EOs are secondary metabolites produced by plants increasingly used as antimicrobial agents; clove, oregano and thyme EOs being among the most promising products. The present work aimed to evaluate bacteria present in the oral microbiota for their capacity to produce biofilms, both in pure and mixed cultures. Distinct growth media, temperatures and times of incubation were tested. Results showed that Brain Heart Infusion medium supplemented with mucin (an in vitro simulation of the oral environment), 48h of incubation and room temperature favoured biofilm formation for pure and polymicrobial bacterial cultures. Subsequently, clove, oregano and thyme EOs for screened for their capacity to control the growth and/or eliminate the oral bacteria. Assays using the microdilution methodology allowed the determination of Minimum Inhibitory Concentrations (MIC), Minimum Bactericidal Concentrations (MBC) and Minimum Biofilm Eradicating Concentrations (MBEC). Clove and thyme showed the most promising results and can be considered good antimicrobial alternatives. To further confirm such an assumption a toothbrush-model was developed. To simulate a toothbrush contaminated with microorganisms, as usually occurs in a common bathroom, sterilized nylon fibers inoculated with the oral bacteria under study were used. After allowing biofilm formation, essential oils at different concentrations were tested for their ability of disrupt the matrix and eliminate the bacteria. Results demonstrated the antimicrobial potential of thyme and clove essential oils, as occurred in the screening assays. Thus, the present study verified that EOs can be considered valid alternatives to traditional disinfectants and other antimicrobial agents. Keywords: Oral microbiota, biofilms, essential oils, antimicrobial activity, toothbrush-model Funding Acknowledgments: Teresa Semedo-Lemsaddek is financially supported by FCT (SFRH/BPD/108123/2015).

100

Poster 55

Ocular brachycephalic syndrome

Costa J1, Steinmetz A2, Delgado E3 1. Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Department of Veterinary Ophthalmology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany; 3. CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal Background: Brachycephalic breeds are well known to have respiratory problems due to anatomical skull changes, which are also responsible, directly or indirectly, for ocular alterations. The most common diseases include entropion, distichiasis, trichiasis, keratoconjunctivitis sicca, ectopic cilia and corneal ulceration. When not treated, they lead to permanent damage which consequently can cause a decrease of the dogs’ vision acuity and compromise their wellbeing. Purpose: This prospective study aims to characterize a sample of forty -seven brachycephalic dogs on what concerns their breed, age, gender, clinical signs and ocular alterations. Materials & Methods: The studied population consisted of 49% females and 51% males, including 45% French Bulldogs, 34% Pugs, 7% English Bulldogs, 4% Boston Terriers, 4% Bullmastiffs, 2% Boxers, 2% Shar-Peis and 2% Shih-Tzus, with an age range between 4 months and 12 years old. The most frequent clinical signs were blepharospasm in 28% of the animals, conjunctivitis in 26%, ocular discharge in 19%, and epiphora in 15% of the cases. After an ophthalmological examination that included the assessment of ocular reflexes and responses, and closer inspection with a slit lamp, some complementary exams were performed. Results: The most commonly used ophthalmic diagnostic exams were Schirmer tear test in 40%, Tonometry in 23% and Fluorescein test in 19% of the cases. The most frequent ocular abnormalities were macroblepharon in 75%, entropion in 45%, corneal ulcers in 30%, corneal pigmentation in 23%, corneal opacity and distichiasis in 21% of the studied animals. There was a higher incidence of corneal pigmentation in Pugs and corneal opacity in French Bulldogs. According to the clinical presentation, the patients underwent medical or surgical treatment. The surgical techniques more frequently used were medial canthoplasty in 28% and electroepilation in 11% of the cases. All the cases had a good recovery and showed no post-operative complications. The medical treatment was often indicated, mostly in cases of superficial ulcers, management of KCS and during surgical recovery. Conclusions: This study allowed the perception of the incidence of ocular alterations related to the brachycephalic conformation. Some differences between breeds were also noted, including a higher incidence of corneal pigmentation in Pugs and corneal opacity in French Bulldogs, which suggests that some brachycephalic breeds may be predisposed to certain ocular abnormalities. The percentage of secondary problems, like ulcers, was high, which alerts to the importance of a regular ophthalmological check- up, so that an early diagnosis of the primary disorders can be achieved, avoiding the development of secondary alterations and permitting the institution of an adequate treatment. Keywords: Ocular Brachycephalic Syndrome; entropion; corneal ulcers; distichiasis Funding Acknowledgements: Funded by Project UID/CVT/276/2013 (CIISA)

101

Poster 56

Medical therapeutic approaches to corneal ulcers in dogs and cats: a prospective study of 112 cases

Martins S1, Resende AP2, Delgado E3

1. Faculty of Veterinary Medicine, University of Lisbon, Alameda da Universidade Técnica, 1300-477 Lisbon, Portugal; 2. Vet Visão – Veterinary Clinic of Benfica, Av. Grão Vasco, nº 46, store 2, 1500-339 Lisbon, Portugal; 3. CIISA - Centre for interdisciplinar Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon

Background: A prospective study of the medical therapeutic approaches applied in corneal ulcer cases in cats and dogs during 15 months at the Veterinary Hospital of the Faculty of Veterinary Medicine and Vet Visão Veterinary Clinic is presented. The purpose of this study was to evaluate the clinical outcomes of the clinical cases followed in response to the medical therapeutic approaches applied. Methods: A total of 112 clinical cases met the inclusion criteria. The incidence of corneal ulcer cases, animal species, sex, age, breed, clinical description of the cases, results of the ophthalmological examination, treatment, healing time, outcome, sequels and relapses were statistically analyzed. Results: The incidence of corneal ulcer cases at the Ophthalmology Consultation at the Veterinary Teaching Hospital FMV-UL between February and July 2016 was 13.5% (42/312). Almost half of the cases occurred in brachycephalic breeds (46.4%), the French Bulldog (17.1 %) and Persian (46.2%) being the most common breeds for dogs and cats respectively. The types of corneal ulcers found in this study were superficial simple corneal ulcers 37.5% (42/112), stromal ulcers 23.2% (26/112), spontaneous chronic corneal epithelial defects 10.7% (12/112), melting ulcers 9.8% (11/112), corneal perforations 7.1% (8/112), descemetoceles 6.3% (7/112), and superficial dendritic ulcers 5.4% (6/112). 58.0% (65/112) of the patients received exclusively medical treatment, the rest received medical plus surgical treatment. In all the cases topical antibiotherapy was employed, tobramycin being the most frequent topical antibiotic applied in clinical cases in dogs (76.3%) and cats (56.3%), followed by chloramphenicol (8.8%) in canine patients and ofloxacin in felines (25.0%). Intensive topical antimicrobial therapy was applied to 73.2% of the cases. Other frequent therapeutic agents administered include mydriatics (65.2%), autologous serum (44.6%) and topical antivirals (28.6%). The mean healing time was 60.9 ± 59.7 days. A positive clinical outcome occurred in 90.2% (101/112) of the cases. The most common sequel was leukoma (51.5%). Relapses occurred in 11.6% (13/112) of the clinical cases. Conclusions: Brachycephalic breeds have a higher risk of developing complicated corneal ulcers. The diversity of corneal ulcer types and its clinical evolution makes it necessary to be flexible in the therapeutic approach chosen for each clinical case. Keywords: corneal ulcer incidence; brachycephalic breeds; ocular topical antibiotherapy; tobramycin Funding: UID/CVT/276/2013 (CIISA)

102

Poster 57

Development and analytical validation of a group-specific real-time RT-PCR assay for the detection of the Simbu serogroup orthobunyaviruses Camarão AAR1, Boinas F1, Quan M2

1. Faculty of Veterinary Medicine, University of Lisbon, Portugal; 2. Vector and Vector-borne Diseases Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa Background: The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses. This group of arthropod-borne viruses has a cosmopolitan distribution and causes clinical disease in humans and domestic animals, however, definitive diagnosis always requires laboratorial confirmation. A novel group-specific TaqMan® real-time RT-PCR assay was developed, optimised and analytically validated for the broad detection of the Simbu serogroup orthobunyaviruses. Methods: The published genomic data of the Simbu serogroup members were evaluated, and a conserved region, situated in the S segment which encodes for the nucleocapsid protein, was used to design a group primer set and a pair of differently labelled TaqMan® minor groove binder probes to distinguish phylogenetic clade A and B of the Simbu serogroup. Seven prototype Simbu serogroup isolates were used for the development of the assay, namely Akabane orthobunyavirus (AKAV), Simbu orthobunyavirus (SIMV), Shuni orthobunyavirus (SHUV), Sathuperi orthobunyavirus (SATV), Shamonda orthobunyavirus (SHAV), Ingwavuma virus (INGV, now classified as Manzanilla orthobunyavirus) and Sabo virus (SABOV, now classified as AKAV). The primer and probe concentrations in the PCR reactions were optimised. To assess the specificity of the assay, a panel consisting of genetically related, causative agents of abortion in ruminants and arthropod-borne viruses was selected for in vitro and in silico analysis. Results: The efficiency of the assay was determined for each one of the viruses: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) and SABOV (110%). The assay was shown to be specific, as no cross-reactions were observed either in vitro or in silico, and sensitive, with a 95% limit of detection ranging from 100,39 to 10-

3,61 TCID50/reaction, for the detection of Simbu serogroup viruses. The repeatability of the assay was evaluated and was shown to be consistent within and between runs. Conclusion: This assay showed more sensitivity, specificity and broad detection capacity when compared with other published methods for detection of Simbu serogroup viruses. Therefore, it can be a useful tool to confirm diagnosis of suspect or clinical cases and to estimate the prevalence of infection or exposure, facilitating risk analysis. Keywords: Akabane, Shuni, TaqMan® Funding Acknowledgements: This research was supported by the Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Pretoria, South Africa.

103

Poster 58

Canine model for sepsis: a contribution to the classification and stratification of septic patients Prata S1, Aguiar S2, Gomes J3, Almeida V2, Bexiga R2, Tavares L2, Aires da Silva F2, Gil S2

1. Faculty of Veterinary Medicine, ULisbon, Portugal, Av. Universidade Técnica, 1300-477; 2. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculty of Veterinary Medicine, ULisbon, Portugal, Av. Universidade Técnica, 1300-477 Lisboa, Portugal, Department of Animal Health (DSA); 3. Hospital Escolar da Faculdade de Medicina Veterinária, Faculty of Veterinary Medicine, ULisbon, Portugal, Av. Universidade Técnica, 1300-477 Lisboa, Portugal Background: Sepsis can affect all species, being a very relevant cause of death among Human beings. Its high prevalence and severity make research a priority for the Medical and Veterinary community. CPV (Canine Parvovirus), a viral disease that can promote bacterial translocation from gut origin, can be a predisposing cause of sepsis. Studies involving naturally CPV infected dogs can be useful when investigating sepsis mechanisms, not only in animals but also in human beings. This work aims to propose a sepsis patient scoring and stratification system based on the PIRO (Predisposition, Infection, Response and Organ dysfunction) model. Two SIRS (Systemic Inflammatory Response Syndrome) diagnostic criteria (SIRS 1991 and SIRS 2001) were compared. Another goal was to study CRP (C-reactive protein) in infected dogs, comparing those results with the SIRS diagnostic criteria. Methods: The 31 dogs in the experimental group were scored according to several parameters regarding Predisposition, Response and Organ dysfunction (all the subjects were given an equal Infection score). Data was retrieved from clinical records of the HEV-FMV-UL Infectious Diseases Unit. They were also subjected to two different sets of SIRS diagnostic criteria. CRP concentration was measured in 13 animals. Results: Blood CRP measurements were higher in CPV infected dogs when compared to dogs in the control group (p<0.001). Based on approved Veterinary Medicine SIRS criteria, a CRP threshold value for the presumptive diagnosis of SIRS was found (83.8 mg/L; AUC=0.85). Results obtained using different SIRS diagnostic criteria, on hospital admission as well as after 48h, were significantly different (p<0,01). Differences between scores obtained on hospital admission and after 48h were not (p=0,187). A correlation between the increase in CRP concentration and the increase in the PIRO score was not found (rs=0,187; p=0,540). Conclusions: The number of animals diagnosed with SIRS is larger when the SIRS 1991 set of criteria is used. Despite the likely loss of sensitivity, this adaptation aims to improve specificity. CPV-infected dogs have higher CRP concentrations than control group dogs. The CRP cut-off value for SIRS (83,8 mg/L) could be a valuable tool, especially when in conjunction with rapid tests. This work represents a contribution towards the development of a classification system for septic canine patients. Keywords: CPV, Sepsis, SIRS, PIRO, CRP, HEV-FMV-UL. Funding Acknowledgements: This work was supported by the Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) (Project UID/CVT/00276/2013).

104

Poster 59

Feline hemoplasmas: evaluation of specific antibodies for molecular and cytological diagnostic

Catarino P1, Hofmann-Lehmann R2, Meireles J1, Nunes T1, Tavares L1, Duarte A1 1. CIISA FMV-UL, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2 Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland

Background: Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMti) are three feline hemoplasmas, responsible for severe hemolytic anemia in felids. The laboratory diagnosis of infection can be performed by cytological examination of blood smears or by quantitative PCR assay (qPCR), which is considered the gold standard method. For a better understanding of the infection pattern and differentiation of acute and chronic infections, an ELISA was developed (1) using a recombinant antigen (rDnaK; Mhmf). In order to evaluate the usefulness of this recombinant ELISA, for the detection of specific hemoplasmas antibodies. Antibody detection was performed in parallel to qPCR and cytological examination of blood smears in natural infected cats. Material and Methods: Total blood samples from 104 stray cats, were collected under a Capture Neuter Release Program implemented by the Lisbon City Hall. The study was approved by the Ethical Committee (CEBEA) of FMV-UL. After collection blood samples were divided in: i) Plasma samples, sent to Vetsuisse Faculty, University of Zurich, Switzerland for antibody detection; ii) total DNA extraction, for qPCR of Mhmf, CMhmm and CMturi (Virology Laboratory, FMV/ULisboa) and iii) blood smears preparation followed by Giemsa staining (Parasitology Laboratory, FMV/ULisboa). Results: The qPCR assay detected 23/104 positive samples, including Mhf (n=2), CMhm (n=12) and CMt (n=2) single and co-infections (n=7). The cytological examination identified 51/104 positive smears. Regarding the ELISA results, 16/104 samples were seropositive and 10/104 samples presented values considered as suspicious. Within the 23 positive qPCR samples, only 16 were also found positive by cytology and only 5 were considered seropositive; 3 were suspicious. Of the 83 negative samples by qPCR, 43 were also negative by cytology, 63 were seronegative and 7 were undetermined. Conclusions: Assuming the higher performance of the qPCR assay, little convergence was observed between the molecular data and the microscopic analysis, which was already expected. Regarding the recombinant ELISA, probably due to the recombinant antigen origin (Mhf), a higher proportion of seropositive animals (n=16) was detected than Mhf DNA positive animals (n=2 Mhf; n=5 co-infections). Further studies are needed to determine the longevity of hemoplasmas specific antibodies and the usefulness of a recombinant ELISA to assess the infection status of susceptible felids. Funding acknowledgments: Funded by Project UID/CVT/276/2013 CIISA (Centro de Investigação Interdisciplinar em Sanidade Animal da Faculdade de Medicina Veterinária da Universidade de Lisboa (CIISA FMV-UL) Keywords: Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum; Candidatus Mycoplasma turicensis; quantitative PCR, ELISA (1) Barker, E.N., et al., Detection of humoral response using a recombinant heat shock protein 70, DnaK, of Mycoplasma haemofelis in experimentally and naturally hemoplasma-infected cats. Clin Vaccine Immunol, 2010. 17(12): p. 1926-1932.

105

Poster 60

Frequency of companion animals infectious diseases at a veterinary hospital isolation unit Machado IC1, Cunha E1,2, Gomes J1, Tavares L2, Almeida V2, Gil S1,2 1. Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Lisbon (VTH), Infectious Diseases Isolation Unit (IDIU); 2. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon. Animal Health Department, Lisbon, Portugal. Background: The Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Lisbon has a multi-species Infectious Diseases Isolation Unit (IDIU) for the admission of animals with confirmed infectious disease (ID) or clinically suspected awaiting diagnosis. Identification and segregation of ID patients is a key procedure for preventing spread of ID. Proper isolation procedures are mandatory to assure best medical practices, namely reducing nosocomial infections. This study aims to characterize the IDIU in-house population, from October 2013 to December 2017, describe ID clinical presentations and calculate survival rates for the most frequent ID in cats and dogs. Methods: Data records were exported from the VTH information system into an Excel, validated and coded. Missing information was taken from patients' record cards and added to database. Only animals with confirmed ID were considered for estimation of disease frequencies and survival rates. Results: Out of 612 animals hospitalized during the study period 316(51.6%) were cats and 296(48.4%) were dogs. From 316 cats, 231(73.1%) had confirmed ID, fulfilling inclusion criteria. 65 had feline leukemia (FeLV), with 50.8% survival rate, 54 had feline immunodeficiency (FIV), with 70.4% survival rate and 11 were coinfected FeLV/FIV with 63.6% survival rate. 45 had upper respiratory tract disease with 77.8% survival rate, 23 had panleukopenia with 65.2% survival rate, 7 had multidrug-resistant bacteria (MDR) with 57.1% survival rate, 18 had coronavirus (FCoV), with 50% survival rate for feline infectious peritonitis (FIP) cases and 62.5% for FCoV positive (but not FIP), 8 cats had other ID. From 296 dogs, 174(58.8%) had confirmed ID, fulfilling inclusion criteria. 66 were confirmed with parvovirosis, with 84.9% survival rate, 37 had leptospirosis with 62.2% survival rate, 24 had distemper with 41.7% survival rate and 15 had MDR with 66,7% survival rate. 8 dogs had dermatophytosis, 4 adenovirus, 3 infectious tracheobronquitis and 17 other ID. All these animals were recovered, discharged and returned to their owners Conclusions: Hospitalization of patients with ID in negative pressure, high security isolation units intend to reduce microbial dissemination, nosocomial infections and improve survival rates. Although ID are important in veterinary medicine, there is an objective lack of clinical studies. In this study, several ID in cats and dogs were investigated with survival rates ranging from 42% to 85%. Keywords: IDIU, survival rates, infectious diseases Funding Acknowledgements: This work was supported by CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) (Project UID/CVT/276/2013), by the Foundation for Science and Technology (FCT)”, Portugal (Fellowship SFRH/BD/131384/2017)

106

Poster 61

The Toxoplasma gondii MOB1 protein is involved in the regulation of the parasite’s replication rate. Delgado ILS1, Tavares A1,2,5, Francisco S1, Coelho J1,6, Santos D1,3, Leitao A1, Soares H2,4, Nolasco S1,4 1. Centro de Investigacao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Universidade de Lisboa, Lisboa, Portugal; 2. Centro de Quimica e Bioquimica, Faculdade de Ciencias, Universidade de Lisboa, Lisboa, Portugal; 3. Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal; 4. Escola Superior de Tecnologia da Saude, Instituto Politecnico de Lisboa, Portugal; 5. Present address: Instituto Gulbenkian de Ciencia, Oeiras, Portugal; 6. Present address: Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal MOB (Mps one binder) proteins form a conserved family of regulator kinases and are a key component of the Hippo pathway, a signalling cascade essential for the regulation of the cell cycle, cell proliferation and apoptosis. Studies on MOB proteins have targeted mostly mammals, drosophila and yeast. However, its involvement in cell division is also known in free-living protozoans (Tetrahymena thermophila and Stentor coeruleus), including parasites (Trypanosoma brucei), and in plants (Arabidopsis thaliana). Toxoplasma gondii is a parasite of great medical and veterinary relevance and a model organism for other Apicomplexa. Its ability to convert from rapidly proliferating tachyzoites to latent bradyzoites contained in cysts is key to the success and progression of infection. Identifying the players regulating the proliferation of T. gondii is a major step towards controlling it. We performed protein-protein BLAST searches against ToxoDB. Protein sequence alignments were performed using M-COFFEE and the phylogenetic tree was constructed using a ML model. We analysed the TgMOB1 gene expression through general and gene-specific cDNA synthesis and qPCR. To study the TgMOB1 protein function we isolated transgenic parasite strains harbouring randomly integrated overexpression constructs of TgMOB1 or a CRISPR/Cas9 disrupted TgMOB1 gene causing a functional knockout of the TgMOB1 protein. We identified a single MOB gene in T. gondii, which presents highest homology with annotated genes of the MOB1 subtype. Phylogenetic analysis showed that T. gondii and other apicomplexan parasites form a distinct group, evolutionarily distant from MOB1 proteins of other organisms. The TgMOB1 mRNA levels dramatically decrease (94%) during parasite replication inside the host cell. Though, this gene presents multiple alternative forms of mRNA and is transcribed from both sense and antisense genomic strands, with only the sense transcripts coding for a putative protein. While the overexpression of TgMOB1 causes a delay in tachyzoite replication, the knockout of TgMOB1 leads to an increased replication rate of tachyzoites. TgMOB1 protein presents a cytoplasmic localization. The results show that TgMOB1 protein expression is tightly regulated at the transcriptional and post-transcriptional levels, suggesting TgMOB1 is involved in the mechanisms of replication control, affecting T. gondii’s replication rate. Keywords: toxoplasma, MOB1, replication. Funding Acknowledgements: Funding from Fundacao para a Ciencia e Tecnologia fellowships SFRH/BD/101619/2014 (I.L.S.D) and SFRH/BD/79423/2011 (S.F.) and grants UID/CVT/00276/2013 and EXPL/CVT-EPI/1945/2013.

107

Poster 62

Establishment of patterns for histopathological lesions and viral loads in vaccinated and non-vaccinated wild rabbits, all positive for rabbit haemorrhagic disease virus

Abade dos Santos F1, Duarte M1,2, Peleteiro MC 1

1. Centre for Interdisciplinary Research in Animal Health (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa. Av. da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. Instituto Nacional de Investigação Agrária (INIAV), Quinta do Marquês, 2780-157 Oeiras, Portugal

Background: The European wild rabbit, Oryctolagus cuniculus (Linnaeus, 1758) subspecies algirus occupies most of the Portuguese territory4. After 2005, the wild rabbit populations declined abruptly in the Iberian Peninsula, strongly due to Viral Haemorrhagic Disease. In Portugal, rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2) was detected for the first time in 2012 and is disseminated both in the continent and in the islands1,2,3. This study aimed to establish a relation between various patterns of histopathological lesions in RHDV2 positive vaccinated and non-vaccinated wild rabbits and the respective viral loads. Methods: Thirty-six naturally dead wild rabbits were studied (15 vaccinated and 21 non-vaccinated). Necropsies and histopathological examination of various organs were performed by standard methods. Statistical analysis was done with Related-Samples Wilcoxon Signed Rank. RHDV2 was investigated by a quantitative RT-PCR as recommended by the OIE (Duarte, 2015). All homogenates were prepared at 10% in PBS. Nucleic acid extraction (BioSprint 96 nucleic acid extractor) and RT-qPCR were performed at the same conditions for all samples. Viral loads were inferred by the Cq values according to the equation y=-3,336x + 43,3863. Results: All 36 rabbits included in this study were positive for RHDV2. Lesions found in the various organs were mainly necrotic (spleen, thymus and kidney) or edematous and hemorrhagic (trachea and lungs). The more severe lesions were significantly more frequent in the non-vaccinated rabbits. The Cq value (cycling quantification, inversely proportional to the viral charge), was higher by 4.55 to 9.46 cycles in the vaccinated group compared to the non-vaccinated animals, reflecting higher viral loads in the latter. Conclusions: The progression of the disease in a vaccinated industrial domestic rabbit population has been previously reported2, but no data are available for wild rabbits. The fact that lesions were systematically more severe in the non-vaccinated group suggests that the disease progressed more rapidly in this group that also showed the higher viral loads. The efficacy of vaccination in wild rabbits should be investigated, as this study showed that a large number of vaccinated rabbits were subsequently infected and died of the disease. Keywords: Rabbit Hemorrhagic Disease; histopathological patters; RHDV 2; viral load Funding acknowledgements This project was supported by FCT project UID/CVT/276/2013. Molecular diagnostic was funded by FUNDO FLORESTAL PERMANENTE (Projeto Avaliação Ecossanitária das Populações Naturais de Coelho-Bravo Visando o Controlo da Doença Hemorrágica Viral, Dispach 4547/2017, May 31)

108

Poster 63

Microbiological and physico-chemical features of artisanal Portuguese PDO cheeses Baptista E1, Crespo MTB2,3, Barreto AS1, Semedo-Lemsaddek T1* 1. Centro de Investigação Interdisciplinar em Sanidade Animal -CIISA, Faculdade de Medicina Veterinária da Universidade de Lisboa, Portugal; 2. iBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 3. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal Background: Following traditional protocols, Portugal produces several cheeses, using with raw ewe’s or goat’s milk. Due to the use of artisanal manufacture procedures, raw materials and production settings, these cheeses present unique organoleptic features. Being part of our cultural heritage, these products have been recognized by the European Community with the attribution of the Protected Denomination of Origin -PDO- status. Despite their economic importance, microbiological and physico-chemical characteristics have been unexploited over the years. Trying to fulfill that gap, in the present work PDO cheeses from several Portuguese regions, namely Azeitão, Nisa and Rabaçal were characterized. Methods: PDO cheese samples were subjected to conventional microbiology procedures to detect the presence of lactic acid bacteria, Enterococcus spp., mesophiles at 30°C, coliforms, Escherichia coli, molds and yeasts, Listeria monocytogenes, Salmonella spp. and coagulase positive Staphylococcus aureus. Using PCR amplification, relevant species frequently identified in cheeses were also detected. Regarding physical-chemical parameters, water activity, pH, humidity, chlorides, ash, total protein, total acidity, fat and water-soluble nitrogen were also analyzed; always following Portuguese Standards (NP), International Organization for Standardization (ISO) or Official Methods of Analysis (AOAC) procedures. Results: Lactic acid bacteria were the dominant bacterial group in all regions under study. Samples from Azeitão, showed the highest microbial counts, followed by Rabaçal cheeses. Nisa presented the lowest counts and also a low microbial diversity. Regarding the physico-chemical approach, Azeitão cheeses revealed the highest humidity and acidity values, results related to its characteristic consistency and Nisa samples showed the lowest humidity values. Conclusions: Results obtained showed distinct features between samples collected at the different geographical regions under analysis. Considering that information on these cheeses is scarce and out-of-date, results hereby presented constitute the basis for the continuation of this investigation. Keywords: Portuguese Traditional cheeses, Protected Denomination of Origin, microbiologic and physico-chemical analysis. Funding Acknowledgments: Experimental work was supported by Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). Teresa Semedo-Lemsaddek is financially supported by FCT (SFRH/BPD/108123/2015). Authors also thanks PDO cheese producers for supplying the samples.

109

Poster 64

Comparative study of two methods in the molecular diagnosis of lymphoma in dogs Valente P, Carvalho S, Peleteiro MC, Correia J, Duarte A, Pomba C Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal Introduction: Lymphoma is considered the main neoplasm of hematopoietic origin in the canine species. The diagnostic technique of malignant clones of lymphocytes performed by polymerase chain reaction (PCR) for the characterization of receptor rearrangements of T and B lymphocyte antigens (PRRA) is widely used in human medicine. However, in veterinary medicine the application of these methods is limited. The aim of the present study is to evaluate different methods in the molecular diagnosis of PRRA in cases of canine lymphomas previously diagnosed by histopathology and immunohistochemistry. Methods: Sixty-four samples of dogs with lymphoma were included in this study, all previously analyzed by histopathology and typed by immunohistochemistry. Of these, 37 were classified as canine lymphoma type B and 27 type T. DNA extraction was performed from paraffin blocks with a commercial extraction kit. PCR products were subjected to 3% agarose gel electrophoresis (method 1) and heteroduplex analysis followed by 5% polyacrylamide gel (method 2). Results: Of the previously typified cases from B-cell lymphoma, 91.9% (n = 34/37) revealed monoclonal rearrangement of type B cells in the PCR products subjected to 3% agarose gel electrophoresis. The same samples, after heteroduplex analysis and electrophoresis in 5% polyacrylamide gel, 70.3% (n = 26/37) demonstrated monoclonal rearrangement of type B cells. Therefore, the sensitivities obtained for the identification of B lymphomas were of 91.9% and 70.3%, in methods 1 and 2, respectively. Of the 27 cases previously identified as T-cell lymphoma, 63.0% (n = 17/27) demonstrated monoclonal rearrangement of T-cells on 3% agarose gel. In the same ones, after heteroduplex analysis and electrophoresis in 5% polyacrylamide gel, 77.8% (n = 21/27) demonstrated clonality for T-type lymphocytes. Showing, 63.0% and 77.8% sensitivity, in methods 1 and 2, respectively. Conclusion: The present study with molecular diagnosis PRRA using two methodologies allowed to verify that, although the technique of heteroduplex and polyacrylamide gel was the most indicated in human studies, this one presented less sensitivity than those submitted to agarose gel in the diagnosis of B-cells lymphomas, which did not occur in cases of T-cells lymphomas. This suggests that more studies need to be carry out to define the best technique to be used. Keywords: Dogs; Lymphomma; PAAR. Acknowledgments: To the financing of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministry of science, technology, innovations and communications, Ciência sem Fronteiras Program, Brazil. To the financing of CIISA and FCT through project UID / CVT / 00276/2013.

110

Poster 65 Putative probiotic lactic acid bacteria isolated from sauerkraut fermentations Semedo-Lemsaddek T1, Touret T2, Oliveira MM1

1. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Nova Lisbon University, Oeiras, Portugal Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Nowadays there is an increased interest in probiotics, in face of their recorded safe use and recognized effects on human health. However, to be selected and applied as probiotics, microorganisms must possess several characteristics: The strain must be safe for human ingestion, with the most important concerns being its virulence potential and the possibility to act as a reservoir for mobile antimicrobial resistance genes. Furthermore, the probiotic candidate must be able to survive the transit through the gastrointestinal tract, in order to maintain viability and potentially colonize the human host. The main objective of this work was the isolation of lactic acid bacteria -LAB- from sauerkraut fermentations and the selection of strain(s) with potential to be used as probiotic microorganisms. After isolation LAB were phenotypically and genotypically characterized and a safety evaluation, based on the search for virulence factors and antimicrobial resistance features, was also performed. Probiotic candidates were further analyzed regarding the aptitude to survive and grow under distinct conditions, as well as the ability to inhibit the growth of pathogens. During this investigation 114 lactic acid bacteria were isolated from four different sauerkraut fermentations and characterized both phenotypically and genotypically. Results obtained allowed the selection of representative isolates, the majority identified as Lactobacillus spp. or Leuconostoc spp. Carefully chosen isolates were then analyzed regarding resistance to eight antibiotics and the ability to inhibit the growth of Listeria monocytogenes and were also tested for resistance to low pH and bile, important characteristics to survive transit though the human GI tract. Overall, data gathered throughout this work allowed the selection of three lactobacilli showing characteristics associated with probiotics. Selection of these strains supports the fact that sauerkraut fermentations are suitable substrates for the isolation of probiotic candidates. Furthermore, these strains may be more appropriate for application in vegetable products, particularly fermented foods such as sauerkraut itself. In fact, due to their origin, they should maintain a high viability in sauerkraut and related products, not leading to a significant change of flavor and other sensory properties, important characteristics for the application of probiotics in non-dairy food products. Keywords: probiotics; lactic acid bacteria; sauerkraut. Funding Acknowledgments Experimental work was supported by Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). Teresa Semedo-Lemsaddek is financially supported by FCT (SFRH/BPD/108123/2015).

111

Poster 66

Ruminal health and performance of feedlot fed Nellore cattle supplemented with live yeast and lipids Sousa S1, Martins CL2, van Harten S 1,2 1. CBIOS, Faculty of Veterinary Medicine,

Lusófona University of Humanities and Technologies, Campo Grande 376, 1749 – 024 Lisboa, Portugal; 2. UNESP- Botucatu, Brazil; 3.

CIISA / Faculty of Veterinary Medicine, University of Lisbon, Avenida da Universidade Técnica, 1300-477, Lisboa, Portugal In intensive bovine production systems, dietary manipulation with additives becomes an indispensable tool in the prevention of diseases and may contribute to prevent the occurrence of acidosis and consequently ruminitis. The effect of supplementation with live yeast (Saccharomyces cerevisiae) and lipids on ruminal health and performance of feedlot fed Nellore cows was studied. This trial was performed at the Department of Animal Nutrition and Breeding of UNESP – Brazil during 109 days. Nellore cows (n= 133; 372kg) were randomly distributed into three groups: Control group (n=20) fed without any additives, live yeast supplemented group (n=17) fed with 2g Saccharomyces cerevisiae (2x1010 UFC/g) and lipid group (n=96), fed with a lipid source. Ruminal epithelium was classified regarding the incidence and level of lesions, the number of papillae, their average and total area, their participation on the total absorptive surface and their mitotic index. Dry matter intake (DMI) fluctuation and weight gain were also determined. Lipid group presented a greater DMI fluctuation, but gained more weight (26,6%) when compared to control and live yeast group. Regarding ruminal health, lipid group cows were, once again, the ones with a superior average and total papillae area (+ 70%) and with a higher participation of the papillae on total absorptive surface area when compared to the other groups. Ruminitis index was, however, the same for all experimental groups (p<0,05). In this study, the inclusion of live yeast in the diet led to a steadier daily intake of dry matter, suggesting a better ruminal fermentation control, as this diet can lead to more cellulitic bacteria and less digestive problems. However, this result did not translate in a higher weight gain, reinforcing the inconclusive evidence of the benefits of yeast in ruminants’ diets. Animals fed with supplemented lipid diet presented more papillae area and mitotic index than the other groups, fact that can be explained by a higher production and absorption of volatile fatty acids. Although all experimental groups presented ruminitis, its index did not differ among groups and it also did not lead to ruminal acidosis, since their level was inferior to two. This work can reinforce the concept of introducing a lipid supplementation instead of yeast addition to fattening cattle due to a higher weight gain and a low level of ruminal acidosis, but more work must be done to confirm the influence of this supplement. Keywords: Additives, Ruminitis, Nellore

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Poster 67

The effect of Arthrospira sp. microalga as a feed supplement on growth performance, pigmentation and meat quality in broiler chickens Pestana JM1, Puerta B1, Lopes PA1, Santos H1, Alfaia CM1, Madeira MS1, Lemos JPC1, Lordelo M2, Prates JAM1 1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa, Portugal; 2. LEAF - Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisboa, Portugal Chickens have been almost the focus of research into Arthrospira sp. (commonly known as Spirulina) in monogastric feed rations. However, chickens cannot digest the intact cell which is composed of cellulosic structure being therefore, most of Arthrospira sp. nutrients unprocessed in gastrointestinal tract. To increase its digestibility, commercial enzymes have been incorporated as feed additives in poultry diet formulation. A total of 120-day old male Ross 308 birds were housed in 40 battery brooders with 3 birds per cage. The experimental period lasted from day 21 to 35. Chickens were given 4 different treatments, as follows: a) corn/soybean meal (control), b) Spirulina diet (replacement of 15% corn/soybean meal), c) Spirulina diet (15%) + Rovabio commercial enzyme mix (0.005%), and d) Spirulina diet (15%) + commercial Lysozyme (0.01%). At 35 days, birds were slaughtered, and breast and thigh collected. For both tissues, the inclusion of 15% Spirulina as a natural pigment resulted in the highest carotenoid deposition. The highest values of b* (yellowness) were observed in chickens fed microalgae. Growth rates were significantly higher in control animals when compared to Spirulina fed chickens. No significant differences were observed in meat quality parameters (pH, shear force and total carotenoids) among treatments. Regarding the sensory analysis, the scores for meat tenderness, flavor, off-flavor and over acceptability were not affected by treatments. In contrast, meat juiciness from control and Rovabio groups was increased relative to others. Taken together, the results reported herein indicate that 15% of dietary Spirulina, alone or in combination with the commercial enzyme Rovabio, may be a good strategy to improve broiler meat without affecting its quality. Keywords: Spirulina, broiler meat quality, sensory traits Funding Acknowledgements: This study was supported by Fundação para a Ciência e a Tecnologia (FCT, Lisbon, Portugal) through PTDC/CVT-NUT/5931/2014 grant, CIISA project (UID/CVT/00276/2013), and individual fellowships to JP (SFRH/BPD/116816/2016), MSM (SFRH/BPD/97432/2013), and PAL (Post-Doc, Portugal2020 08/SI/3399/2015).

113

Poster 68

Unveiling new histone deacetylase- and microtubule-based strategies for treatment of feline mammary tumors Almeida F, Gameiro A, Ferreira F

Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, 1300-477, Portugal Background: Feline mammary tumors (FMTs) are predominantly malignant and metastases occur in most cases, leading to a poor prognosis and a short life expectancy. Thus, new molecular targets are needed for the development of more efficient therapeutics. Histone deacetylases (HDACs) play an important role in the regulation of gene expression at the chromatin level, and have been associated with the development of cancer. HDACs have been attractive candidates for new therapeutic targets, with the development of HDAC inhibitors (HDACis). HDACis inhibit deacetylating enzymes causing uncontrolled expression of essential genes and consequently cytotoxicity for the tumor cell. Microtubules (MTs) are dynamic structures composed of tubulin polymers that play an important role in cell growth, division and intracellular trafficking, and have been the focus of research for cancer treatment. MT inhibitors (MTis) interfere with the polymerization of microtubules, causing disruption of the microtubule cytoskeleton, followed by cell cycle arrest and apoptosis of the tumor cell. Very few studies on the use of HDACis and MTis in the domestic cat have been published. In this project we aimed to evaluate HDACis and MTis as antitumor agents for the development of new therapeutic options for FMTs. Methods: We evaluated the cytotoxicity of seven HDACis (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) and four MTis (colchicine, nocodazole, paclitaxel, and vinblastine) in three cell lines representative of FMT, and in one cell line representative of human breast cancer. The different cell lines were incubated with increasing concentrations of each inhibitor for 72h, and cell viability was measured using the WST-1 cell proliferation assay. Results: All HDACis and MTis exhibited dose-dependent inhibitory effects on cell viability in the three FMT cell lines tested, and results were in agreement with the human breast cancer cell line. However, for one feline cell line, we observed a pattern indicative of resistance to some inhibitors. The IC50 values for HDACis were in the µM range and for MTis were in the nM range. Among the eleven inhibitors studied, panobinostat (HDACi) and colchicine (MTi) showed the highest cytotoxicity activity. Discussion: Our results validate HDACis and MTis, in particular, panobinostat and colchicine as promising novel anti-cancer therapies for FMTs. Keywords: Feline mammary cancer, Histone deacetylase inhibitors, microtubule inhibitors, cell viability, therapeutics Funding Acknowledgements: This work was supported by Fundação para a Ciência e a Tecnologia (FCT/MCTES) through grant PTDC/CVT-EPI/3638/2014 and by the Centre for Interdisciplinary Research in Animal Health (CIISA), which is financed by national funds from FCT/MCTES (UID/CVT/00276/2013). FA is supported by a post-doc fellowship (PTDC/CVT-EPI/3638/2014) and AG by a PhD fellowship (SFRH/BD/132260/2017).

114

Poster 69

First report of Neotrombicula inopinata infestation in domestic cats from Portugal

Ramilo DW1*, Monteiro C2*, Carreira M3, Pereira da Fonseca I1, Cardoso L4

1. CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 3. Master in Veterinary Medicine Student, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 4. Department of Veterinary Sciences, University of Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal Background: Larvae of Trombiculidae mites parasitize a wide variety of terrestrial vertebrates throughout the world, including cats and humans, causing several skin lesions and pruritus. Trombiculidae family includes Neotrombicula genus, which is associated with trombiculosis outbreaks in cats reported in some European countries. Neotrombicula autumnalis is frequently incriminated by default in clinical cases of animal trombiculosis, although other species, such as Neotrombicula inopinata can also be the agent of this parasitosis. Accurate taxonomic identification is essential to better understand the epidemiology of the disease considering that some of these mites are reservoirs, if not vectors, for humans and other vertebrate hosts, of different pathogens such as Ehrlichia, Rickettsia and Borrelia. Methods: Several trombiculid specimens were collected from two domestic cats attended at the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon. Mites were immersed in Hoyer’s medium, examined under a compound microscope with phase contrast optics (Olympus BX41 Phase Contrast Microscope), being the specimens identified at the species level. Results: The observed specimens were compatible with Neotrombicula genus, due to palpal claws three-pronged, nude galeal setae and subpentagonal or subhexagonal scutum wider than long, with rounded posterior margin. Furthermore, the dorsal idiosomal setae were arranged as (8-9)-(8-9)-(7-8)-6-4-2, which denotes a unique morphological characteristic of N. inopinata. Conclusions: To the best of our knowledge, this is the first report of N. inopinata in mainland Portugal, a fact that highlights the need of a correct taxonomical identification of these mites. Further studies are needed to understand the role of this species as reservoir/vector of other pathogens. Keywords: Neotrombicula inopinata, mites, cat, Portugal. Funding Acknowledgements: Centre for Interdisciplinary Research in Animal Health, Project UID/CVT/00276/2013; Fundação para a Ciência e Tecnologia, Post-doctoral Grant SFRH/BPD/115202/2016.

115

Poster 70

Prevalence of antibodies to Neospora spp. in horses in Portugal Waap H1,2, Oliveira U3, Gomes J1,2, Schares G4 1. Laboratório de Parasitologia, Instituto Nacional de Investigação Agrária e Veterinária, Av. da República, Quinta do Marquês, 2780-157 Oeiras, Portugal; 2. CIISA, Faculdade de Medicina Veterinaria, Universidade de Lisboa, Av. da Universidade Tecnica, 1300-447 Lisboa, Portugal; 3. Departamento de Ciencias Agrarias e Ambientais, Universidade Estadual de Santa Cruz (UESC), Ilheus, Bahia 45662-900, Brasil., Bahia 45662-900, Brazil; 4. Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, 17493, Greifswald-Insel Riems, Germany Background: Equine neosporosis is caused by Neospora spp. (N. hughesi or N. caninum). Parasites of this genus are obligate intracellular cyst-forming coccidia, closely related to Toxoplasma gondii and Sarcocystis neurona. Though infection has been linked worldwide to neurological disease, abortion and reproductive failure, the epidemiology of this disease is still unclear. This study aimed to evaluate the seroprevalence of Neospora spp. in horses in Portugal. Methods: A panel of 384 sera from natural exposed horses originating from different geographical areas was screened at 1:50 dilution by the Immunofluorescent Antibody Test (IFAT) and positive results were confirmed by Western blot. Results: A total of 82 horses (21.4%) were found to be positive by IFAT. Endpoint titres were distributed as follows: 1:50 (51 sera), 1:100 (15 sera),1:200 (9 sera),1:400 (6 sera) and 1:800 (1 serum). Confirmation of Neospora spp results by Western blot showed infection in 35 horses, while 15 were considered inconclusive, resulting in an overall seroprevalence of 9.1% (95% CI:6.6-12.4%). There was no statistical relationship between seroreactivity to Neospora spp. and age, gender and breed. Conclusions: To the best of our knowledge, this is the first evidence of natural exposure to Neospora spp. in horses in Portugal. Further investigations in the clinical context are needed in order to discriminate the parasite species involved and evaluate the importance of Neospora spp. in the differential diagnosis of neurological and reproductive disorders in horses. Keywords: Neospora spp., seroprevalence, horses, Portugal

116

Poster 71

Molecular detection of Theileria species infecting sheep: a retrospective study Gomes J1,3, Costa M2, Pereira da Fonseca I3

1. National Institute for Agrarian and Veterinarian Research, Oeiras, Portugal; 2. Faculty of Sciences and Technology, Universidade NOVA de Lisboa, Caparica, Portugal; 3. CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal BACKGROUND: Theileriosis is one of the most prevalent infectious diseases of sheep worldwide and can be due to different Theileria species. Theileria ovis and T. lestoquardi are known to occur in Europe and T. luwenshuni and T. uilenbergi are described in China. Some species are more pathogenic than others and can cause a high rate of morbidity and mortality. Since there is no information on the occurrence and distribution of Theileria spp. infecting sheep in Portugal, a molecular survey study was carried out to detect T. ovis and T. lestoquardi in animals from different regions of mainland Portugal. MATERIAL AND METHODS: A retrospective molecular study was performed in a total of 126 DNA samples from INIAV biobank. DNA samples were extracted from blood collected in 2008 and sent to INIAV for different diagnosis and kept frozen for further studies. PCR for the 18S rRNA gene and a PCR for the merozoite surface protein were used for T. ovis and T. lestoquardi, respectively. RESULTS: T. ovis was detected in 42 sheep samples (33.3%; 95% CI: 25.2–42.3%) but no cases of T. lestoquardi were detected in this population. CONCLUSIONS: T. ovis is widespread in Portugal with variable prevalence detected in different regions. This species is considered by some authors as non-pathogenic but, as in other Theileria species, this has to be thoroughly studied. This species is transmitted by Rhipicephalus ticks and the occurrence area of the infection reflects the widespread distribution of these ticks. T. lestoquardi is described as pathogenic and transmitted by Hyallomma ticks that are also widespread in Portugal. Further studies are needed to clarify the current situation of theileriosis in small ruminants in Portugal. Keywords: Theileria, theileriosis, sheep Funding Acknowledgements: This work was supported and funded by the National Institute for Agrarian and Veterinary Research (INIAV) and Centre for Interdisciplinary Research in Animal Health, Project UID/CVT/00276/2013.

117

Poster 72

Equine piroplasmosis by Theileria equi in military horses from Portugal

Alho AM1, Kayikci F2, Shahi Barogh B2, Rosa H3, Tomás J3, Rocha H3, Madeira de Carvalho LM1, Fuehrer HP2

1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisbon, Portugal; 2. Institute of Parasitology, University of Veterinary Medicine, Vienna, Austria; 3. Guarda Nacional Republicana, Lisbon, Portugal

Background: Vector-borne diseases (VBDs) have been increasingly reported in horses worldwide, although few data is currently available regarding working and military horses in Portugal. Military horses may constitute a risk group for the agents causing these diseases, as they frequently work outdoors in different areas and are thus exposed to vector arthropods. The aim of this study was to identify the presence and prevalence of the most significant VBDs in Portuguese military horses, using molecular detection and sequencing of the DNA products. Methods: During 2017, blood samples were collected from 101 apparently healthy horses belonging to the Portuguese Republican Guard (GNR), stabled at the guard facilities in Lisbon. DNA was extracted using a Chelex based DNA extraction techniques (Instagene Matrix, BioRad) and examined for the presence of DNA of Anaplasmataceae, filarioid helminths, Piroplasmida and Rickettsiales, using established protocols. Results: In total 60 male and 41 female horses with an age ranging from 4 to 24 years (median: 12 years) were included. Overall, 32.7% (33/101) of the samples were positive for Theileria equi. Sequence results showed that the genetic variance was limited within the SSU rRNA gene (31 – genotype 1; 2 – genotype 2). No positive results were recorded for Anaplasma spp., Babesia spp., Rickettsia spp. or any filarioid helminths. Conclusions: These data confirm a relatively high prevalence of T. equi in military horses in Portugal. As equine piroplasmosis is a severe infectious tick-borne disease responsible for significant losses in equine production and with numerous impacts in the international movement of horses, treatment and preventive measures are needed to reduce exposure and future infections. Keywords: Horses, Military, Theileria, equine piroplasmosis, Portugal Funding acknowledgements: Project UID/CVT/276/2013 (CIISA). TD1303 COST Action - Short Term Scientific Mission (STSM).

118

Poster 73 Topographic study of Trypanosoma brucei parasites

Alexandre-Pires G1, Dias-Guerreiro T2, Palma-Marques J2, Mourata-Gonçalves P2, Valério-Bolas A2, Gabriel A2, Pereira da Fonseca I1, Sousa-Silva M3, Santos-Gomes G2 1. CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary medicine, University of Lisbon, Av. Universidade Técnica, 1300-477 Lisbon, Portugal; 2. Unidade de Ensino e Investigação de Parasitologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal; 3. Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisbon, Portugal Background: African trypanosomiasis is a neglected parasitic disease that still has a considerable public health relevance. Is a vector-borne disease caused by protozoan parasites of Trypanosoma genus, including the species Trypanosoma brucei, which is transmitted by Glossina spp. (tsetse fly) during a blood meal. Like most eukaryotic cells, trypanosome also secretes extracellular vesicles that may be involved in a number of cellular processes. In the case of trypanosomes, the secreted extracellular vesicles, such as exosomes, are 20-150 nm nanovesicles released into the extracellular space upon fusion with the plasma membrane. Thus, this study aims to characterize topographically T. brucei and nanovesicles released by this extracellular parasite. Materials and Methods: Crioconserved T. brucei G.V.R. 35 parasites were obtained and maintained in culture. Manipulation of parasites was done at containment level 2, ensuring biosafety and biosecurity in compliance with EU directive 2000/54/EC. To obtain nanovesicles, parasites were maintained in nanovesicle-free culture medium and nanovesicles were isolated and purified using appropriate Spin Columns (MW 3000). For topographic studies, T. brucei trypomastigote forms and parasite vesicles adhered to coverslips covered with poly-D-lysine. Then, the critical‐point drying (CPD) method was used or the hexamethyldisilazane (HMDS) solvent was applied. Coverslips were coated with gold palladium, mounted on stubs. Parasites were observed under an ultra-high scanning electron microscope (SEM) and images were acquired. Results: Trypomastigote forms were identified based on parasites dimensions, which can range between 16-42 µm. However, when the CPD drying method was used the parasites showed signs of degradation, only the flagellum remained intact. HMDS application reduced the aggressivity of the method, and it was possible to observe the intact parasite. T. brucei isolated nanovesicles presented a spherical form and diameter ranging between 40-100 nm. Conclusion: The fragility of the parasitic body and the well-preserved flagellum indicates that T. brucei trypomastigote exhibits a faint microtubule cytoskeleton, contrary to other trypanosomatids or other eukaryotic cells. The fragility evidenced by parasite cell body associated with flagellum preservation may be related to the ability of the parasite to cross the blood-brain barrier and invade the central nervous system, causing sleeping disease. Although CPD is the most used method for drying biological specimens for SEM evaluation, in the particular case of T. brucei drying by evaporation (HMDS) seems to be a better alternative to preserve the entire parasite. In addition, this study demonstrates that in vitro T. brucei parasites release exosomes. These parasite nanovesicles can have a significant role in modulating host immune responses. Further studies are need to evaluate these hipothesis. Keywords:Trypanosoma brucei, microtubule-cytoskeleton, exosomes, CPD versus HMDS Support: This study was supported by RedVLP (CYTED ref 216RT0506), and CIISA - UID/CVT/267/2013 and GHTM – UID/Multi/04413/2013.

119

Poster 74

Use of Cistus ladanifer condensed tannins to improve the nutritional value of ruminant edible fat Guerreiro O1,2, Alves SP2,3, Soldado D1, Cachucho L1, Francisco A2,4, Costa M2, Duarte MF1,5, Santos-Silva J2,4, Jerónimo E1,5, Bessa RJB2,3

1. Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL), Instituto Politécnico de Beja (IPBeja), 7801-908 Beja, Portugal; 2. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 3. Faculdade de Medicina Veterinária, Universidade Lisboa, Avenida Universidade Técnica, 1300-477 Lisboa, Portugal; 4. Instituto Nacional de Investigação Agrária e Veterinária, Pólo Investigação da Fonte Boa (INIAV-Fonte Boa), 2005-048 Santarém, Portugal; 5. Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Évora, Portugal Cistus ladanifer L. (rockrose) is a tanniferous shrub quite abundant in Mediterranean countries. Cistus ladanifer is able to modulate ruminal biohydrogenation (RBH) and, when associated with forage-rich diets supplemented with PUFA-rich vegetable oils, increased the ruminal production of vaccenic acid (t11-18: 1), resulting also in a higher concentration of conjugated linoleic acid isomer (CLA) - rumenic acid (c9,t11-18:2) in lamb meat. Due to beneficial health effects of CLA isomers, RBH modulation has been target of numerous works in order to increase ruminal production of t11-18:1 and, thus the t11-18:1 availability for conversion to c9,t11-18:2 in tissues/mammary gland. To explore the use of C.ladanifer as a way to improve the fatty acid (FA) profile of ruminant fats, were developed two trials with the objective to: i) elucidate which type of C.ladanifer compounds might be responsible for RBH modulation; and ii) evaluate if a specific C.ladanifer fraction is able to induce the same effect of C.ladanifer plant on FA profile of fat. In order to clarify which C.ladanifer fractions might modulate RBH was performed an in vitro study, where five fractions of C.ladanifer – essential oil, dichloromethane extract, total phenolics, non-tannin phenols and condensed tannins (CT), were incubated with ruminal fluid for 6 h. Volatile FA production was not affected by C.ladanifer fractions. Fraction of CT was the most active on RBH modulation, leading to highest t11-18:1 accumulation. To evaluate if the inclusion of C.ladanifer CT extract in lamb diets can induce the same effect of C.ladanifer aerial part on the FA profile of fat, two levels of C.ladanifer CT (1.25 and 2.5%) and two ways of CT supply (aerial part vs. CT extract from C.ladanifer) were tested in a productive trial with 36 crossbred Merino Branco ram lambs. A diet without CT was also tested. Basal diet was composed of dehydrated lucerne with soybean oil (6%). Lambs stayed in trial for 5 weeks. Inclusion of C.ladanifer CT extract in diet (1.25% CT) led to the highest t11-18:1 concentration in intramuscular and subcutaneous fats. However, in this diet the c9,t11-18:2 content was similar to diet without CT incorporation, which may be due to downregulation of stearoyl-CoA desaturase activity, the enzyme that convert t11-18:1 to c9,t11-18:2. Cistus ladanifer CT extract might be a good approach to enhance the nutritional value of ruminant fat, but is essential to ensure the endogenous synthesis of c9,t11-18:2. Keywords: Cistus ladanifer; condensed tannins; fatty acids; biohydrogenation; ruminants Funding Acknowledgements: “CistusRumen” project (ALT20-03-0145-FEDER-000023) funded by Alentejo2020 through the FEDER, and FCT by research grants of O. Guerreiro (SFRH/BD/84406/2012), S.P. Alves (SFRH/BPD/76836/2011) and M. Costa (SFRH/BD/90468/2012).

120

Poster 75

Spirulina in piglet diets: effects on growth, feed conversion and muscle, liver and intestinal metabolomics profiles using NMR (nuclear magnetic resonance) Martins CF 1, Matzapetakis M3, Ribeiro DM1, Prates JAM2, Almeida AM1, Freire JPB1 1. DCEB, LEAF (Linking Landscape, Environment, Agriculture and Food), Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisboa; 2. CIISA (Centre for Interdisciplinary Research in Animal Health), Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa; 3. ITQB NOVA (Instituto de Tecnologia Química e Biológica António Xavier), Universidade Nova de Lisboa, Oeiras, Portugal This work aimed to study the use of spirulina as an ingredient for piglet´s diets on their growth performance and metabolic system. The study was conducted during 4 weeks with 40 post-weaning piglets. Animals were divided into 4 groups of 10 animals, allocated two by two in pens and fed ad libitum. Four isoproteic and isoenergetic diets were formulated, replacing soybean meal by spirulina and adding two different enzymes: Control diet, Spirulina (SP), Spirulina with Rovabio® Excel (SP+R) and Spirulina with Lysozyme (SP+L). Piglets were weighted weekly (from 12 kg to 29 kg of average live weight). At the end of the study, animals were sacrificed and LD muscle, liver and intestinal samples collected and snap-frozen in liquid nitrogen until further analysis. For metabolomics profiling, metabolites were extracted using the methanol/chloroform method. Proton (1H) NMR spectroscopy was performed on an 800 MHz Bruker Advance II+ spectrometer (Ettlingen, Germany). Spectra were acquired, processed and analysed using TopSpin 3.2 (Bruker Biospin, Billerica, MA, USA). Identification and quantification of metabolites was performed by using the Chenomx NMR Suite 8.0 software (Chenomx Inc., Edmonton, Canada). To evaluate any clustering behavior through metabolomic multivariate analysis, an unsupervised Principal Component Analysis (PCA) was applied to all sets of data for each sample type (muscle, liver and intestine). Growth performance showed significant differences (p<0.05) between the control group and the other groups fed with spirulina. For animals fed with control, SP, SP+R, SP+L diets, final weights of 31.0, 28.3, 28.4 and 27.8 kg were respectively obtained. The average daily gain was 677, 593, 582 and 567 g, respectively, whilst feed conversion ratio was respectively 1.48, 1.62, 1.62 and 1.69. Regarding the metabolite profiling, no grouping could be observed in the PCA plot, suggesting that there were no global differences among the different treatments. As the PCA did not reveal any clustering between groups, a supervised Partial Least Squares Discriminant Analysis (PLS-DA) was subsequently performed, again revealing minor changes between the different profiles. Finally and given the lack of information on metabolite quantification in these tissues and species, we have performed an overall metabolite identification in samples from the control group. It rendered the identification of approximately 60 metabolites. In conclusion, dietary Spirulina seems to decrease piglet growth performance without change the metabolic system. Keywords: Metabolites, NMR, piglets Funding Acknowledgements: This study was supported by FCT (UID/CVT/00276/2013) and Portugal2020 (08/SI/3399/2015) projects.

121

Poster 76

Beef consumption in the near future: what do we need to change? Fontes MA1, Viegas I2, Madureira L3, Nunes LC4, Santos JML5

1. CIISA- Faculdade de Medicina Veterinária – ULisboa, Pólo universitário da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal; 2. ICAAM/ABG, University of Évora, 7002‐554 Évora, Portugal; 3. UTAD (Universidade de Trás-os-Montes e Alto Douro), CETRAD (Centro de Estudos Transdisciplinares para o Desenvolvimento), 5001-801 Vila Real, Portugal; 4. Nova SBE - Universidade Nova de Lisboa, Lisbon, Portugal; 5. Centro de Estudos Florestais (CEF) /Forest Studies Centre – Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal

Animal production is often seen as a societal concern, mainly for environmental and ethical reasons, contributing for the pressure this sector has been undergoing. It is recognized that meat consumption is expected to reach 466 million tons in 2050 (FAOstat, 2010), due to human population growth and increasing wealth. Today`s world is facing the huge challenge of how to cope with higher food demand within the framework of the sustainability triangle of simultaneously managing: economic competitiveness; environmental protection; and social acceptability. Sustainability, defined as meeting the needs of the present without compromising the needs of future generations, is a widely used and important concept that characterises today`s worldview. Within the meat sector, the beef value chain has been under great pressure and huge challenges. There is a need for innovation and for competitiveness improvement. This is particularly relevant in a global market where production and processing have to focus on satisfying increasing demand from sophisticated markets in developed economies that require higher quality products. This quality, which includes production in an environmentally friendly manner, respect for animal welfare, amongst other attributes, should also represent good value for money, and should allow players to remain competitive. Not an easy task…Beef consumption is becoming increasingly contested in affluent regions (Henchion et al, 2016; Miele and Evans, 2010). As such, European consumers are becoming more aware of the environmental and social impact of beef production with growing concerns of the personal health impact of beef consumption. However, evidence suggests that many consumers prioritise personal and family health motives over environmental consideration. The quality-quantity dynamic is shifting where some consumers are seeking higher quality meats in smaller quantities. Healthier meats produced in a more sustainable manner have the potential to command a premium on the market. Hence, based on research undertaken, we want to understand if quality perceptions towards beef are being translated in buying decisions. How are consumers perceiving some credence attributes, namely animal welfare, food safety and the environment, in the beef consumed. Based on consumers’ willingness to pay (WTP) for safer, cleaner and animal friendlier beef we elaborate on future beef consumption trends. Our results lead us to conclude that though consumers are becoming more aware of the need for safer, cleaner and animal friendlier beef on the market, this awareness is not yet, translated into immediate buying choices amongst Portuguese consumers, because price and income considerations are still overwhelming in the buying decisions. Keywords:Sustainability, beef, consumer perceptions. Funding Acknowledgements: We would like to thank Fundação para a Ciência e Tecnologia, through Grant SFRH/ BD/37715/2007 and CIISA.

122

Poster 77

Relationships between rumen protozoa, biohydrogenation and bioactive fatty acids deposition on lamb muscle

Francisco AE1,2*, Santos-Silva J1,2, Portugal P1, Alves SP2,3, Bessa RJB2,3

1. Instituto Nacional de Investigação Agrária e Veterinária, Pólo Investigação da Fonte Boa (INIAV-Fonte Boa), 2005-048 Santarém, Portugal; 2. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 3. Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal

Protozoa can comprise up of half of rumen biomass and their membranes present a high content on polyunsaturated fatty acids (PUFA) and on biohydrogenation (BH) intermediates with healthy properties, such as vaccenic (t11-18:1) and rumenic (c9,t11-18:2) acids. By this reason, protozoa can act as potential reservoirs of such fatty acids in rumen, which can flow out and be deposited in meat and milk. Ciliate protozoa may also stabilize rumen pH by regulating starch fermentation in rumen, what may help to control the changes on the normal pattern of BH that occur when ruminants are fed with high-starch diets and that favors the synthesis of the unhealthy t10-18:1 instead of t11-18:1 (trans-10 shift). This study investigated the associations between the abundance of protozoa in rumen and the proportion of t10-18:1, t11-18:1, c9,t11-18:2 and of PUFA in the whole rumen content and in the meat of growing lambs. Data were derived from four production trials performed by our research team (N=116 lambs) and ciliate protozoa abundance (cells/ml rumen liquor) was assessed by microscopic counting. Global principal component analyses and Spearman correlation coefficient were applied. A large variation between lambs was observed for protozoa abundance, that ranged between 0 (N=11 lambs) and 2.79 × 106 ciliates/ml rumen liquor. Nine genera of ciliates were identified: Isotricha, Dasytricha, Entodinium, Diplodinium, Eudiplodinium, Polyplastron, Diploplastron, Epidinium and Ophryoscolex, being Entodinium the most abundant genus, averaging 4.94 × 105 cells/ml of rumen liquor. In general, ciliates were positively correlated with t11-18:1; c9,t11-18:2 in rumen and meat and negatively correlated with t10-/t11-18:1 ratio in rumen and meat and with t10-18:1 and PUFA in rumen. For t11-18:1 and c9,t11-18:2, in both rumen and meat, the highest positive correlations were found with the genus Entodinium (P<0.001). Also, t10-18:1 was mainly negatively correlated with Entodinium (ρ= -0.63, P <0.001) in rumen and t10-/t11-18:1 ratio in both rumen (ρ= -0.70, P <0.001) and meat (ρ= -0.21, P =0.035). The results suggest an association between rumen fauna and meat nutritional quality and that the intensity of that link may depend of the ciliates community structure. Rumen protozoa are positively linked to the deposition of bioactive fatty acids that are benefits to the human health and negatively related to the occurrence of the trans10-shift. Keywords: Rumen, protozoa, biohydrogenation, bioactive fatty acids, lamb meat Funding acknowledgements: Financial support through the European Fund for Regional Development (ERDF) (ALT20-03-0145-FEDER-000040-ValRuMeat- Valorização da carne de ruminantes em sistemas intensivos de produção), the PTDC/CVT/103934/2008, and UID/CVT/00276/2013 projects and individual fellowships to A. Francisco (SFRH/BD/68773/2010) and S. P. Alves (SFRH/BPD/76836/2011), from Fundação para a Ciência e Tecnologia are acknowledged.

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Valrumeat - How to reconcile the intensive production of ruminants with the production of meat with high nutritional value Santos-Silva J1,2*, Francisco A1,2, Dentinho MT1,2, Portugal P1, Almeida J1, Jerónimo E4, Soldado D4, Alves SP2,3, Bessa RJB2,3

1. INIAV Fonte Boa, 2005-048 Santarém, Portugal; 2. CIISA, Av. Universidade Técnica, 1300-477 Lisboa, Portugal; 3. FMV-U.Lisboa, Av. Universidade Técnica, 1300-477 Lisboa, Portugal; 4. Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo CEBAL/IPBeja, Beja, Portugal; 5. ICAAM, Universidade de Évora, Évora, Portugal Ruminants from intensive meat production systems are fed with low fibre and high energy diets, usually rich in cereals. In such conditions, the rumen environment changes and the pattern of rumen biohydrogenation is modified, compromising the synthesis and deposition in meat of healthy vaccenic (t11-18:1) and rumenic (c9,t11-18:2) fatty acids (FA) (known as trans-10 shift). The causes of trans10-shift are still not known but has been associated to diets with high starch content and to low rumen pH. ValRuMeat aims to study the main factors related to trans-10 shift and define the main lines for the formulation of diets to enhance t11-18:1 and c9,t11-18:2 levels in meat. Three trials with Merino Branco lambs were carried out for 6 weeks each to study: a) the source of neutral detergent fiber (ground alfalfa vs soybean hulls) (Trial 1); b) the forage particle size (ground vs chopped hay); c) the cereal replacement in the concentrate fraction of the diet (100, 65, 35 or 0%) (Trial 2); d) the forage species (alfalfa vs ryegrass); and e) the proportion of buffer (sodium bicarbonate) in the diet (0.5 vs 2.0%) (Trial 3). All these trials considered growth performance and carcass and meat quality traits, including FA composition of Longissimus thoracis muscle and subcutaneous fat. These 3 trials showed a high individual variability in the resistance do trans-10 shift. The causes of this variation are poorly understood and probably related to the interaction between the host and rumen microbiome. In addition, the nature of the main energy component of the diet (fiber or starch) does not appear to be mandatory for trans-10 shift occurrence. When formulating diets for growing lambs, a minimum 40% of good quality forage should be included (Trial 1), namely alfalfa (Trial 3). The replacement of cereals by low-starch agro-industrial by-products reduced the t10/t11-18:1 ratio but depressed growth performance, with 35% of replacement being the best compromise solution (Trial 2). Increasing the buffer capacity of the rumen content, including 2% of sodium bicarbonate in the concentrate, increased t11-18:1 (P=0.030) and c9,t11-18:2 (P=0.021) proportions, reducing t10/t11-18:1 ratio (P=0.049) (Trial 3). Based on these series of results we will test, in a final trial with growing beef, an experimental diet combining 40% of forage, with 40% replacement of cereals by low-starch by-products and 2% inclusion of sodium bicarbonate, which will be compared with a conventional diet (20% forage and 100% cereals). Keywords: Rumen biohydrogenation, trans10-shift, bioactive fatty acids, lamb meat Funding acknowledgements: This work was supported by European Fund for Regional Development (ERDF) (ALT20-03-0145-FEDER-000040 ValRuMeat- Valorização da carne de ruminantes em sistemas intensivos de produção). Also, research grant to S. P. Alves (SFRH/BPD/76836/2011) and UID/CVT/00276/2013 project from Fundação para a Ciência e Tecnologia (FCT) are acknowledged.

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Poster 79

Edible animal by-products as a source of valuable compounds with potential sustainable applications Alfaia CM1, Madeira MS1, Pestana JM1, Toldra F2, Prates JAM1 1. CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa, Portugal; 2. Instituto Agroquímica y Tecnología de Alimentos, Paterna, Valencia, Spain Edible animal by-products have received worldwide attention as value-added ingredients like essential amino acids, fatty acids, minerals and vitamins. Therefore, new opportunities for exploiting the inherent value of animal by-products are required to warrant that this under-employed resource can provide compounds with targeted high value and functionality to a multiplicity of applications including meat industry, animal feed, cosmetics, biomedicine and pharmaceutical purposes. However, due to serious safety concerns, research on animal by-products regarding nutrients value, environmental considerations and potential risks to consumers’ health are ongoing. A deeper knowledge of the nutritional composition of these alternative dietary sources would contribute to increase their consumption and represent new challenges to meet nutrient requirements. Herein, we hypothesised that deposition of fatty acids is distinct among beef by-products and it could be modulated by diet composition. To test this hypothesis, fatty acid composition, including conjugated linoleic acid isomers (CLA), of the most relevant beef by-products (brain, heart, kidney, liver, pancreas and tongue) from young bulls fed different silage levels was assessed. The results showed that beef by-products, in general, had high contents of cholesterol, saturated fatty acids and trans fatty acids, as well as high levels of CLA, which has been reported to exert some anticarcinogenic effects and other benefits on lipid metabolism. In addition, high grass silage diet relative to low silage diet promoted an increase of n-3 polyunsaturated fatty acids and CLA in some by-products. Accordingly, and from a nutritional point of view, it is recommended to consume these beef by-products in small amounts and integrated in a balanced diet (main findings have been published in Food Sci Technol Int, 2017, 23:3, 209-221). Our near future perspective is to increase both the nutrients content and the added-value of typical Mediterranean edible by-products from beef, lamb and fish origin, thus contributing to improve their nutritional quality and sustainability. To funding this research line, we have submitted a proposal to the PRIMA EU Call, that has already succeded to the second phase, involving partners from six Mediterranean Countries. Keywords: edible by-products, beef, diet, fatty acids, nutritional quality Funding Acknowledgements: This study was supported by Fundação para a Ciência e a Tecnologia (FCT, Lisbon, Portugal) through PTDC/CVT/2006/66114 grant and UID/CVT/00276/2013 CIISA project.

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Poster 80

The composition of the lipid and proteic fractions of dried salted cod obtained from the Atlantic cod (Gadus morhua) and the Pacific cod (Gadus macrocephalus) Quaresma M1, Antunes I1,2, Montoito M2, Jardim A3, Bandarra N4, Carvalho A5, Santos C5, Roseiro C5 1. Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário Alto da Ajuda, 1300-477 Lisboa, Portugal; 2. Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. 3Divisão de Alimentação e Veterinária de Setúbal, Direcção Geral de Alimentação e Veterinária, Lisboa, Portugal; 4. Division of Aquaculture and Upgrading, Portuguese Institute for the Sea and Atmosphere (IPMA, IP), Lisbon, Portugal; 5. Food Technology and Safety Division, National Institute for Agricultural and Veterinary Research (INIAV, IP), Quinta do Marquês, Oeiras, Portugal Cod is part of the human diet since ancient times, and part of the Portuguese diet since the XV century. Within the traditional Portuguese cuisine, it is impossible to find a more versatile ingredient and one that gathers higher approval among consumers. In Portugal, two species are sold as cod, the Atlantic cod (Gadus morhua) and the Pacific cod (Gadus macrocephalus), nonetheless, there is a considerable difference between the market value of these two species. Therefore, the aim of this study was to compare the amino acid and fatty acid profiles of Atlantic and Pacific cods, to evaluate whether the two species are similar in nutritional terms.Cod muscle samples were obtained from dry salted cod of both the Atlantic (n=20) and Pacific (n=20) cod, from the inner portion of the dorsal muscle. The fatty acid (FA) profile of the intramuscular lipid fraction was analysed by Gas Chromatography, whereas the amino acid profile and the combined quantification of cholesterol and tocochromanols was analysed by High-Performance-Liquid-Chromatography. The comparison of Atlantic and Pacific cods showed no significant differences on total FA content and FA partial sums (P>0.05). The n-3 PUFA (also known as omega-3 FA) were for both cods responsible for 49.1-50.4% of total FA. Among individual FA, the docosahexaenoic (DHA), palmitic and the eicosapentaenoic (EPA) acids were prime FA in both species (accountable for 68.7-70.4% of total FA) and showed no significant differences in-between.The total cholesterol content revealed no significant differences between the cod species, averaging 69.7 mg/100 g of cod flesh). On the other hand, the Pacific cod presented a significant higher content of α-tocopherol, than was observed on Atlantic cod (8.0 versus 4.9 µg/g of cod flesh). Regarding the amino acid (aa) profile, we can only present preliminary results, since the derivatization method used did not allow us to obtain the complete profile. Nevertheless, it was observed similar amounts of essential aa and non-essential aa. The comparison of the two species showed significant differences in all essential aa (P<0.01), exception made for tryptophan. Among non-essential aa, highly significant differences between the 2 species were observed on all of them (P<0.001), except for glycine. The difference in market value between the two cod species does not seem to be explained by the composition of fatty acid and amino acid profiles. Keywords: Gadus morhua; Gadus macrocephalus; fatty acid profile; amino acid profile; total cholesterol Acknowledgments: This study was funded by the Projects Pest-OE/AGR/U10276/2014 and UID/CVT/276/2013 (both funded by CIISA), cod samples were offered by Mr. João Alves (Riberalves)

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Poster 81

Response to local pain in Brava de Lide cows Mendes-Jorge M1,2, Lucas S1, Valença A1, Mendes MG1, Ferraz C1, Mateus L1, George Stilwell G1, Brito-Paes V2

1. CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa; 2. AMVAT - Associação de Médicos Veterinários de Actividades Taurinas Brava de Lide breeding females are selected in a functional test, known as tenta, where animals are ranked based on the score attributed according to their morphology and behavioral response to different stimuli. This study aimed to determine the relationship between scores obtained after nociceptive stimuli in sorte de varas and serum cortisol levels. In this work, 2-years-old females of the bovine Brava de Lide breed (n=20) were studied. Blood samples were collected immediately before (n=6) and after tenta (n=20), centrifuged at 2500 G for 10 minutes and serum stored at -20ºC. Cortisol concentration was determined by chemiluminescence (Immulite 1000, Siemens Healthcare Diagnostics, Lda., Amadora, Portugal), using a commercial kit (Immulite 1000 cortisol kit, Siemens). Our results showed that there was a strong relationship between cortisol levels and scores attributed in sorte de varas. Animals with lower cortisol levels showed better performance in response to nociceptive stimuli, obtaining the highest scores. Blood cortisol concentration is commonly used as an indicator of stress and pain, and it has been considered that, in general, low levels of cortisol correspond to low levels of stress and pain, and vice versa. In this study, animals that showed a proactive behavior in sorte de varas, obtaining higher scores, presented lower cortisol levels, suggesting a different response to nociceptive stimuli. Given the complexity of pain response, further studies are necessary in order to understand the mechanisms involved in the response to local pain in these animals. Key words: Brava de Lide breed, pain response, cortisol Funding: This study was supported by CIISA - UID/CVT/267/2013 and ProToiro-Federação Portuguesa de Tauromaquia.

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Poster 82

Genome-wide association analysis and gene interaction networks to identify genes associated with carcass traits, meat quality and fatty acid profiles Bressan MC1,2, Amaral AJ2, Almeida J3, Bettencourt C4, Moreira O3, Sá J5, Gama-Carvalho M5, Bessa R2, Gama LT2 1. Universidade Lusófona, Lisbon, Portugal; 2. Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 3. Instituto Nacional de Investigação Agrária e Veterinária, Santarém, Portugal; 4. Direcção Regional de Agricultura e Pescas do Alentejo, Serpa, Portugal; 5. BioISI- Biosystems and Integrative Sciences Institute, Lisbon, Portugal The Alentejano breed is a strain of Iberian pigs that is characterized by excellent meat quality and slow growth rate, very well adapted to harsh climate conditions and usually raised outdoors in oak-tree forests. In this study, a sample (n=60) of Iberian pigs from the Alentejano strain was randomly allocated to be raised either indoors or outdoors throughout the finishing period. Information on a total of 153 different traits was collected, including carcass, meat cuts, meat physicochemical and sensory properties, fatty acid composition and derived metabolic indexes. DNA was isolated from blood and genotyping was performed with PorcineSNP60 v2 Beadchip to identify markers and candidate genes associated with the traits analysed. Standard quality control was performed using GenABEL package in R environment, and SNPs were removed from further analysis if they didn’t fulfil the established criteria. After quality control, 31,434 SNPs remained for further analysis. The association testing was conducted using the GenABEL toolset accounting for stratification using Principal Component Analysis with the egscore function, adding finishing system and/or slaughter weight as fixed factors in the model. To explore the biological role of the revealed associations, SNPs significant in GWAS which overlapped introns or gene regulatory regions were used to identify related genes, by generating gene interaction networks covering an enlarged and connected list of candidate genes. In our analyses, we identified novel associations for the index of desaturase and elongase activity of n-3 c18:3 to c20:5 and for the content of cis-vaccenic acid in intramuscular fat, for the content of lauric acid in subcutaneous fat and for meat moisture and heart weight. The identified SNPs of interest were located within introns or within regulatory regions of 11 relevant genes, namely CRISPLD2, LDLRAD3, PPIL4, SOGA3, ME3, FLRT3, PPIL4, DOPEY2, CELC3A, and DSCAM. Based on these SNPs, we have further identified direct interactions with other genes and the biological processes involved in the regulation of the significantly-associated traits. Our results show that the study of a local pig breed known for its exceptional meat quality allowed the identification of new genomic regions and candidate genes associated to several traits, and some of these are related with the levels of fatty acids that occur in high abundance in Iberian pigs and contribute positively to consumer health. Keywords: GWAS, gene networks, pig, meat quality, fatty acids Funding Acknowledgements: AJA is supported by a Post-doctoral fellowship within IMAGE H2020 project (REF 677353-2). This work was funded by FCT grants PTDC/CVT/116729/2010 and UID/CVT/00276/2013.

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Poster 83

Biochemical characterization of a bi-functional glycoside hydrolase family 5 from Ruminococcus flavefaciens Centeno MSJ1, Pires VR1, Fontes CMGA1,2

1. CIISA-Centro Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa; 2. NZYTech Lda, 1649-038 Lisboa

Many enzyme activities relevant to biomass analysis and conversion are found in Glycoside Hydrolase family 5 (GH5) e.g., cellulases, mannanases, xylanases, galactanases, and xyloglucanases. Due to this great enzymatic diversity many GH5 members have been assigned to subfamilies based on primary sequence properties. It is known that GH5 subfamilies 4 are predominantly endo-E-1,4-glucanases (EC 3.2.1.4) while GH5s of subfamily 7 predominantly display mannanase activity (EC 3.2.1.78). Ruminococcus flavefaciens (Rf) is an anaerobic cellulolytic bacterium found in the rumen and in the hindgut of monogastric mammals. Rf is a major contributor to the hydrolysis of structural carbohydrates in the rumen of herbivores. In this work we report the biochemical characterization of a bi-functional Rf enzymes containing two GH5 catalytic domains belonging to GH5_4 and GH5_7 subfamilies. To investigate the substrate specificity of two catalytic domains, the genes encoding GH5_4 and GH5_7 domains were isolated through PCR into pHTP1 expression vector, generating pGH5_4 and pGH5_7. A third gene encoding the full-lenght protein was also obtained subcloned into the pHTP1 vector, generating pGH5_4-Rf2–GH5_7 (named pMG) which encodes a complex module protein GH5_4-CBM/Rf2-GH5_7 (named MG). The recombinant purified GH5_4; GH5_7 and MG proteins were analysed by SDS-(10%)-PAGE, revealing their molecular mass of 41177; 35287 and 89445 Da, respectively. A preliminary specific catalytic activity for these three enzymes was evaluated against eight substrates incubated at 37ºC. The data revealed that GH5_4 had a higher activity towards xyloglucan and CMC while GH5_7 was particular active against galactomannan and, as expected, MG could cleave both xyloglucan and galactomannan. All the three enzymes showed an optimal temperature for about 55-60ºC, an optimal pH at 6,5-7 and a termostability at 50ºC. The data presented here reveal the importance of bi-functional enzymes to degrade complex polysaccharides. Keywords: Biomass, mannanase, endo-xyloglucanase, cellulosome, glycoside hydrolase; polysaccharide; Ruminococcus flavefaciens. Funding acknowledgements: This work was supported by Fundação para a Ciência e Tecnologia, through projects IF/01621/2013/CP1183/CT0002. VMRP is an assistant researcher supported through an IF-FCT contract (2013 FCT investigator).

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Poster 84

A structural-based approach to unravel all protein-protein interactions supporting Acetivibrio cellulolyticus cellulosome assembly

Alves VD1, Cameron K1, Bule P1, Smith SP3, Gilbert HJ4, Bayer EA2, Najmudin S1, Fontes CMGA1

1. CIISA – Faculdade de Medicina Veterinaria, ULisboa, Polo Universitario do Alto da Ajuda, Avenida da Universidade Tecnica, 1300-477 Lisboa, Portugal; 2. Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot 76100 Israel; 3. Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ONT K7L 3N6, Canada; 4. Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom

Protein-protein high-affinity interactions play a pivotal role in the assembly of the cellulosome, one of nature’s most intricate nano-machines that specialized anaerobic bacteria utilize to degrade complex structural plant polysaccharides. Cellulosome assembly involves two main interactions: i) the integration of cellulosomal carbohydrate-active enzymes (CAZymes) that use their type I dockerin (Doc) modules to bind specific cognate partners called cohesin (Coh) modules, populating non-catalytic structural proteins termed scaffoldins; and ii) the attachment of those enzyme loaded primary scaffoldins to the bacterial cell-surface, via anchoring scaffoldin subunits through type II Coh-Doc interactions. Docs can bind Cohs in two different orientations, a process known as dual-binding mode, by rotating 180° around their two symmetry-related binding sites, while the absence of a compatible two-fold symmetry will only support a single-binding mode interaction. The archetype cellulosome of Clostridium thermocellum revealed a dichotomy between a type I dual-binding mode enzyme assembly versus a type II single-binding Coh-Doc cell-surface cellulosome anchoring. Acetivibrio cellulolyticus is an anaerobic mesophilic bacteria that produces a cellulosome comprising an additional degree of complexity by involving an adaptor scaffoldin (ScaB) besides the typical primary scaffoldin (ScaA) and the cell surface anchoring scaffoldins (ScaC, ScaD and ScaF). Since ScaB Cohs are highly similar to those from scaffoldins ScaD and ScaF, the elucidation of the CohScaB–DocScaA complex would constitute a generic example of the type II interactions recruiting the cellulosome to the cell surface. Additionally, scaffoldin ScaB presents a divergent type I Doc that interacts specifically with the three type I Cohs from ScaC. This atypical type I CohScaC-DocScaB would thus complete the overall picture of A. cellulolyticus cellulosome attachment. Lastly, the structural elucidation of the mechanism of CAZymes recruitment to the cellulosome was the missing piece on all the A. cellulolyticus Coh-Doc specificities. In this report all representative Coh-Doc complex specificities from A. cellulolyticus were produced and crystallized to generate 3D structural data, allowing a comprehensive panorama of its cellulosome assembly and anchoring. The data shows that A. cellulolyticus presents an ubiquitous highly flexible dual-binding Coh-Doc pairing for all its cellulosome protein-protein interactions, revealing an ever-increasing number of subtle intricacies governing their specificity, affinity and flexibility. Keywords: cellulosome, cohesin, dockerin, protein-protein complexes, Acetivibrio cellulolyticus

130

Poster 85

Modulation of aquaporins gene expression by n-3 long-chain polyunsaturated fatty acids (LCPUFA) lipid structures in white and brown adipose tissue from hamsters Lopes PA1, Martins R2, Silva IV2, Madeira MS1, Prates JAM1, Soveral G2

1. CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa; 2. iMed.ULisboa-Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa

Eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3) fatty acids have weight-reducing properties with physiological activity depending on their molecular structure, that is, as triacylglycerols (TAG) or ethyl esters (EE). Aquaporins (AQP) are membrane protein channels recognised as important players in fat metabolism, but their differential expression in white adipose tissue (WAT) and brown adipose tissue (BAT), as well as their modulation by dietary n-3 long-chain polyunsaturated fatty acids (LCPUFA), such as EPA and DHA, has never been investigated. The transcriptional profile of AQP3, AQP5, AQP7 and selected lipid markers of WAT (subcutaneous and visceral) and BAT (interscapular) from hamsters fed diets containing n-3 LCPUFA in different lipid structures, fish oil (FO, rich in EPA and DHA in the TAG form) and FO-EE (rich in EPA and DHA in the EE form) were used and compared with linseed oil (LSO) as the reference group. A clear effect of fat depot was observed for AQP3 and leptin, with the lowest values of mRNA found in BAT relative to WAT. The opposite occurred for PPARα. AQP7 was affected by diet, with FO-fed hamsters having higher mRNA levels compared with LSO-fed hamsters. The relative gene expression of AQP5, adiponectin, GLUT4 and PPARγ was influenced by both fat tissue and diet. Taken together, we reported differential AQP3, AQP5 and AQP7 gene expression across subcutaneous WAT, visceral WAT and interscapular BAT, which may reflect adipose tissue depot’s own location and metabolic function. We also found different patterns of AQP and markers of lipid metabolism expression in both WAT and BAT in response to feeding n-3 LCPUFA in two different structural formats: TAG versus EE. Among the AQP isoforms, AQP7 stands out as the more promising target for developing new anti-obesity drugs owing to its conservative role across WAT and BAT. Keywords: Aquaporins; Markers of lipid metabolism; n-3 LCPUFA; White adipose tissue; Brown adipose tissue Acknowledgements: This study was supported by the Spanish Ministry of Science and Innovation through “Structured lipids: novel dietary strategies for improving human health” grant AGL/25807/2011 and by Fundação para a Ciência e a Tecnologia, Portugal, through projects UID/CVT/00276/2013 and 2016 INOV-20 to CIISA, and UID/DTP/04138/2013 to iMed.ULisboa. Funding Acknowledgements: This work was supported by Fundacao para a Ciencia e a Tecnologia (Lisbon, Portugal) through grants EXPL/BIA-MIC/1176/2012 and PTDC/BIA-MIC/5947/2014.

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Poster 86

The CBMomes of cellulolytic bacteria colonizing different ecological niches present distinct carbohydrate specificities Brás JLA1,2, Ribeiro D3, Romão MJ3, Carvalho AL3, Chai W4, Liu Y4, Feizi T4, Prates JAM1,2, Ferreira LMA1,2, Palma AS3, Fontes CMGA1,2

1. NZYTech - genes & enzymes - Estrada do Paço do Lumiar, Campus do Lumiar, Edif. E, R/C, 1649-038 Lisboa, Portugal; 2. CIISA – Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477 Lisboa, Portugal; 3. UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 4. Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom Background: Plant cell walls, predominantly composed of cellulose, are the most abundant source of organic carbon on earth. The energetic constraints posed by anaerobic ecosystems lead to the evolution of a remarkable highly efficient supramolecular enzyme complex, termed cellulosome. Cellulosomes are multi-enzyme complexes of Carbohydrate Active enZymes (CAZymes) and play a pivotal role in the degradation of plant cell wall carbohydrates. Clostridium thermocellum and Ruminococcus flavefaciens FD-1 are two highly efficient cellulolytic bacteria that produce very complex but well characterized cellulosomes. The two bacteria colonize different, highly dynamic and populated ecological niches, the soil and the rumen of mammals, respectively. In the present work we aim to study all known CBMs, here designated the ‘CBMome’, of two cellulolytic bacteria that colonize different ecological niches, R. flavefaciens and C. thermocellum. Our main goal was to investigate novel CBM-carbohydrate interactions and novel CBMs ligand specificity. Methods: The CBMs from the two bacteria R. flavefaciens and C. thermocellum (150 CBMs in total), were cloned, expressed and purified by using high-throughput platforms. 90 genes from C. thermocellum and 60 genes R. flavefaciens were amplified and cloned in an Escherichia coli expression vector. Immobilized metal affinity chromatography (IMAC) was performed to purify the recombinant proteins. Oligosaccharide and polysaccharide specificity of the two CBMomes was analysed using a carbohydrate microarray platform derived from target polysaccharide glycomes. Results: The majority of the CBMs were readily expressed and purified through. The data revealed that the two bacteria present CBMomes expressing different carbohydrate-binding capacities. Conclusions: Overall this work suggests that the physico-chemical properties of different ecological niches modulate the evolution of CAZymes presenting distinct ligand specificities. Keywords: CBMomes, High-throughput, Microarray Funding Acknowledgements: This work was supported by the Portuguese Science and Technology Foundation (FCT-MEC) through grants UID/Multi/04378/2013, PTDC/QUI-QUI/112537/2009, RECI/BBB-BEP/0124/2012 and SFRH/BD/100569/2014.

132

Poster 87

The mechanism by which family 56 Carbohydrate-Binding Modules recognize complex β-1,3-glucans

Pires VMR1, Correia M2, Centeno MSJ1, Bastos J1, Alves VD1, Ferreira LMA1,3, Fontes CMGA1,3

1. CIISA, Faculdade de Medicina Veterinária, Pólo Universitário da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 3. NZYTech genes & enzymes, Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E - R/C, 1649-038 Lisboa, Portugal

The critical importance of structural polysaccharide degradation, which is orchestrated by a cohort of carbohydrate-active enzymes (CAZymes) produced microbial organisms, has been appreciated for decades. Identification of new organisms that produce biomass-degrading enzymes is of considerable interest, especially in the renewable chemical industry. The aptitude of thermophilic microbes and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability and decreased risk of contamination (1), although most of their enzymes remain largely unknown or uncharacterized. Here we report the heterologous expression and subsequent biochemical and structural characterization of a putative multi-modular endo-B-1,3-glucanase (PLamGH16) from Paenibacillus sp Y412MC10 (GenBank: ACX65777.1), composed of a catalytic domain of the GH16 family at the N-terminus, followed by an internal Ig-like module and an uncharacterized C-terminal CBM module classified into family 56. Gene synthesis was used to obtain the DNA encoding PLamGH16 (residues 39-872; Accession no. ACX65777.1). Gene fragments encoding truncated derivatives of PLamGH16, namely the catalytic module PGH16 (residues 39-682) and the CBM, PCBM56 (residues 777-872), were amplified by PCR using appropriated primers. All genes were cloned into pHTP1 and al recombinant proteins contained N-terminal His6 tags to promote affinity purification. PCBM56 displayed high and exclusively affinity for insoluble (curdlan) and soluble (laminarin) β-1,3-glucans, but lacks affinity for their respective β-1,3-glucooligosaccharides (methods in reference 2). The native PCBM56 structure was determined by Se-SAD using synchrotron radiation, and crystals diffracted up to 1.7 Å resolution and belonged to C2 space group. Crystal structure analysis of PCBM56 accompanied with sequence alignment of some CBM family 56 members revealed that 13 residues display a role in ligand recognition. These residues were mutated to alanine by site direct-mutagenesis. The F28A, D30A, H32A, Y33A, Q40A, N42A and F69A mutations resulted in a substantial reduction in affinity for laminarin. Further, PLamGH16 and PGH16 (with the CBM56 deleted) were assayed for enzyme activity (3), revealing that both proteins displayed higher activity for laminarin and curdlan, thus displaying exclusive β-1,3-glucanase specificity. Our findings reinforce the concept that catalytic modules and their cognate CBMs have complementary specificities including targeting of soluble or insoluble polysaccharides. Keywords: Carbohydrate-Binding Modules (CBMs), Carbohydrate Active enZymes (CAZymes), β-1,3-glucans. Funding Acknowledgements: This work was supported by Fundação para a Ciência e Tecnologia (FCT), through projects IF/01621/2013/CP1183/CT0002 and Individual fellowship SFRH/BPD/64917/2009 (MASC) and VMRP is an assistant researcher supported through an IF-FCT contract (2013 FCT investigator).

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Poster 88

Larval cyatostominosis in domestic (Equus caballus) and wild equids (Equus quagga) in Portugal: pathology and epidemiology in outbreaks with a high mortality rate Correia JJ1, Lamas L1, Cosinha C2, Mousinho C3, Carneiro C1, Oliveira M1, Peleteiro MC1, Madeira de Carvalho L1

1. CIISA, Faculty of Veterinary Medicine, University of Lisbon; 2. Clínica Veterinária Lusopecus; 3. Médica Veterinária

Background: Strongyles of the subfamily Cyathostominae are the most important parasites of equids due to their prevalence and associated mortality. The cyathostomins are parasites of the cecum and colon, inducing nodule formation due to hypobiosis and are associated with anthelmintic resistance. Cyatostominosis is of increasing importance in equines despite the improvement of husbandry, hygiene and health conditions. Methods: We report four cases of larval or type II cyathostominosis involving three Lusitano horses, aged one year, under grazing regime in Ribatejo and regularly dewormed, and a Common zebra from Estremadura. After sudden death, equids necropsy were performed at FMV-ULisbon and samples were collected for histopathological, bacteriological and parasitological analysis. Results: At necropsy, all animals revealed abdominal distension due to severe hydrohemoperitoneum. Large colon and cecum presented parasitic nodular enteritis, with oedematous thickening of the wall and presence of free cyathostomins in the lumen and encysted (L4-L5). Lymphadenomegaly was identified in the caecocolic lymphnodes. Histopathological exam revealed oedematous thickening of the cecum and large colon wall, with lymphangiectasia, and presence of cyathostomins in mucosa and submucosa nodules, involved by granulomatous inflammatory reaction. The thymus, intestine and spleen presented lymphoid hypoplasia compatible with immunodepression. Bacteriological characterization of the samples allowed the isolation and identification of Clostridium sordelli in several organs from one horse and also of Streptococcus equi ssp. zooepidemicus in a lymphnode and of Escherichia coli and Clostridium sp. in the peritoneal effusion from another horse; in the zebra Escherichia coli was isolated from several organs. The agents were considered secondary to parasitic infection, although playing an important role in the death of animals. Parasitological exams identified adult cyathostomins and eggs in the faecal samples from all animals. Conclusions: Cyathostomins have a high prevalence in equids, even when regularly dewormed, as the reported cases in the three domestic equids and zebra. Being an emerging helminthiasis with high morbidity and mortality, cyathostominosis requires further investigation of its biology, pathology, diagnosis and control in the Iberian Peninsula. Keywords: cyatostominosis, horse, zebra, outbreaks, pathology, epidemiology, Portugal. Funding Acknowledgements: this research was funded by CIISA-FMV-ULisbon, Project UID/CVT/00276/2013, Fundação para a Ciência e a Tecnologia.

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Poster 89

Hemoparasites of birds of prey and other avian hosts kept in a Wildlife Rehabilitation Center in Lisbon, Portugal Madeira de Carvalho LM1, Alho AM1, Breuer H2, Shahi Barogh B2, Zittra C2, Harl J3, Caetano I4, Brazio E4, Mira M4, Fuehrer HP2

1. CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisbon, Portugal; 2. Institute of Parasitology, University of Veterinary Medicine, Vienna, Austria; 3. Institute of Pathology, University of Veterinary Medicine, Vienna, Austria; 4. Centro de Recuperação de Animais Silvestres de Lisboa (LxCRAS), Camara Municipal de Lisboa, DMEVAE/DEV/DGPFM, Lisbon, Portugal Background: Avian parasitism have been increasingly reported worldwide. Birds of prey may constitute a risk group for the agents causing these diseases, considering their migrations where they are thus exposed to vector arthropods. The aim of this study was to identify the presence and prevalence of the most significant hemoparasites in birds of prey in the main wildlife rehabilitation center of Lisbon, using molecular detection and sequencing of the DNA products. Methods: During 2017, 137 blood samples were collected from wild birds, mainly birds of prey, of the following families: Falconidae (34 Falco tinnunculus), Strigidae (31 Athene noctua, 17 Strix aluco, 1 Bubo bubo), Laridae (17 Larus fuscus, 6 Larus michahellis, 2 Larus sp.), Tytonidae (15 Tyto alba), Corvidae (3 Garrulus glandarius, 2 Corvus corone corone), Anatidae (1 Aix galericulata, 1 Alopochen aegyptiacus, 1 Anas platyrhynchos), Accipitridae (2 Accipiter gentilis, 1 Aquilla pennata), Rallidae (1 Fulica atra), Columbidae (1 Columba palumbus), Ardeidae (1 Egretta garzetta). DNA was extracted using a Chelex based DNA extraction techniques (Instagene Matrix, BioRad) and examined for the presence of DNA of Anaplasmataceae, filarioid helminths, Piroplasmida and Rickettsiales, using established protocols. Results: Overall, 25.5% (35/137) of the birds were infected with a vector-borne agent, 25.7% (26/101) in birds of prey. In total, 17.5% (24/137) were positive for Leucocytozoon sp. (genotypes Leucocytozoon sp. STAL1-LIKE, ATNO1, CIAE02, STAL5, AEMO02, COCOR09 and Leucocytozoon ziemanni BUBO01); 6.6% (9/137) positive to Haemoproteus sp. (Haemoproteus sp. LISBOA01 and Haemoproteus syrnii) and 1.5% (2/137) positive to Plasmodium relictum. A new lineage was described: Haemoproteus sp. LISBOA01, in gulls. No positive results were recorded for filarioid helminths and trypanosomatid protozoa. Conclusions: These results show that one quarter of the total surveyed birds and of the raptors, was infected with vector borne protozoans. Considering the conservation status of some of these birds, the wide migrations routes and the close contact with competent vectors, preventive measures such as mosquito populations control for avian malaria at this wildlife rehabilitation center are needed, to reduce exposure and future infections, namely with potential pathogenic and widespread species of avian malaria. Keywords: Birds of prey, Portugal, Leucocytozoon, Haemoproteus, Plasmodium Funding acknowledgements: Project UID/CVT/276/2013 (CIISA). TD1303 COST Action - Short Term Scientific Mission (STSM).

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Poster 90

Ocular sequelae of heminopter bites in dogs - 6 clinical cases Esmeralda Delgado CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal Background: Heminopter bites can induce severe allergic reactions, and can even be life-threatening sometimes. When they occur in the periocular region, they can induce ocular lesions secondary to self-induced trauma or corneal lesions. Ocular injuries can occur following bites by bees, wasps, and ants which belong to the group hymenoptera. The effects are both local and systemic, and are mainly due to the toxins elaborated by the sting. So far, the eye-related injuries reported in humans are conjunctivitis, corneal infiltrates, cataract formation, iritis, hyphaema, lens subluxation, and optic nerve damage. Corneal injury is the most commonly reported ocular injury. Recently, ischaemic optic neuropathy and cilio-choroidal detachment have also been reported. We present the ocular sequelae of 6 cases of periocular heminopter bites in dogs. Methods: All patients were presented with severe unilateral acute swelling of the affected periocular region. All owners described that the animals had been stung by heminopters some hours before. Ophthalmic examination of the ocular globe was impossible in all cases due to intense swelling of the affected periorbital region. Contralateral eye was normal. Intravenous medication with corticosteroids was performed at the initial presentation and oral anti-histaminic drugs were prescribed along with topical treatment. The swelling substantially decreased in a week time in all cases but there was still chemosis and conjunctival hyperemia present. Results: Two out of five patients developed corneal ulceration due to self-inflicted trauma and one developed corneal degeneration one month later. The patients with corneal ulcers were treated with topical antibiotics, midriatic cicloplegic drugs and elizabethan colar. The remaining four cases were treated with topical prednisolone ointment for a week time. One month later one of these dogs presented with a mid stromal degeneration, lipidic and calcic in nature, occupying the center of the conea of the previously affected eye. CBC profile and biochemistry of this animal including kidney, liver, pancreas, lipids, cholesterol and calcium profiles were within normal values. The lesion did not disappear nor changed in appearance after 12 months of follow-up, the other ocular globe remaining assymptomatic. Conclusions: Heminopter bites can be very dangerous, especially when it is the first time the animal is being stung. The poison can vary between heminopter species. In canine patients topical corticospteroids should be used with caution in such cases because there is the potential for the development of a corneal ulcer, and also because the topical use of prednisolone could potentially result in corneal lipid and mineralization, exacerbating a corneal degeneration. An Elizabethan colar to prevent self-trauma is advised in all cases. Keywords: Heminopter bites, ocular injuries, dogs, corneal degeneration Funding acknowledgements: Funded by Project UID/CVT/276/2013 (CIISA).

136

Poster 91

Effects of a 15o variation in poll flexion during riding on the respiratory system and on behaviour in pure blood Lusitano high dressage horses Tilley P, Simoes J, Sales Luis, JP CIISA (Centro de Investigacao Interdisciplinar em Sanidade Animal / Centre for Interdisciplinary Research in Animal Health), Department of Clinics, Faculty of Veterinary Medicine, University of Lisbon, Portugal Background: Based on previous studies about the impact of hyperflexion on horse welfare, the ISES position statement advised for further research to be done on physiological and psychological effects of lesser degrees of poll flexion. We evaluated the effect of 2 riding poll flexion positions with only 15° difference on the respiratory system and on behaviour. Methods: 20 Lusitano high dressage horses underwent a 40 min. standardized ridden test with 85o ground angle1 in the 1st evaluation and 100° in the 2nd and conflict behaviour was registered. Overground endoscopy was performed for the last 5 min. of canter to classify upper airway dynamic collapse2,3. After each test arterial oxygenation and lactate, intrathoracic pressure (oesophageal catheter) and pharyngeal diameter (lateral pharyngeal radiographs4) were evaluated. HR/RR were measured immediately before/after both tests. Mixed model for repeated measures, Wilcoxon and Friedman tests were used according to the design of experiments and/or errors normality. Results: Conflict behaviour (tail swishing, head shaking, mouth opening, open/close jaw movement, teeth grind, excessive salivation/drooling) and rider encouragement were significantly higher with 100° and relaxation behaviour (ear play, ears turned forward) with 85°. Upper airway abnormalities were more common with 100° (aryepiglottic folds axial deviation; palatal instability/dysfunction; pharyngeal lymphoid hyperplasia; vocal fold, nasopharyngeal, intermittent bilateral arytnoid cartilage and cricotracheal collapse). Blood lactate (0.438+/-0.12 mmol/L at 85° and 0.574+/-0.19 mmol/L at 100°) and intrathoracic pressure difference (ΔIP, 17.95+/-1.14 cm H2O at 85° and 22.15+/-1.04 cm H2O at 100°) were significantly higher, but pharyngeal diameter (2.028+/-0.65 cm at 85° and 1.162+/-0.5 cm at 100°) was significantly less with 100°. HR/RR were significantly lower at the beginning of the 2nd test (41.5 +/-6.08 bpm and 20.3 +/- 6.02 rpm) when compared to the 1st (46.9 +/-6.39 bpm and 23.4+/-7.17 rpm), but significantly higher at the end of the 2nd test (108.25+/-14.67 bpm and 79.4+/-14.63rpm) when compared to the end of the 1st (93.95+/-11.32 bpm and 65.35 +/- 18.19 rpm). Conclusions: The significant differences identified between 2 very close positions in the ridden high dressage horse, support the idea that as little as 15° variation in poll flexion can have negative effects on the respiratory system and behaviour, therefore, on the horse’s welfare. ISES = International Society for Equitation Science’s Keywords: poll flexion, dressage, horse, welfare Funding acknowledgements: This research was supported by CIISA – Centre for Interdisciplinary Research in Animal Health hosted by the Faculty of Veterinary Medicine, University of Lisbon (CIISA- FMV-ULisboa) through the CIISA-Coordenacao. 1 Go et al. (2014) BMC Veterinary Research (10) 118. 2 McGivney et al. (2017) Equine Veterinary Journal (49) 433-437. 3 Holcombe et al. (2005) AAEP Proceedings Vol.51. 4 Linford et al. (1983) American Journal of Veterinary Research, 44 (9) 1660-1666.

137

Poster 92

Bioprotective solution against pathogens: selection of protective starter cultures for meat products

Fraqueza MJ, Chaves A, Rebelo C, Goncalves S, Martins A, Fernandes MH, Fernandes MJ, Barreto AS

CIISA - Centro de Investigacao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Universidade de Lisboa, 1300-477 Lisboa

The potential presence of pathogens on meat products needs to be controlled. The main control measures are the implementation of HACCP method coupled with technological strategies namely bioprotective solutions by the use of protective starter cultures. The aim of this work was to select presumptively safe Lactobacillus strains isolated from traditional dry fermented sausages regarding their potential production of bacteriocins. Lactobacillus sakei (n= 32) and Lactobacillus plantarum (n=82) were isolated from meat products, equipment surfaces and ingredients on different meat industries. All isolates were assessed regarding antibiotic susceptibility. Disc susceptibility testing was performed according to CLSI for nine antibiotics belonging to different classes. The PCR detection for tet(L), tet(K) and tet(M) genes was performed. Genomic typing was done by PCR-fingerprinting using the primers (GTG)5 and M13. L. plantarum and L. sakei isolates genetic background to express phenotypic bacteriocinogenic ability was achieved by applying PCR for known sakacin (sakA, P and Q) and plantaricin genes. Bacteriocinogenic potential was also assessed by Skalka qualitative test. The majority of isolates L. plantarum and L. sakey were susceptible to tetracycline (75% and 81%) and erythromycin (71% and 97%). The presence of tetM and tetL genes linked to mobile elements, were not frequent. However, among L. plantarum the tetK gene was detected on 44% of the isolates. The potential for producing Sakacin was detected only in three L. sakey isolates (L1B8, L3B8 and P2B3) with sakA and sakQ genes while the majority of the L. plantarum presented genes plnC, N, R, K, L for Plantaricin. L. plantarum P3B8 was selected to further studies as a safe strain with plantaricin genes and high bacteriocinogenic activity against L. monocytogenes and S. aureus. Keywords: Bacteriocins, Protective starter, lactobacilli, Safety, Meat products This study was supported by Fundacao para a Ciencia e a Tecnologia (FCT, Lisbon, Portugal) through CIISA project (UID/CVT/00276/2013) and project PTDC/AGR- ALI/119075/2010.

138

Poster 93

Effect of genotype and salt reduction on the quality of traditional Portuguese sausages Laranjo M1,2, Potes ME1,3, Véstia J1, Rodrigues S1, Agulheiro-Santos AC1,4, Charneca R1,3, Fraqueza MJ5, Elias M1,4 1. ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; 2. IIFA-Instituto de Investigação e Formação Avançada, Universidade de Évora, Ap. 94, 7006-554 Évora, Portugal; 3. Departamento de Medicina Veterinária, Escola de Ciências e Tecnologia, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; 4. Departamento de Fitotecnia, Escola de Ciências e Tecnologia, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; 5. Departamento de Produção Animal e Segurança Alimentar, Faculdade de Medicina Veterinária, CIISA, Universidade de Lisboa, Lisboa, Portugal Background: The Portuguese autochthonous swine breeds Alentejano (Al) and Bísaro (Bi) are traditionally bred in extensive systems. Al is a fat pig breed related to the Iberian pig breed, from the group of Mediterranean pigs, while Bi belongs to the group of Celtic pigs. The main objective of the work was to evaluate differences in product quality between genotypes, while trying to reduce the salt content without compromising the safety or acceptability of the products by consumers. Methods: Traditional standard (6% NaCl) and low-salt Portuguese meat products (2% NaCl) were produced in a local production unit using four different pig genotypes: Al, Bi and the hybrid genotypes BiAl and AlBi. Three independent batches were produced, and two sausages per batch were analysed. Quality of products was assessed by physicochemical (pH, aW-water activity, and lipid content), microbiological and sensory parameters. Results: Significant differences in product composition were observed between genotypes. Products from Bi and BiAl pigs had a higher total lipid content (P<0.001). Furthermore, pH was significantly lower, while aW was significantly higher in low salt products (P<0.05). The total content of biogenic amines was higher (P<0.05) in Al products than in other products, mainly because of higher amounts of putrescine and cadaverine. Lactic acid bacteria counts are not influenced by genotype or salt content. Generally, staphylococci numbers increase with salt content, while the number of enterobacteria decrease. Conclusions: The instrumental and sensory analyses show that reduction of the salt content did not decrease the quality or acceptability of the sausages. Keywords: Autochthonous breeds; salt content; microbiota; biogenic amines; sensory acceptability. Funding acknowledgements: This work was funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634476 (Project acronym: TREASURE) and by national Funds through FCT-Fundação para a Ciência e a Tecnologia under Project UID/AGR/00115/2013. M. Laranjo also acknowledges a Post-Doc research grant from FCT (SFRH/BPD/108802/2015). The authors thank Paladares Alentejanos, Lda for their collaboration.

139

Poster 94 Isolation of bioactive compounds from Arbutus unedo “medronheiro” and evaluation of antimicrobial properties against bacteria from food related environments Barreto AS, Félix V, Semedo-Lemsaddek T

CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal For many decades, natural products have been a wealthy source of antimicrobials and more recently academic drug discovery in this research field has been accentuated. Traditional methods start with the extraction of the natural product from the source, followed by screening against a specific target and further characterization of the bioactive properties. Arbutus unedo, known as the strawberry tree “medronheiro”, grows spontaneously in the South region of Portugal. This plant is known to produce a variety of chemical compounds however, a comprehensive characterization of its antimicrobial potential is far from being complete. Our study aims to reinforce the potential applicability of natural compounds extracted from the “medronheiro” as antimicrobial agents against foodborne bacteria. Bioactive compounds from various plant parts of Arbutus unedo at different stages of ripening were isolated and evaluated regarding their antimicrobial potential against food-related bacteria (e.g., Pseudomonas, E. coli, S. aureus and L. monocytogenes) in planktonic and biofilm-state (at distinct temperatures and growth times). Bioactive compounds were isolated from roots, stems, leaves and fruits using water, methanol and acetone. “Spot on lawn” was used as an initial screening approach for the selection of the most promising bioactive compounds. Subsequently, the microdilution method was used to determine Minimum Inhibitory Concentrations (MIC), Minimum Bactericidal Concentrations (MBC), Minimum Biofilm Inhibitory Concentrations (MBIC) and Minimum Biofilm Eradicating Concentrations (MBEC). Results demonstrated a low antimicrobial activity for most of the extracts under study. Considering that such molecules are secondary metabolites produced by plants, we must consider that the abnormal ripening time for medronho observed last year, later than usual, could be related with the low production of bioactive compounds. Overall, this study optimized extraction protocols from various plant materials and showed the efficacy of microbiological procedures to determine the antimicrobial activity of the obtained compounds. Furthermore, the interest of Arbutus unedo as a plant of interest should not be overlooked and samples from distinct years and regions of production must be analyzed for comparison purposes and subsequent on food-related models. Keywords: Arbutus unedo; bioactive compounds, antimicrobial activity Funding Acknowledgments: Experimental work was supported by Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013). Teresa Semedo-Lemsaddek is financially supported by FCT (SFRH/BPD/108123/2015).

140

Poster 95

Estimation of inbreeding using pedigree or genomic information in Ramo Grande cattle Gama LT1, Pavão AL2, Amaral AJ1 1. CIISA- Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária da Universidade Lisboa. Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; 2. Direção Regional da Agricultura, Secretaria Regional da Agricultura e Florestas da Região Autónoma dos Açores. Vinha Brava, 9701-861 Angra do Heroísmo, Portugal In local breeds, often with reduced census, controlling inbreeding is one of the major concerns, as high inbreeding levels lead to reduction of genetic diversity and to inbreeding depression. The Ramo Grande cattle breed was developed in the Azores, from a founder population brought by the Portuguese, possibly derived from animals of the Alentejana, Minhota, Mirandesa and Algarvia breeds. Currently, Ramo Grande has a small population that counts with 77 sires and 1330 dams. The on-going management program started 20 years ago, and the Herdbook is open to the registration of new animals, and demographic information has been used for management decisions. The availability of high density single nucleotide polymorphism (SNP) arrays has enabled the identification of long stretches of homozygous genotypes, known as runs of homozygosity (ROH) that represent an estimate of the degree of autozygosity at genome-wide level. The current study aims to quantify the genomic inbreeding coefficient (F) derived from ROH (FROH) in the Ramo Grande cattle breed. Also, F was estimated from pedigree (FPED), and from the proportion of homozygous genotypes (FHOM). A total of 44 sires with 10 or more offspring were genotyped with the 50K v3 Illumina Beadchip. We used PLINK 1.9 to perform quality control (calling rate, Hardy-Weinberg, minor allele frequency, SNPs in linkage disequilibrium) and filtering of samples and genotypes, and the detectRUNS package of R to estimate ROH and F(ROH). After quality control 42 samples remained and a total of 21,884 SNPs remained for the analyses. A total of 1186 ROH were identified, of which 53% correspond to ROH segments of 2.5-6Mb (related with ancient inbreeding) whereas 23% correspond to ROH segments >12Mb, which are assumed to represent recent inbreeding. Chromosomes 5, 8 and 11 displayed the highest levels of mean FROH. The mean for individual FROH, which corresponds to the proportion of the genome which is in the form of ROH, was 11.78±7.74. On the other hand, the FPED (which is highly dependent on pedigree depth) had a mean of 4.03±6.72, and the correlation between FROH and FPED was 0.64, a moderate estimate which is probably due to the reduced pedigree information available for some animals. The results represent a crucial contribution of genomic tools for improving the on-going management program of the Ramo Grande breed, in order to control inbreeding and maintain genetic diversity. Keywords: Local cattle breed, runs of homozygosity, inbreeding, SNP chip. Funding Acknowledgements: AJA is supported by a Post-doctoral fellowship within IMAGE H2020 project (REF 677353-2). This work was funded by UID/CVT/00276/2013 and by Direção Regional da Agricultura da Secretaria Regional da Agricultura e Florestas dos Açores.

141

Poster 96

Correlation between histological grading and KIT expression patterns in eighty canine mast cell tumors Peleteiro MC1, Borges I2, Carvalho S1, Pissarra H1, Carvalho T3

1. CIISA, Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Av. Universidade Técnica, 1300-477 Lisbon, Portugal; 2. CEDIVET, Rua Antero de Quental, 991, 2ºD, 4200-071 Porto, Portugal; 3. Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal Background: The objective of this work was to establish the relation between grading and the KIT pattern expression in canine mast cell tumor (MCT) and if both data could be enough to establish a prognosis and help treatment decisions. Methods: Eighty cases of canine MCTs were selected from the archives (2017-2018). All tumors were classified in low and high grade and submitted to immunohistochemistry (DAKO polyclonal c-kit antibody counterstained with Gill2 hematoxylin) for classification in patterns 1, 2 or 3 for KIT expression. The follow up of nine high grade and six low grade MCTs in which the prognosis could have been influenced by the KIT expression was performed and the results analyzed accordingly. Results: MCTs analysis showed the following distribution: 42.5% of the tumors (34) were low grade and 57.5% (46) high grade. Of the low grade group 55.9% were pattern 1, 23.5% pattern 2 and 20.6% pattern 3. Of the high grade group 28.2% were pattern 1, 34.8% pattern 2 and 37% pattern 3. In low grade tumors the percentage of patterns 2 and 3 was high (44.1%), eventually aggravating the prognosis. In high grade tumors, 28.2% expressed pattern 1, alleviating the prognosis. For all low grade MCT that showed pattern 2 or 3 for KIT expression, surgery was curative, except for one that developed a new nodule far from the first tumor. After a second surgery this dog remains well up to the end of August ‘18. In the case of the nine high grade MCTs that labeled according to pattern 1, five were euthanized one to 12 months after the first diagnosis, three having been submitted to chemotherapy. The remaining four are still alive and well up to the end of August ’18, three of them having never been submitted to chemotherapy. Conclusions: Our data corroborate the fact that in low grade MCTs KIT expression pattern 1 is the most common, and that presence of pattern 2 or 3 does not seem to change the prognosis. For high grade tumors the patterns were more evenly distributed, with a higher percentage of pattern 3 cases. If the KIT expression pattern in high grade tumors is restricted to the membrane (pattern 1), our recommendation is that the grade should be reevaluated. Keywords: Canine mast cell tumors, immunohistochemistry, KIT expression pattern, Funding Acknowledgements: Work supported by the FCT project UID/CVT/276/2013.

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Poster 97

Diptera fauna associated with cat corpses’s decomposition as a possible entomological forensic tool

Pereira da Fonseca I1*, Diz S1*, Loução C1*, Santos M1*, Rebelo MT2*

1. CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 2. Centre for Environmental and Marine Studies (CESAM), Department of Animal Biology, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal * these authors contributed equally for this work. Background: Many Diptera species play an important role as decomposers and scavengers of animal and human remains. As the decomposition of a corpse takes place, so do distinct insect species appear, colonize the remains, and eventually move on when conditions are no longer favorable being excellent indicators for forensic purposes. The study of the entomofauna succession on two cat corpses was the object of this work. Methods: The remains of 2 female cats (C1/C2) were placed outdoors in separated modified Malaise traps. C1 and C2 had different characteristics: age (C1=2 months; C2=14yrs), weight (C1=1kg; C2=1.7 kg) and cause of death (C1=permethrin poisoning; C2=intestinal lymphoma). The remains were placed on Aug 2014, observed regularly while arthropods were collected and environmental temperature and humidity were recorded. Identification was based on morphological features and, whenever possible, done to species level. DNA from Diptera specimens reported for the first time in Portugal was extracted by the Chelex method, amplified (COI gene) and sent to StabVida for sequencing. Results were submitted to GenBank database and phylogenetic trees were built using Neighbour-Joining method and Maximum Likelihood algorithm. Results: A total of 4274 arthropods were collected (of which 3801 Diptera). Significant differences were detected: C1-3636 Diptera vs C2-165. Dead arthropods were occasionally found in and around C1, attesting that the insecticide was still having effect on this fauna several weeks p.m. In C1 there were two main peaks in Diptera activity however in C2 most Diptera were collected in the first weeks. Genus Pollenia (species identification performed by Dr. Knut Rognes, Univ. Stavanger, Norway) was surprisingly prevalent being possible that some chemical compound present in the poisoned cat carrion had an attractive effect on these flies, or that some unknown detail of their life cycle causes them to seek out carrion in advanced stages of decay. Four species not yet reported in Portugal were found: Lucilia cuprina (Wiedemann, 1830), Sarcophaga aratrix Pandelle, 1896, Pollenia leclercqiana (Lehrer, 1978) and Sarcophila meridionalis Verves, 1982. Conclusions: The number of collected arthropods can be explained by differences in body score condition and cause of death. Genus Pollenia was highly prevalent in the poisoned carrion pointing to a possible forensic indicator in criminal cats poisoning cases. Four Diptera species were reported for the first time in Portugal. Keywords: Diptera, species, cat, decomposition, forensic. Funding Acknowledgements: CIISA-FCT Project UID/CVT/00276/2013. Thanks are also due to CESAM (UID/AMB/50017 - POCI-01-0145-FEDER-007638), to FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.

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Poster 98 Dogs and Cats dermatophytosis in Portugal: a 16-year study Lança A, Nikonov V, Ramos A, Tavares L, Bernardo F, Oliveira M Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Portugal Background: Dermatophytes are a group of fungi capable to invade keratinized tissues of animals and humans, including skin, hair and nails. They are associated with dermatophytosis or ringworm disease, which are cutaneous infections usually restricted to nonliving cornified layers; however, symptoms may range from mild to severe, depending on the fungus virulence potential, infection location and the host immune system. Tricophyton, Microsporum and Epidermophyton are the most frequent genera associated with dermatophytosis, being often isolated from pets. Due to their well-established zoonotic potential they represent a major public health concern. Methods: In this study, 2006 dogs and 801 cats with clinically suggestive ringworm lesions were investigated for the presence of dermatophytes by fungal culture, the gold standard diagnosis technique. Samples were collected during a 16-year period (2001-2016) and included hairs and scales plucked from the lesions periphery. All samples were inoculated in Sabouraud dextrose agar supplemented with cycloheximide and chloramphenicol, incubated for 21 days at 28ºC and observed daily for the presence of dermatophytes. Species identification was performed by micro and macroscopic examination. Results: In spite of presenting dermatophytosis characteristic lesions, only 185 (9.2%) of the dogs were tested positive for the presence of dermatophytes, with Microsporum canis (53%), Trichophyton mentagrophytes (13.5%) and Microsporum gypseum (7.6%) being the most frequently identified pathogens. Regarding cats, the percentage of positive animals was higher (160 animals, 19.98%). In these cases, M. canis (73.8%), T. mentagrophytes (11.3%) and Microsporum nanum (4.4%) were the most frequently identified species. Conclusions: Results from this long-term study are in agreement with previous studies and confirm the importance of pets as major vectors for human dermatophytosis. Considering the “One Health” concept and the strong relations between humans and pets, growing globalization, reduction of restriction criteria for the circulation of people and goods and dermatophytes ubiquity, there is a clear necessity for conducting more comprehensive epidemiological studies and for developing rapid and precise diagnostic methods, aiming at contributing for managing programs and reducing zoonotic transmission to humans. Keywords: Dermatophytosis; Tricophyton; Microsporum; Epidermophyton; zoonotic Acknowledgments: Work was supported by the Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon (Project UID/CVT/00276/2013).

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Poster 99

The experience of forensic pathology of the Laboratory of Pathological Anatomy of the Faculty of Veterinary Medicine, University of Lisbon (2015-2018) Carvalho S, Pissarra H, Afonso F, Luís MR, Correia J, Ferreira da Silva J, Peleteiro MC CIISA, Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Av. Universidade Técnica, 1300-477 Lisbon, Portugal Background: With the implementation of new legal rules in Portugal regarding animal abuse, the Portuguese Courts of Law soon had to identify which institutions could provide specialized and scientific support for decision making. The Laboratory of Pathological Anatomy (LPA) of the Faculty of Veterinary Medicine, University of Lisbon, has been asked to perform laboratory investigation, mostly necropsies and the respective complementary exams, in order to identify possible situations of crime requested by Courts of Law located within the Lisbon area and further south up to Odemira (Alentejo). Methods: The total of forensic necropsies performed was retrieved from the archives of the LPA ranging from 2015 to the middle of 2018. Only cases for which a final diagnosis was issued were considered, even if the cause of death was undetermined. Results: One hundred and fifty eight cases were included in this study. The Court of Law that required a larger number of necropsies was Setúbal (37%). Dogs were by far the most common species necropsied (73%). The most common situation was unnatural death, partly accidental (11%) and partly premeditated (28%), caused by poisoning, drowning, trauma and gunshot. Conclusion: A new challenge has arisen within the scope of the activities of the veterinary pathologist, creating a specialization that most developed countries seek in order to prevent and punish animal abuse. Although sometimes gruesome and difficult to perform, forensic necropsies are also challenging. The fact that a large number of the necropsies performed in the LPA proved situations of animal abuse is an incentive to go on helping Justice to be fairly applied. Keywords: Forensic Veterinary Pathology, Necropsy, Animal Abuse, Portugal Funding Acknowledgements: Work supported by the FCT project UID/CVT/276/2013

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Poster 100

Chemical hazards screening in dry fermented sausages from Mediterranean influence countries

Fernandes MJ1, Alves SP1, Alfaia CM1, Skrbic BD2, Zivancev JR2, Bessa RJB1, Fraqueza MJ1

1. CIISA - Centro de Investigacao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Universidade de Lisboa, 1300-477 Lisboa; 2. University of Novi Sad, Faculty of Technology, 21000 Novi Sad, Serbia

Chemical compounds such as polycyclic aromatic hydrocarbons (PAHs), biogenic amines (BAs) and heavy metals are hazards identified and related with meat products. The introduction of these different compounds in dry fermented sausages (DFS) could be linked to raw materials and auxiliary ingredients used, to a specific step of their technological process, or by environmental contamination. This work aims to evaluate the presence of chemical hazards (PAHs, BAs and heavy elements) on commercially-available dry fermented sausages from different origins and provide preventive recommendations to assure products safety. Samples (n=20) were purchased at retail level in different markets of Lisbon (Portugal) and Novi Sad (Serbia). The levels of PAHs were determined by gas- chromatography coupled to mass spectrometry (GC–MS) while BAs and heavy elements were analyzed using reversed-phase high performance liquid chromatography (RP-HPLC) and atomic absorption/emission spectrometry, respectively. The most abundant PAHs in DFS were those with low molecular weight, comprising more than 90% of total PAHs. Serbian and most of the Portuguese DFS well meet the EU legislation for the PAHs content. The levels of BAs in DFS from distinct origin (Portugal and Serbia) show a wide variation, however, the major BAs found were tyramine (32.6–206 mg/kg and 5.0–246 mg/kg, respectively) and putrescine (3.5–401 mg/kg and 1.4–291 mg/kg, respectively) followed by cadaverine (0.6–202 mg/kg and 1.6–291 mg/kg, respectively). Histamine never reached considerable levels in both DFS. Contents of heavy elements in DFS from Portuguese and Serbian markets indicated that these potential hazards were present but at low levels. The most prevalent heavy element in DFS from both origins was lead (0.01–0.34 mg/kg). In summary, the data demonstrated that the health risks derived from the intakes of these chemical compounds present in both Portuguese and Serbian DFS is not a major concern for consumers. Nonetheless, additional research still has to be done regarding the selection of autochthonous starters and smoking step optimization to minimize the contents of potential toxic PAHs and BAs in dry meat sausages. Keywords: Dry fermented sausage; polycyclic aromatic hydrocarbons; biogenic amines; minerals; safety. Funding Acknowledgements: This study was supported by the Ministry of Education, Science and Technology Development of the Republic of Serbia through Project No. 172050, by Fundacao para a Ciencia e a Tecnologia (FCT, Lisbon, Portugal) through CIISA project (UID/CVT/00276/2013), Project Agreement FCT – 6818 and by PTDC/AGR-ALI/119075/2010 and by Ministerio da Agricultura, do Mar, do Ambiente e do Ordenamento do Territorio through PRODER-PA Project No. 13017. Individual fellowship to SPA (SFRH/BPD/76836/2011) from FCT is gratefully acknowledged.

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Poster 101

The ability of Ruminococcus albus and Ruminococcus flavefaciens to share important components of their cellulosome systems Andre LA1, Pires VMR1, Fontes CMGA1,2

1. CIISA – Faculdade de Medicina Veterinaria, ULisboa, Polo Universitario do Alto da Ajuda, Avenida da Universidade Tecnica, 1300-477 Lisboa, Portugal; 2. NZYTech genes & enzymes, Estrada do Paco do Lumiar, 1649-038 Lisboa, Portugal Background: Within the gastrointestinal tract of herbivorous animals, the process of plant fibre degradation by commensal bacteria is orchestrated by an exquisite system called cellulosome. Ruminococcus flavefaciens and Ruminococcus albus are both anaerobic cellulolytic bacteria that colonize the rumen of such animals. Interactions between cohesins and dockerins are responsible for cellulosome assembly. Cohesin-dockerin specificity and organizational flexibility confer stability but also flexibility in adapting the cellulosome to different environmental contexts. Moreover, the complexity and architecture of these systems may differ according to species. R. flavefaciens, for example, has one of the most complex cellulosomes currently known. Intriguingly, several R. albus enzymes contain C-terminal dockerin domains, although the genome sequence of this bacterium revealed no apparent sequence encoding cohesin modules. This study aims to understand if dockerins from R. albus and R. flavefaciens share similar cohesin specificities. Methods: The genes encoding 48 dockerins from R. albus and R. flavefaciens and 8 cohesins from R. flavefaciens were isolated through PCR and cloned into the bacterial expression vector pHTP2. The resulting 56 recombinant proteins were expressed in Escherichia coli and purified through Immobilized Metal-Affinity Chromatography. The interactions between the 8 cohesins versus the 48 dockerins (48 x 8 = 384) were explored though native acrylamide gel electrophoresis. Results: A significant fraction of the 56 rumen modules were expressed in the soluble form in E. coli. Data will be presented describing the cohesin-dockerin specificities of the different modules. Conclusions: A thorough understanding of the cellulosome components is crucial in order to explore the potential of this intricate cellular organelle. The ability of different cellulosome systems to share various of their components will be discussed. Keywords: Dockerin, cellulosome, PCR, recombinant DNA. Funding Acknowledgements: This study acknowledges financial support from Fundacao para a Ciencia e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-MIC/5947/2014.