2nd International Conference on Necrotic Enteritis

July 11-12, 2018Denver, Colorado, USA

Program

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July 11-12, 2018, Denver, Colorado, USA

GENERAL INFORMATION

Welcome to the 2nd International Conference on Necrotic Enteritis.

We are pleased to welcome our national and international attendees composed of researchers, graduate students, pharmaceutical company professionals, industry professionals and stakeholders interested in gaining knowledge about the production of poultry with less/no antibiotics. We know you will find much of interest in the program including the following:

Meeting Highlights

Guest Speakers

• Dr. Rob Moore, Royal Melbourne Institute of Technology, Australia • Dr. Joan Smyth, University of Connecticut • Dr. Micheal Kogut, ARS-USDA • Dr. Filip Van Immerseel, Ghent University Belgium • Dr. Allen Byrd, Diamond V • Dr. Todd Applegate and Cristiano Bortoluzzi, University of Georgia • Dr. Marie Archambault, University of Montreal

Student Poster Session

Graduate students participating in the poster competition will present their posters on Wednesday, July 11 from 10:15 am to 11:15 am in the South Convention Lobby at the Sheraton. The winners of the competition will be announced at the closing dinner, Thursday, July 12 at 6:00 pm in the Sheraton’s Grand Ballroom 1.

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Closing Dinner

We have a fantastic dinner full of western USA flavors that is included in the attendee registration fee. Meeting attendees who are interested in bringing a guest to July 12 dinner can purchase tickets for $20 at the Registration Desk on July 11 and until noon on July 12.

Thank you to our Sponsors

This conference would not be possible without the generous support of our sponsors. Sponsors are recognized throughout the conference on signage, in the printed program and on posters and banners. Many thanks to the individuals who worked to make the sponsorships possible.

Registration

The Registration Desk is located in the South Convention Lobby at the Denver Sheraton Hotel. The desk will be open as follows:

Tuesday, July 10 3:00 pm to 6:00 pm

Wednesday, July 11 7:00 am to 4:30 pm

Thursday, July 12 7:00 am to 4:30 pm

Poster Information

1. Poster Dimension: 35.4 inches (90cm) wide by 46.5 inches (118cm) tall.

2. The poster area is the South Convention Lobby in the Denver Sheraton Hotel. Posters must be up by 7:30 am on July 11. Scheduled time to put posters on the boards is Tuesday, July 10 from 3:00 to 6:00 pm and Wednesday July 11 from 7:00 to 7:30 am. The posters are numbered in the program and presenters should find the corresponding number on the poster boards to place their poster. Please check with the registration desk to find the placement of any posters that are not listed in the program. Posters can be removed at 4:30 pm on July 12.

3. Student posters will be identified by a “Golden Chicken” sign. Get the sign at the registration desk. All student posters in the competition must have the sign with their poster.

4. Poster Session Wednesday, July 11 10:15 to 11:15 am Student Poster Competition and poster viewing.

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A Special Thanks to the Scientific Committee

Dr. Martine Boulianne Faculte De Medecine Veterinaire Université de Montréal Saint -Hyancinthe, Quebec Canada martine.boulianne@umontreal.ca Dr. Charles L. Hofacre Southern Poultry Research Group Watkinsville, Georgia United States clhofacre@thesprgroup.com Dr. Rob Moore Biosciences and Food Technology School of Science Cluster RMIT University, GPO Box 2476 Melbourne VIC 3001 Australia rob.moore@rmit.edu.au Dr. Filip Van Immerseel Ghent University Faculty of Veterinary Medicine Department of Pathology, Bacteriology and Poultry Diseases Salisburylaan 133, D5 ingang 78 9820 Merelbeke Belgium filip.vanimmerseel@ugent.be Dr. Audrey McElroy Texas A&M University Department of Poultry Science 107E Kleber Center, 2472 TAMU College Station TX 77842 amcelroy@tamu.edu Dr. Marie Archambault Faculté de médecine vétérinaire Département de pathologie et microbiologie 3200, rue Sicotte St-Hyacinthe, Qc, J2S 2M2 Canada marie.archambault@umontreal.ca

Dr. Francisco Uzal UC Davis 105 West Central Ave San Bernardino, CA 92408 United States fuzal@cahfs.ucdavis.edu Dr. Huyn Lillehoj USDA-ARS Bldg. 1043, Rm. 107, Barc-East Usda, Ars, Barc, ABBl Beltsville, Maryland 20705 United States hyun.lillehoj@ars.usda.gov Dr. Joshua Gong Senior Research Scientist Guelph Research and Development Centre Agriculture and Agri-Food Canada Government of Canada 93 Stone Road West Guelph, Ontario N1G 5C9 Canada Joshua.gong@agr.gc.ca Dr. Julian Rood Monash Biomedicine Discovery Institute Monash University Building 76, Level 1 19 Innovation Walk, Clayton VIC 3800 Australia julian.rood@monash.edu Dr. Karl Pedersen National Veterinary Institute Technical University of Denmark Kemitorvet Building 202, room 2037 2800 Kgs. Lyngby Denmark kape@vet.dtu.dk Dr. Vasileios Tsiouris Unit of Avian Medicine Clinic of Farm Animals School of Veterinary Medicine Faculty of Health Sciences Aristotle University, Thessaloniki Greece Biltsiou@vet.auth.gr

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PROGRAM

SHERATON DENVER DOWNTOWN, GRAND BALLROOM 2

July 11th 2018

7:00 Breakfast courtesy of Zoetis, Grand Ballroom 1

8:00 Welcome: Charles Hofacre and Martine Boulianne

8:10 Opening note: John Smith, President AAAP

8:30 Guest speaker: Genomics overview on Clostridium perfringens Rob Moore, Royal Melbourne Institute of Technology, Australia

Biology of Clostridium perfringens

9:15 Proteogenomic analysis of netB positive non-disease producing and netB positive necrotic enteritis producing Clostridium perfringens strains. Neha Mishra, University of Connecticut

9:30 Challenges with different Clostridium perfringens strains alter the expression of genes encoding proteins for apoptosis, mucin production and tight junction in broilers. Shu-Biao Wu, University of New England

9:45 The biofilm of Clostridium perfringens. Marie Archambault, University of Montreal

10:00 Antimicrobial peptides secreted by Clostridium perfringens. Martine Boulianne, University de Montreal

10:15 Break and Posters session (for graduate students competition) in South Convention Lobby.

Pathogenesis and virulence of Clostridium perfringens

11:15 Guest speaker: Understanding the pathogenesis of Clostridium perfringens. Joan Smyth, University of Connecticut

11:45 Comparison of pathogenicity of several Clostridium perfringens strains with Eimeria co-infection in inducing necrotic enteritis lesions in broiler chickens. Charles Li, ARS-USDA

Noon Lunch courtesy of Evonik, Grand Ballroom 1.

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Pathologies caused by Clostridium perfringens

1:15 Pathology and diagnosis of necrotic enteritis of chickens. Francisco Uzal, California Animal Health and Food Safety Lab, University of California, Davis

1:30 Non-enteric lesions of necrotic enteritis in broiler chickens. H. L. Shivaprasad, University of California, Davis

Epidemiology of necrotic enteritis

1:45 Industry-wide survey of Clostridium perfringens Type A populations in US broilers and turkeys. Dan Karunakaran, Arm & Hammer

2:00 Necrotic enteritis and toxin analysis of Clostridium perfringens isolated from turkeys in California. H. L. Shivaprasad, University of California, Davis

2:15 Persistence of a Clostridium perfringens isolate in a poultry farm over a three-year period. Audrey Charlebois, University of Montreal

2:30 An epidemiological approach to studying the co-occurrence of Eimeria oocysts and Clostridium perfringens spores in litter from commercial broiler farms. Mark Jenkins, Animal Parasitic Diseases Laboratory, NEA, ARS, USDA

2:45 Beverage Break courtesy of Phibro, South Convention Lobby.

Immunity and Vaccination

3:15 Guest speaker: The role of gut immunity in the prevention of necrotic enteritis. Micheal Kogut, Agricultural Research Service-United States Department of Agriculture

4:00 Does early exposure to Clostridium perfringens provide immunity against an oral Clostridium perfringens challenge at 17 days in a necrotic enteritis challenge model? Stephen Davis, Colorado Quality Research, Inc.

4:15 Attenuated Salmonella delivering three clostridial antigens to control necrotic enteritis. Kenneth Roland, Arizona State University

4:30 Plant-based vaccine to prevent necrotic enteritis in chickens. Amanda Tafoya, Arizona State University

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July 12th 2018

7:00 Breakfast courtesy of Zoetis, Grand Ballroom 1

Raising antibiotic free chickens

8:00 Guest speaker: Controlling the necrotic enteritis threat without antibiotics, a European experience Filip Van Immerseel, Ghent University, Belgium

8:45 Life Without Antibiotics: Field Lessons Learned the Hard Way. Timothy Cummings, Zoetis

Necrotic enteritis experimental models in research

9:15 Guest speaker: Necrotic enteritis: the pros and cons of experimental models. Allen Byrd, Diamond V

9:55 Effects of salmonella exposure times in a necrotic enteritis challenge model. Sam Hendrix, Colorado Quality Research

10:15 Beverage Break courtesy of Phibro, South Convention Lobby.

Control of necrotic enteritis with additives:

10:30 From in vitro to in vivo, an approach to assess organic acids and botanicals on necrotic enteritis. Ester Grilli, Dimevet, University of Bologna, Italy; Vetagro Inc.

11:00 Comparing a feed and drinking water application of butyric acid in broilers challenged with necrotic enteritis. Haci Bayir, University of Georgia

11:15 Probiotic, 1-monglycerides, and feed acidification improve performance and reduce necrotic enteritis when fed to broiler chickens challenged with Clostridium perfringens. Greg Mathis, Southern Poultry Research, Inc.

11:30 Effect of replacing in-feed antibiotics with synergistic organic acids, with or without trace minerals and/or water acidification, on growth performance and health of broiler chickens under a Clostridium perfringens type A challenge. Yanming Han, Trouw Nutrition Agresearch

11:45 Effect of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative status of broiler chickens under Clostridium perfringens type A challenge. Yanming Han, Trouw Nutrition R&D

Noon Lunch courtesy of CHR Hansen, Grand Ballroom 1.

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1:00 Evaluation of different doses of a novel Bacillus multi-strain DFM to control Clostridium perfringens induced necrotic enteritis (NE) in broiler chickens. Alfred Blanch, Chr. Hansen, A/S

1:15 Comparative efficacy of a novel multi-strain Bacillus-based DFM and each one of its single strains for the control of necrotic enteritis caused by Clostridium perfringens in broiler chickens. Dorthe Sandvang, Chr. Hansen, A/S

1:30 Evaluating the performance of Bacillus subtilis DSM 32315 under different necrotic enteritis models in broiler chickens. Adebayo Sokale, Evonik Corporation, Kennesaw, GA, USA

1:45 Effect of dietary supplementation of Bacillus subtilis DSM 32315 on the performance and intestinal microbiota of broiler chickens under Clostridium perfringens challenge. Cristiano Bortoluzzi, University of Georgia

2:00 Synbiotic (probiotic + prebiotic) as a potential alternative to ionophore to alleviate the effects of coccidiosis and necrotic enteritis in broiler chickens. G. Raj Murugesan, BIOMIN America Inc.

2:15 Use of Alquermold Natural as an effective manner to control Clostridium perfringens induced necrotic enteritis in poultry. Elvira Cano, Biovet, S.A.

2:30 Effects of valeric acid glycerides on performance, intestinal morphology, and protective effects during necrotic enteritis challenge. Jacob Lum, Perstorp Feed and Food

2:45 Necrotic enteritis prevention and control in broilers: The effects of an animal-use only antibiotic compared to shared-class antibiotics. Jaime Ruiz, Poultry Technical Consultant - Elanco Animal Health

3:00 Beverage Break courtesy of Phibro, South Convention Lobby.

3:15 Nutritional modifications to decrease risks of necrotic enteritis. Todd Applegate and Cristiano Bortoluzzi, University of Georgia

3:45 Antimicrobial effect of selected yeast fractions on Clostridium perfringens. Giuseppina Avantaggiato, ISPA-CNR, Italy

4:00 Efficacy of trace mineral-amino acid complexes in a necrotic enteritis challenge model. Charles Hofacre, Southern Poultry Research Group, Inc.

4:15 The use of in-feed mineral supplementation to control the impact of Clostridium perfringens on performance, lesion scores and necrotic enteritis associated mortality in various necrotic enteritis challenge models in broilers. Daniel Moore, Colorado Quality Research

4:30 Closing remarks

6:00 Banquet courtesy of Elanco and best graduate student poster awards, Grand Ballroom 1.

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POSTERS

Sheraton Denver Downtown, South Convention Lobby

Biology of Clostridium perfringens

1: Characterization of Clostridium perfringens netB+tpel+ Type A Strains Isolated from Necrotic Enteritis-Afflicted Broiler Chickens. Xianghe Yan, Beltsville Agricultural Research Center, ARS/USDA

2: Molecular characterization of type IV pilus operons in chicken Clostridium perfringens strains reveals a highly conserved organizational structure and a low level of gene sequence conservation. Marie-Lou Gaucher, University de Montreal

Pathogenesis and virulence of Clostridium perfringens

3: Viability of the Greater wax moth larvae (Galleria mellonella) as a model organism for the investigation of Clostridium perfringens associated disease (CPAD). Sammy Kay, Arden Biotechnology

Epidemiology of necrotic enteritis

4: Pulsed field gel electrophoresis and toxin type comparison of Clostridium perfringens isolates from birds with varied health statuses. Joseph Brown, Arden Biotechnology

5: Clostridium perfringens populations in the gastrointestinal tracts of healthy broilers in Asia. Evan Hutchison, Arm and Hammer

6: Prevalence of Clostridium perfringens isolates containing the netB gene in UK environmental sources. Joseph R. Edwards, Arden Biotechnology

Risk factors and their control

7: High body weight gain is a risk factor for the development and severity of necrotic enteritis lesions. Evelien Dierick, Ghent University

8: Interaction of necrotic enteritis severity and live salmonella vaccination on salmonella prevalence and load in broilers. Manuel Da Costa, Zoetis

9: Effect of selected Eimeria maxima strains on pathology and subsequent development of necrotic enteritis in a model system. Juan Latorre, University of Arkansas, Department of Poultry Science

10: Poultry feed linked with a clinical case of chicken necrotic enteritis in Quebec, Canada. A. Charlebois, E. Parent, D. Venne, M. Boulianne and M. Archambault, University of Montreal

11: Presence of NetB positive isolates of Clostridium perfringens in gizzards of broiler chickens at slaughter. A. Charlebois, A. Hayer, S. Lemire, M. Boulianne, and M. Archambault, University of Montreal

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Diagnostic of Necrotic Enteritis

12: Optimization of an in vitro hemolysis assay for the detection of Clostridium perfringens NetB toxin in culture supernatant. Lore Van Damme, Ghent University

13: Evaluation of Media for the Enumeration of Clostridium perfringens from Poultry Feces. Martina Husta, Ghent University

Control of necrotic enteritis with additives

14: Evaluation of different doses of a novel Bacillus multi-strain DFM to control Clostridium perfringens induced necrotic enteritis (NE) in broiler chickens. Alfred Blanch, Chr. Hansen, A/S

15: Comparative efficacy of Alquermold Natural in a necrotic enteritis model as a treatment in drinking water and as a preventative in feed. C. Domenech, C.L. Hofacre, C.S. Hofacre, J. Pié, E. Cano, A. Tesouro, BioVet

16: In vitro evaluation of the potential of lactic acid bacteria isolated from chicken ceca to inhibit Clostridium perfringens. Nuria Vieco-Saiz, Phileo Lesaffre Animal Care

17: Effects of NeutraPath™ with or without nicarbazin on the performance of broiler chickens during simulated field challenge conditions. Hongyu Xue, Amlan International

18: In vitro comparison of the growth inhibition concentration of two agents (essential oil blend and antibiotic) against 8 probiotics and Clostridium perfringens from monogastric gut flora. Isabelle Deuve Riou, Phytosynthese

19: Protective Effect for Broiler Chickens of Valerins and the Antibiotic BMD in Necrotic Enteritis Challenge Model. Charles Hofacre, Southern Poultry Research Group, Inc.

20: Evaluation of the in vitro and in vivo antibacterial effects of NeutraPath™. Dongping Wang, Amlan International

2ND INTERNATIONALCONFERENCE ON NECROTIC ENTERITIS

July 11-12, 2018 Denver, Colorado USA

ABSTRACTS

July 11th 2018 GUEST SPEAKER

Genomics overview of necrotic enteritis causing isolates of Clostridium perfringens

Jake Lacey1,2,3, Theodore Allnutt4, Ben Wade4, Anthony Keyburn2,5, Honglei Chen5, Ben Vezina6, Thi Thu Hao Van6, Torsten Seemann3, Thomas Stent1,2, Xiaoyan Han1, Julian Rood1,2, Priscilla

Johanesen1, Dena Lyras1, and Robert Moore1,2,6

1 Department of Microbiology, Monash University, Clayton, Australia 2 Poultry Cooperative Research Centre, Armidale, Australia

3 Doherty Institute, University of Melbourne, Melbourne, Australia 4School of Medicine, Deakin University, Waurn Ponds, Australia

5Australian Animal Health Laboratory, CSIRO, Geelong, Australia 6School of Science, RMIT University, Bundoora, Australia

Introduction C. perfringens isolates that express NetB toxin (Type G isolates) are able to reliably and reproducibly induce necrotic enteritis in suitably predisposed and challenged broiler chickens. NetB is encoded on a large conjugative plasmid and chicken isolates of C. perfringens are often found to carry a number of other large conjugative plasmids. Plasmid carriage appears to be essential for virulence but the contribution of the genomic background is less well understood. We determined the genomic sequence of chicken-derived C. perfringens isolates and compared them to non-chicken isolates in an effort to gain some understanding of the variation present within the C. perfringens strains circulating in chickens and determine whether there is any indication of host species specificity of C. perfringens isolates. Materials and Methods The genomic sequences of a collection of C. perfringens isolates, of known virulence in a necrotic enteritis disease induction model, were compared and contrasted with the genomes of non-pathogenic chicken isolates and non-chicken isolates. We used gene content and SNP analysis of the core genome to investigate the evolutionary relatedness of isolates. Results Phylogenetically distinct clades of necrotic enteritis causing C. perfringens were identified by differential carriage of variable chromosomal regions. The functions encoded by the variable regions included capsule production, adhesion, and metabolism. Phage integrations and remnants accounted for substantial proportions of the inter-isolate genomic differences. The pathogenic isolate fell into three distinct clades. Interestingly, the clade structure, as identified by chromosomal analysis also reflected the combinations of large conjugative plasmids that were carried by the isolates. There was no evidence that specific phylogenetic lineages were associated with particular host, although the C. perfringens sequence database is currently dominated by chicken isolates and it would be of value to have a wider representation of isolates from other hosts. Conclusions The genome of C. perfringens has been modified by gene acquisition and loss and there doesn’t appear any reliable chromosomal “signature” that defines a chicken isolate. Carriage of a netB-encoding plasmid remains the best indicator of necrotic enteritis disease-causing potential.

BIOLOGY OF Clostridium perfringens

Proteogenomic analysis of netB positive non-disease producing and netB positive necrotic enteritis producing Clostridium perfringens strains.

Neha Mishra and Joan A. Smyth

Department of Pathobiology and Veterinary Science, University of Connecticut, 61

North Eagleville Road, Storrs, CT 06269, USA. Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The mechanism for virulence of C. perfringens in the context of NE is still under investigation. In 2008, a pore-forming toxin (NetB) was discovered, which was implicated as a major virulence factor for the C. perfringens to cause NE in chickens. However, we have a netB positive strain which produces minimal intestinal pathology, and then only in a small percentage of challenged birds. Proteogenomic analysis was performed to examine the entire proteome and genome of a netB positive non-disease producing and four netB positive NE producing strains of C. perfringens. The proteomics analysis identified 387 secreted and 230 bacterial cell proteins which were produced at least 2 fold greater by NE producing netB+ strains. The proteogenomic method identified 23 candidate virulence factors of C. perfringens in the context of NE. Many of these proteins were related to adhesion, chemotaxis, VirR/VirS regulatory system and NE pathogenicity loci. The genomic analysis showed that the environmental information processing pathway which includes membrane transport, abc transporters, bacterial secretion system and signal transduction were upregulated in the netB positive necrotic enteritis producing strains. These candidate virulence factors should be the subject of future investigations.

Challenges with different Clostridium perfringens strains alter the expression of genes encoding proteins for apoptosis, mucin production and tight junction in broilers

K. Gharib Naseri, S.K Kheravii, C. Keerqin, R.A. Swick, M. Choct, N. Morgan, and S.-B Wu*

School of Environmental and Rural Science, University of New England, Armidale, Australia, 2350 The impact of two different strains of Clostridium perfringens (Cp) (NE-18 and NE-36) on mRNA expression of genes encoding proteins involved in cell apoptosis, intestinal tight junction and production of immunoglobulins and mucin in broiler chickens was investigated. A total of 468 male Ross 308 broilers was assigned to a 2 x 3 factorial arrangement of treatments. Factors were antibiotics (+/-), necrotic enteritis (NE) and challenge (-/ NE-18/ NE-36). A significant interaction between antibiotic treatment and challenge was observed on the expression of gene CASP3 (P < 0.05); NE-18 upregulated CASP3 only in the presence of antibiotics, while NE-36 upregulated this gene regardless of antibiotic supplementation. Interestingly, both NE strains upregulated CASP8 compared to the control treatment, but NE-36 showed higher CASP8 expression than observed with the NE-18 treatment (P < 0.001). It was shown that challenge significantly downregulated OCLD (P < 0.001), ZO-1 (P < 0.05) and MUC2 (P < 0.001). CLDN1 was significantly upregulated (P < 0.001) by both the NE-36 and NE-18 challenge, but NE-36 showing comparatively greater effects compared to NE-18. A significant antibiotic × challenge interaction on MUC5ac was observed (P < 0.05), where both NE- 18 and NE-36 upregulated MUS5ac when antibiotics was applied, but only NE-36 showed such upregulation in the absence of antibiotics. IgG and IgM were significantly downregulated (P < 0.001) by both strains of Cp. These results indicate that NE challenge alters the expression of genes investigated and different strains of Cp have varying impacts, which has a direct impact on severity of the disease. * Author for correspondence and presentation.

GUEST SPEAKER The biofilm of Clostridium perfringens

Marie Archambault,

Swine and Poultry Infectious Diseases Research Center (CRIPA), Département de pathologie et

microbiologie vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, St-Hyacinthe, Québec, Canada J2S 2M2

Clostridium perfringens is an opportunistic pathogen that can cause necrotic enteritis in

poultry, food poisoning in humans and various enterotoxemia in other animal species. Recently, it was shown to form mono-species biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. Because very little is known on the biofilm of C. perfringens, this topic will be the focus of this presentation.

Morphology examination of the biofilm of C. perfringens revealed flat and mushroom-like biofilms. Matrix composition analysis showed the presence of proteins, beta-1,4 linked polysaccharides and extracellular DNA, but no poly-beta-1,6-N-acetyl-D-glucosamine. Biofilm was also found to protect C. perfringens bacterial cells from exposure to high concentrations of tested antimicrobials. In addition, it was shown to protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite solution was effective on C. perfringens biofilms. Data demonstrates that C. perfringens biofilm is an effective protection mechanism to disinfectants commonly used on farms and in food processing environments.

More recently, to gain insights into the differences between free-living C. perfringens cells and those in biofilms, we used RNA sequencing. In total, 25.7% of genes showed differential expression in the two growth modes; about 12.8% of genes were upregulated and about 12.9% were down-regulated in biofilms. We have shown that 772 genes were significantly differentially expressed between biofilms and planktonic cells from the supernatant of biofilms. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in virulence, energy production, amino acid, nucleotide and carbohydrate metabolism, and in translation and ribosomal structure. Genes up-regulated in biofilm cells were mainly involved in amino acid and carbohydrate metabolism, transcription, inorganic ion metabolism and in defence mechanisms. This data provides new insights into the transcriptomic response of C. perfringens during biofilm formation.

Work is now underway in our laboratory to understand the contribution of biofilm in the

necrotic enteritis disease process.

Antimicrobial peptides secreted by Clostridium perfringens.

Martine Boulianne and Nicolas Deslauriers

Chair in Poultry Research, Centre de Recherche en Infectiologie Porcine et Avicole (CRIPA), Faculté de médecine vétérinaire, University of Montreal, Canada

Numerous risk factors, such as coccidiosis and poorly digestible cereals, have been associated with NE outbreaks, all of which favor Clostridium perfringens (Cp) colonization. Interestingly, our laboratory has observed that, depending on the predisposing factor used, i.e. coccidia or high wheat diet, a different Cp strain took over in a mixed Cp strains model. Cp must compete against an established bacterial community in order to proliferate. Indeed, Cp is able to produce antimicrobial peptides called bacteriocins, which inhibit the growth of closely related strains. Perfrin, a chromosomally-encoded bacteriocin has been identified in pathogenic netB-positive NE-causing strains and is thought to play an important role. This might explain why during an outbreak a single pathogenic strain will become dominant. We compared the pathogenicity of various strains with our ligated intestinal loop model. Their ability to inhibit the growth of other Cp strains was later tested with an agar spot assay where antimicrobial activity is shown by the absence of growth of the bacterial lawn around the colony or on the location of the so-called spot. Numerous strains including a commensal one, showed a marked antimicrobial activity in vitro, but many were negative for perfrin. This demonstrates that bacteriocin production is not only a prerogative of pathogenic Cp strains. Indeed, the action of many probiotic bacteria on competitive exclusion is exerted through the secretion of bacteriocins. Overall, these antimicrobial peptides may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains such as pathogens, modulate the microbiota composition and even appear to influence the host immune system. The use of purified bacteriocins could therefore be a potential alternative to conventional antibiotic use in the control of necrotic enteritis.

PATHOGENESIS AND VIRULENCE OF Clostridium perfringens

GUEST SPEAKER Understanding the pathogenesis of necrotic enteritis caused by Clostridium perfringens

Joan A Smyth

Department of Pathobiology and Veterinary Science, University of Connecticut

The disease necrotic enteritis (NE) caused by Clostridium perfringens was first recognized in 1961 and it was long considered that the disease developed when conditions in the gut favored proliferation of C. perfringens and elaboration of alpha toxin in the lumen of the small intestine, since alpha toxin infused directly into the duodenum had been shown to significantly damage the mucosa of the small intestine. In 2006, the role of alpha toxin in disease development was called into question by Keyburn et al. based on their finding that alpha toxin knockout mutants could cause necrotic enteritis. In 2008, the same group reported the discovery of a new toxin NetB (necrotic enteritis B -like toxin) which based on their studies, was a critical virulence factor for development of disease in that NetB knockout mutants failed to produce disease, re-complementation with netB restored disease producing capability, and the gene was only identified in strains isolated from chickens with NE. After this important discovery, researchers worldwide have been examining the role of NetB in the development of necrotic enteritis, and its potential as a vaccine target. There are many studies now published on the role of NetB, which have unfortunately produced conflicting results; some support the role of NetB in the pathogenesis of NE, but others report that some netB negative strains produce NE in experimental models. Result to date with NetB targeted vaccines have produced at best only partial protection but at this time, it is not clear if the disappointing results are due to universal difficulty in eliciting a robust enough response to protect a mucosal surface. However, it is noteworthy that one netB positive strain that produces active NetB toxin was unable to produce disease in an experimental model. Several other candidate virulence factors and immunogenic targets such as a hypothetical protein which has B cell epitopes, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, fructose-1,6-bisphosphate aldolase, to mention a few, have been identified by other groups. Once again, vaccines produced against some of these proteins have at best, produced only partial protection. Relatively little consideration has been given to date, to non-toxin bacterial factors such as ability of C. perfringens strains to degrade mucin and as to whether or not C. perfringens needs to be able to adhere to the mucosa in order to be able to produce disease. A few studies have examined for an association between disease producing capability and ability to adhere, and an association was found between disease producing ability and ability to adhere to some extracellular matrix molecules, and more recent genomic and proteomic studies have confirmed the presence of genes and proteins predicted to be involved in adhesion. However, the finding that C. perfringens adhered predominantly to extracellular matrix components, raises the question as to whether the C. perfringens are simply adhering to the matrix exposed by toxin-induced death of epithelial cells. Both original and more recent data as they pertain to potential processes occurring at both the bacterial and host levels that could lead to disease, will be considered.

Comparison of pathogenicity of several Clostridium perfringens strains with Eimeria maxima co-infection in inducing necrotic enteritis lesions in broiler chickens

Charles Li1, Liheng Liu1, Xianghe Yan1, Hyun Lillehoj1, Zhifeng Sun1, Hongyan Zhao1, Zhezi

Xianyu1, Stephen Melville2 and Youngsub Lee1

1Beltsville Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, MD, USA; 2Department of Biological Sciences, Virginia Tech, Blacksburg,

Virginia, USA.

Necrotic enteritis (NE) and coccidiosis, caused by Clostridium perfringens (CP) Type A strains and Eimeria spp., are two important enteric infectious diseases in poultry, responsible for the annual loss of 6 and 3 billion dollars to worldwide poultry industry, respectively. The virulence of CP strains is important in inducing NE, but less elucidated in dual infections. In this study, five strains from different sources and genetic backgrounds, N11 (isolate from healthy chicken intestine), Strain 13 (natural isolate from soil), SM101 (isolate from food poisoning), Del1 (field netB+ CP strain isolate from NE outbreak in a chicken farm) and Tpel 17 (field netB+tpel+ CP strain isolate from NE outbreak in a chicken farm) were compared with respect to pathogenicity when co-infected with 5000 oocytes of Eimeria maxima (EM) in 2-wk-old broiler chickens. In the birds infected by EM followed by 2 consecutive oral administrations of CP (1~ 2 x 109 cfu of CP per chicken, EM/2x CP), 40% and 10% mortalities were observed in the groups of EM / 2x CP (Tpel 17), and Eimeria / 2x CP (Del1) at Day 2 post CP infection, respectively. No mortality was observed in EM alone or CP alone groups. In the birds infected with EM followed by one time dosing of CP (EM/1x CP), only the group of EM/1x CP (Tpel 17) had a mortality of 25%, and had severe lesion scores. In all the groups of EM/CP dual infections, lesions were broadly observed, no matter the source of these strains. This study suggested that the Eimeria pre-infection may destroy the intestine epithelial integrity, and provide necessary conditions for C. perfringens outgrowth as a predisposing factor for NE induction. Strain with NetB and Tpel toxins (Tpel 17) is a very virulent strain, causes very severe lesions, and should be used as a good challenge strain when co-infected with Eimeria spp. in studies of CP pathogenesis or development of alternatives to antibiotics.

PATHOLOGIES CAUSED BY Clostridium perfringens

Pathology and diagnosis of necrotic enteritis of chickens

Francisco A. Uzal1,Carlos Daniel Gornatti Churria2, Gabriel Sentíes Cué1, HL Shivaprasad

1

1California Animal Health and Food Safety Laboratory System, School of Medicine, UC Davis;

2Faculty of Veterinary Sciences, National Univeristy of La Plata, Argentina Clostridium perfringens necrotic enteritis (NE) diagnosis in poultry can be challenging, because this organism is usually found in normal chickens and also because the pathology of the disease overlaps with that of other intestinal infections. The diagnostic features of 65 cases of NE in chickens, that were submitted to the Turlock, Tulare and San Bernardino branches of the California Animal Health and Food Safety Laboratory, between 2004 and 2013 are described. These birds had gross lesions in different parts of the intestine, but these were most common in the jejunum. Microscopic intestinal lesions were observed most frequently in the jejunum-ileum (61%), duodenum (43%) and the ceca (17%). Microscopic lesions consisted necrosis of the intestinal mucosa, which in some cases extended to the sub-mucosa and muscularis. Heterophils were the dominant inflammatory cells in the initial stages of the disease, but mononuclear cells are also present in more chronic lesions. Large numbers of gram positive rods, usually grouped in clusters, were seen associated with the necrotic lesions. Immunohistochemistry for C. perfringens performed in small intestine of 10 of the birds with NE revealed the presence of strongly positive intralesional rods in all the birds tested by this technique. C. perfringens type A was isolated from the 24 (100%) cases in which anaerobic culture of the intestine was attempted. Seven (29%) of these 24 isolates carried the gene encoding for beta 2 toxin, while 2 (8%) each of those isolates were positive for the genes encoding enterotoxin and Net B toxin, respectively. Coccidiosis was diagnosed by fecal floatation and/or histopathology in 50% of the cases and it was the most frequent predisposing factor. Diagnosis of NE cannot be based on gross examination alone and an acceptable level of certainty should be achieved by combining several diagnostic tests including histopathology, immunohistochemistry and anaerobic culture followed by PCR typing of C. perfringens isolates. Although NetB seems to be associated with many cases of NE around the world, our results suggest that this toxin is not necessary for NE to occur.

Non-enteric lesions of necrotic enteritis in broiler chickens

H. L. Shivaprasad, M. Crespo, F.A. Uzal and S. Stoute

California Animal Health and Food Safety Laboratory System, University of California, Davis

Necrotic enteritis (NE) is a bacterial infection affecting primarily broiler chickens and turkeys, characterized by necrosis of the intestinal mucosa and occasionally by cholangiohepatitis, resulting in increased mortality and/or significant loss of production. NE is caused by Clostridium perfringens, an anaerobic, gram-positive spore-forming rod. C. perfringens has been known to produce at least 20 different extracellular toxins including alpha (CPA), necrotic enteritis toxin B-like (NetB), beta2 (β2), enterotoxin (CPE) and others. Non-enteric lesions of NE in broiler chickens are rare. The objective of this paper is to document extra-intestinal lesions in 28 cases of NE in broilers, occurring between 2007 and 2017 and sub mitted to the California Animal Health and Food Safety laboratory. Clinical signs included loose droppings, depression, reluctance to move and deaths. The age of the chickens ranged from 14 to 70-days with an average of 33 days. Gross and microscopic lesions included necrotizing enteritis (100%), mild to severe necrotizing hepatitis (79 %), cholecystitis (21 %), splenitis (14 %), bursitis of the bursa of Fabricius (24 %) and gizzard erosions (7 %). These lesions were associated with intralesional gram-positive rods which were positive for C. perfringens by immunohistochemistry. C. perfringens was isolated from most of the livers (92 %), intestine (73 %) and 2/2 gall bladders. Concurrent diseases in the chickens included coccidiosis (59 %), bursal lymphoid depletion (72 %), colibacillosis (38 %), cystic enteritis (17 %), bursal cryptosporidiosis (10 %) and others (3 %). Two isolates of C. perfringens typed by PCR were positive for CPA, NetB and β2, and negative for CPE.

EPIDEMIOLOGY OF NECROTIC ENTERITIS

Industry-wide survey of Clostridium perfringens Type A populations in US broilers and

turkeys.

Dan Karunakaran1, Evan Hutchison1, Tom Rehberger1

1Arm & Hammer

Clostridium perfringens-associated necrotic enteritis (NE) is an economically important disease in the United States poultry industry. As regulatory and consumer demands have limited the use of antibiotic growth promoters in the US, there is fear that NE will spike. Generally, NE rates increase following antibiotic growth promoters (AGP) removal in commercial poultry systems, as observed after AGP ban in Scandinavian countries and then in the European Union in 1995 and 1999, respectively. In order for poultry producers to understand a growing NE challenge, it is becoming important to characterize and track C. perfringens populations within commercial production systems. To this end, the gastrointestinal tract (GIT) of over 4000 broilers was sampled over a three-year period. More than 2,500 C. perfringens strains were isolated and genotyped using RAPD-PCR fingerprinting. Results showed that while many birds had undetectable levels of C. perfringens, the average US broiler GIT harbors 4.4E+4 CFU/g of C. perfringens and that the levels peak at 3 weeks of age, which is consistent with the NE timeline associated with the coccidiosis cycle. The observed C. perfringens populations are highly diverse, breaking into over 200 genotypes by RAPD-typing, although many of these clusters contain a single isolates. Population shifts over time and across age will be discussed as well as the possible role that AGPs play in ever-changing C. perfringens landscape. These results help further our understanding of C. perfringens population dynamics in order to aid in the development of targeted, preventative measures.

Necrotic enteritis and toxin analysis of Clostridium perfringens isolated from turkeys in California

H. L. Shivaprasad1, N. Mishra2, M. Crespo1, J Ochoa1, S. Stoute1 and J. Smyth2

1California Animal Health and Food Safety Laboratory System, University of California, Davis.

2Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs Necrotic enteritis (NE) is an acute bacterial infection of primarily chickens and turkeys characterized by necrosis of the intestinal mucosa resulting in increased mortality. NE is caused by Clostridium perfringens, an anaerobic, gram-positive rod, spore-forming bacterium that has been known to produce at least 20 different extracellular toxins including CPA, NetB, β2-toxin, enterotoxin, TpeL and others. Most of the papers published on NE are in chickens but not in turkeys. The objective of this paper is to summarize the data on the incidence of NE in commercial turkeys in California from 2008 to 2017 and analyze 62 isolates of C. perfringens isolated from the intestine of turkeys with NE for toxins. There were 65 outbreaks of NE in turkeys during this ten-years period and age ranged from 16 to 98 days with an average age of 45 days. Clinically birds were depressed, lethargic with ruffled feathers and experienced rapid death. Mortality ranged from 0, 05 % to 0.5% per day in flocks of about 5000 turkeys per flock. Necropsy revealed distended small intestine with brownish mucoid contents and/or diffuse fibrinonecrotic exudate which sometimes extended deep in to the mucosa. Microscopically there was necrosis of the mucosa with frequent ulcerations and fibrinoheterophilic inflammation associated with numerous gram-positive rods which were positive for C. perfringens by immunohistochemistry. Concurrent diseases in turkeys included coccidiosis (34), hemorrhagic enteritis virus infection (32), crop mycosis (15), colibacillosis (12), aspergillosis (3) and others (3). All 62 isolates of C. perfringens tested were of type A and PCR for toxin genes revealed that 54 (80.6%) were positive for β2 gene and 1 (1.5 %) was positive for cpe gene. None were positive for netB and tpe genes.

Persistence of a Clostridium perfringens isolate in a poultry farm over a three-year period

Audrey Charlebois1, Eric Parent1 Marie-Pierre Létourneau-Montminy2 and Martine Boulianne1

1Chaire en recherche avicole et Centre de Recherche en Infectiologie Porcine et Aviaire (CRIPA), Département de sciences cliniques, Faculté de Médecine Vétérinaire, Université de

Montréal, Saint-Hyacinthe, Canada; 2Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec, Canada

Clostridium perfringens, a commensal of the intestinal tract of many animal species, is also responsible for causing necrotic enteritis (NE), an economically significant poultry disease. C. perfringens is known to survive in the environment for extended periods of time through the formation of spores. These spores have the potential to be transmitted to subsequent flocks. Persistence of a single C. perfringens strain in a poultry farm environment has however been poorly documented. The aim of this study was to compare multiple isolates of C. perfringens collected over time in a single farm with recurrent episodes of necrotic enteritis. Isolates were recovered from the intestines of chickens affected with NE (2014 and 2016 outbreaks) and from healthy chickens (2017), and from environmental samples (2016). PCR characterization of those isolates showed that all sampling groups contained netB-positive isolates except for the environmental samples. Moreover, all environmental isolates were positive for the cna adhesin, whereas other groups had lower numbers of cna-positive isolates. Pulsed field gel electrophoresis analyses showed that one clone was present in every sampling group with the exception of the 2014 outbreak. However, one clone found in the latter group was highly similar, having 94 % similarity with the persistent C. perfringens isolate. This study describes for the first time the persistence of a C. perfringens isolate on a poultry barn over a three-year period.

An epidemiological approach to studying the co-occurrence of Eimeria oocysts and Clostridium perfringens spores in litter from commercial broiler farms.

Mark C. Jenkins

Animal Parasitic Diseases Laboratory, Beltsville

Agricultural Research Center, ARS, USDA, Beltsville The purpose of this study was to examine the co-occurrence of Eimeria oocysts and C. perfringens spores in litter from commercial broiler farms, particularly the levels of viable E. maxima and C. perfringens prior to chick placement. Clostridium perfringens strains were also evaluated for containing netB and alpha toxin genes known to be related to the appearance of necrotic enteritis in chicks less than 3 weeks of age. Litter was collected prior to chick placement (day 0) and at 2 and 4 weeks growout. Eimeria oocysts were isolated for performing E. maxima and non-E.maxima counts by microscopy and for species determination by ITS1 PCR and metagenomics using the Cox1, Cox3, and cytB mitochondrial gene sequences. Eimeria oocysts were also inoculated into susceptible chicks for viability testing. Clostridium spores were isolated for DNA extraction and PCR analysis for C. perfringens and for the netB and alpha toxin genes. Clostridium perfringens positive samples were also evaluated for viability by culturing on selective agar followed by PCR analysis of colonies exhibiting typical Clostridium morphology. Viable Eimeria oocysts and C. perfringens spores were observed in over 50% of poultry houses prior to chick placement. Depending on the coccidiosis control program, numbers of Eimeria oocysts and C. perfringens spores increased over time of growout. These data suggest that litter management, such as crusting off and wind-rowing, may not destroy Eimeria and C. perfringens during down-time between sets of chicks. Studies are underway to compare the levels of viable Eimeria and C. perfringens to chick mortality at 2-3 weeks growout.

IMMUNITY AND VACCINATION

GUEST SPEAKER

The relationship between gut health, the intestinal ménage à trois, and necrotic enteritis

Michael H. Kogut

Southern Plains Agricultural Research Center USDA-ARS

College Station, TX The gastrointestinal tract (GIT), or “gut”, regulates homeostasis of the microbiological, physiological, and physical functions that allows the host to endure infections and other environmental stressors that it encounters. Because the gut has the greatest surface area separating the environmentally exposed lumen and the internal subepithelial tissue, the GIT is constantly exposed to infectious and non-infectious stressors making it an active immune organ containing more resident immune cells than any other organ in the host. The mucosal immune system, a highly regulated network of innate and acquired elements, provides a remarkable ability to respond and modify to these extremely diverse encounters via two distinct functions: the ability to respond to pathobionts (potential pathogenic microbes), invasive pathogens, and microbial products while also maintaining a state of tolerance to the diverse and beneficial commensal intestinal microbes. The development of the different divisions of the immune response has corresponded with the acquisition and maintenance of a symbiotic microbiota. The microbiota trains, stimulates, and functionally adjusts the different features of the immune system. Together the immune system, the microbiota and the host nutritional/metabolic systems form the ‘intestinal ménage à trois’ which provides the maintenance of for optimal gut health. In this presentation, we will detail the role of each component of the intestinal ménage à trois in host defense/pathogenesis of necrotic enteritis (NE). Further, we will provide a perspective on what the removal of antibiotic growth promoters has had on the intestinal ménage à trois, especially the microbiota, which has led to an increase in incidence of NE. Finally, we will discuss the use of alternatives to AGP’s to reduce the incidence of NE by improving gut health by mediating functional changes in the intestinal microbiota.

Does early exposure to Clostridium perfringens provide immunity against an oral Clostridium perfringens challenge at 17 days in a necrotic enteritis challenge model?

Stephen Davis, D. Moore, S. Hendrix

Colorado Quality Research, Inc.

Wellington, CO 80549 It has been reported from commercial broiler field cases of necrotic enteritis, that cases occurring in new farms or recently cleaned out farms with new litter, experience more severe cases of the disease compared to broiler flocks that are placed on the same farms or other farms with used litter in the brood area. A series of floor pen studies were conducted to compare the severity of necrotic enteritis (NE) lesion scores, mortality and performance impacts when day old broiler chicks received various methods of early exposure to Clostridium perfringens (Cp) prior to a severe oral challenge of Clostridium perfringens at 17 days-of-age. Early Cp exposure with used litter, new litter inoculated with Clostridium perfringens and oral gavage were compared to determine if the early Cp exposure would provide immunity protection against the necrotic enteritis challenge model. Results, discussion and conclusions from these studies will be presented.

Plant-based vaccine to prevent necrotic enteritis in chickens

Kenneth L. Roland, Amanda Tafoya, Shyra Wilde, Andrew Diamos, Joseph Hunter and Hugh Mason

The Center for Immunotherapy, Vaccines, and Virotherapy at the Biodesign Institute and

School of Life Sciences, Arizona State University, Tempe, AZ 85287 Necrotic enteritis (NE) is caused by type A strains of Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over 2 billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for disease control. NE strains produce two major toxins, -toxin and NetB. Immune responses against -toxin (Plc) are partially protective, although the toxin has not been demonstrated to play a direct role in NE. The NetB toxin is responsible for the NE-associated symptoms and anti-NetB antibodies are partially protective. We developed an antigen fusion protein combining a non-toxic carboxy-terminal domain of -toxin (PlcC) and a toxoid form of NetB (NetB-W262A) to immunize poultry against NE. We utilized a DNA sequence codon-optimized for plants to enable high expression in a tobacco relative, Nicotiana benthamiana. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity, and used to immunize broiler birds. Immunized birds produced a strong serum IgY response against the plant produced His-PlcC-NetB protein and also against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens. Our results demonstrate that this plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

Attenuated Salmonella delivering three clostridial antigens to control necrotic enteritis.

Amanda Tafoya, Shyra Wilde, Jamie Horsman, Miranda Yousif, Luis Armando Vazquez, Yanlong Jiang and Kenneth L. Roland

The Center for Immunotherapy, Vaccines, and Virotherapy at the Biodesign Institute, Arizona

State University, Tempe, AZ 85287 Necrotic enteritis (NE) is caused by type A Clostridium perfringens strains. The incidence of NE has increased as a result of recent restrictions on use of growth promoting antibiotics in feed. NE strains produce two major toxins, alpha-toxin and NetB. The NetB toxin causes intestinal lesions typical of NE. The role of alpha toxin (encoded by plc) in NE has not been established, but immune responses against it are protective. Fructose-1,6-bisphosphate aldolase (Fba) is a metabolic enzyme secreted by some pathogenic C. perfringens strains. We used a single live attenuated Salmonella vaccine lysis strain to deliver all three C. perfringens antigens. Salmonella vaccine strains with the lysis phenotype are engineered to lyse in host tissues after several rounds of replication. Immunization of broiler birds with the triple-antigen Salmonella lysis strain elicited strong mucosal and cellular responses against all antigens and provided significant protection against NE after challenge with virulent C. perfringens. A lysis strain delivering Fba alone also provided significant protection. We present evidence that Fba resides on the surface of C. perfringens, indicating that it may ‘moonlight’ as an adhesin, possibly explaining its role as a protective antigen.

July 12th 2018 RAISING ANTIBIOTIC FREE CHICKENS

Guest speaker

Controlling the necrotic enteritis threat without antibiotics, a European experience

Filip Van Immerseel, Evelien Dierick, Jill Derix, Lore Van Damme, Martina Husta, Evy Goossens, Richard Ducatelle

Ghent University, Faculty of Veterinary Medicine, Department of Pathology, Bacteriology and

Avian Diseases. Reductions in the use of antibiotics of any kind (antimicrobial growth promoters, therapeutics, ionophores) have forced the poultry industry to develop novel solutions for intestinal health problems that have emerged. Necrotic enteritis is an extreme manifestation of gut health problems related with microbial imbalances, in which harmful pathogens outcompete beneficial microbes in specific segments of the intestinal tract, followed by production of toxins and enzymes that damage host cells. As necrotic enteritis only develops when predisposing factors are present, these are a key target in prophylaxis of necrotic enteritis. High energy diets and coccidiosis are the main predisposing triggers, but a variety of other factors (mycotoxins, feed composition, immune status, …) play a role. Questionnaires and reports from the field indicate that, in addition to biosecurity and coccidiosis control, a variety of feed additives are in use to prevent the disease. Most of these are not specifically developed to control necrotic enteritis, and scientific literature reports plenty of tools that can have good effects, including probiotics prebiotics, acids, essential oils among others. To choose and develop efficient feed additives, it is essential that the targets are identified. Antibacterial products (against C. perfringens) are a logical tool, but prevention of predisposing intestinal damage is a key target for this kind of disease, in which pathogenic strains take benefit of epithelial barrier loss by consuming molecules that leak out of the gut wall and by producing toxins that can enter cells and matrix components beneath the epithelial layer. A variety of factors affect intestinal integrity in broilers so this is a challenge. For this, it is of utmost importance that the intestinal microbial ecosystem is as such that it produces metabolites that support intestinal integrity. As an example, butyrate and thus intestinal butyrate producing bacteria have been associated with strengthening of the epithelial barrier and control of necrotic enteritis, and these anaerobic populations seem depleted when the disease develops or when predisposing factors are applied in animal models. Strikingly, many of the control tools that are in use, either or not deliberately, support colonization by these bacteria. Also feed additives with direct effects on intestinal epithelial layer integrity and inflammation are potential tools, and some are already marketed. Vaccines are not in place, but might be a future tool when live vector systems are developed that can yield mucosal antibodies against key virulence factors, including, but not limited to, the NetB toxin. In the EU, necrotic enteritis in layers in rather common, while in broilers, because of a higher use of a variety of the above mentioned feed additives, the prevalence is rather low in general.

Life without antibiotics: field lessons learned the hard way

Dr. Tim Cummings

Zoetis

The poultry industry has been trending towards reducing overall antibiotic usage in recent history due to marketing pressures and consumer concerns, but it has not been without consequences. One of the primary diseases which integrators contend with in this brave new world without antibiotics is necrotic enteritis. Over the years, the industry is learning how to come to grips with this NE challenge and as such, an informal survey of integrator experiences and practices was conducted to see how poultry companies were managing this challenge. General trends and practices which help poultry companies minimize the field NE challenge will be presented.

NECROTIC ENTERITIS EXPERIMENTAL MODELS IN RESEARCH

Necrotic Enteritis: The Pros and Cons of Experimental Models

Allen Byrd

Diamond V

Necrotic enteritis is a complex disease where a pathogenic strain of Clostridium perfringens must encounter the ideal conditions to compete effectively against the established gut microbiota, proliferate and cause the typical intestinal damages observed during an episode. There are numerous experimental models used to reproduce necrotic enteritis and they all require a predisposing factor to precipitate clinical signs and lesions. The key is to provide Clostridium perfringens with the ideal environment for its growth. Damages to the intestinal epithelium following a coccidial challenge, changes to the diet including high levels of poorly digestible non-starch polysaccharides or proteins, stress and immunosuppression are all means to favor the pathogen’s proliferation in the intestines. The pros and cons of various experimental NE models will be discussed.

Effects of salmonella exposure times in a necrotic enteritis challenge model

Sam Hendrix1, Dan Moore1, Steve Davis, Kalen Cookson2, Jon Schaeffer2, and Don Bade3

1Colorado Quality Research, Wellington, CO, 2Zoetis US Poultry, Durham, NC 3Microbiological Research, Inc., Fort Collins, CO

The symbiotic relationships of various bacteria that make up the gut microbiome is poorly understood and research focusing on their interplay has resulted in mixed findings. Development of appropriate challenge models for testing natural products and their effects on multiple gut flora is necessary. The results and findings presented in this discussion were recorded as part of a pilot challenge study to determine the effect of three different salmonella exposure time points in addition to a Clostridium perfringens (CP) challenge at 18 days of age on salmonella shedding and bird performances in a 43-day growth period. For the Clostridium perfringens challenge, all birds were orally gavaged with precocious strains of Eimeria acervulina, E. maxima and E. tenella oocysts at 14 days of age, then challenged with CP on the feed at 18 days of age. For salmonella exposure, all birds were orally gavaged with a nalidixic resistant strain of S. Heidelberg (SH) at either 4, 18 or 21 days of age. The average necrotic enteritis (NE) lesion score at study day 21, NE related mortality from study days 18 to 43, growth performances, impact on feed efficiency, and salmonella shedding levels were recorded. These results, conclusions, and discussion will be presented.

CONTROL OF NECROTIC ENTERITIS WITH ADDITIVES

From in vitro to in vivo, an approach to assess organic acids and botanicals on necrotic enteritis

Ester Grilli

Dimevet, University of Bologna, Italy

Non-pharma feed additives like organic acids (OA) and botanicals (B) are gaining considerable attention because of the sudden change in antibiotic use and regulation worldwide. Despite the general consensus that these additives are effective in increasing growth performances of healthy birds, there is still a lack of experience and a paucity of scientifically validated approaches to test their efficacy in pathologic conditions –like necrotic enteritis (NE)- and in non-antibiotic-ever type of production. In order for OA and B to be effective in preventing or improving NE- associated mortality and loss of performances they need to have these fundamental characteristics: 1) exhibit antimicrobial power to prevent Clostridium perfringens overgrowth; 2) prevent Eimeria invasion and replication in the host; 3) boost immunity and gut health; 4) retain all of these “functions” in the intestine, where pathogen colonization and problems occur. The purpose of this presentation is 1) to review organic acids and botanicals biological properties and 2) to describe a method to screen and assess properties in vitro, from anti-microbial assays to cell cultures, ex-vivo models, and to in vivo challenge and in-field studies. Results from OA and B antimicrobial assays against C. perfringens will be reviewed, as well as the effects of OA and B on epithelial cell cultures. Finally, in vivo studies with NE challenge model with or without antibiotics will be also presented.

Comparing a feed and drinking water application of butyric acid in broilers challenged with Necrotic Enteritis

H.O. Bayir*1, J.D. Liu1, G. Mathis2, B. Lumpkins2, and J. Fowler1

1University of Georgia; Department of Poultry Science; Athens, GA 30602

2Southern Poultry Research, Inc.; Athens, GA 30607 Butyric acid has shown potential for managing necrotic enteritis (NE) in broilers raised without antibiotics. This study evaluated the effects of butyric acid (SmartFeedsUSA) when added in either feed or water on growth performance and the alleviation of NE. A basal diet was fed to a challenged and non-challenged control treatment. Two additional treatments received butyric acid either in the feed or the drinking water. Cobb male chicks were obtained on the day of hatch and randomly distributed among treatment pens (8 birds/pen). Birds from challenged treatments were given ~5000 E. maxima oocysts on day 14. On day 19, 20 and 21, challenged pens also received 108 cfu/ml C. perfringens. Body weight (BW) and feed intake were recorded on day 14, 21 and 28, and mortality was recorded daily. On day 21, 3 birds per pen were randomly selected and scored for intestinal lesions. Data were analyzed using one-way ANOVA via SPSS, with significant differences (p≤0.05) separated by Duncan’s Multiple Range Tests. On day 21, all challenged treatments showed lower BW and higher feed conversion ratio (FCR) compared to the non-challenged control. For weight gain post-challenge, the water-added treatment was higher than the challenged control, and both forms of butyrate showed improved FCR. On both day 21 and 28, both treatments with butyrate showed lower mortality than the challenged control. Butyric acid offered benefits to growth and gut health in NE- challenged birds. Further, a drinking water application showed a greater effect than when added in the feed.

Probiotic, 1-monglycerides, and feed acidification improve performance and reduce necrotic enteritis when fed to broiler chickens challenged with Clostridium perfringens

Greg Mathis1, Peter Ader2 Mike Coelho3 and Charles Hofacre4

1Southern Poultry Research, Inc., 96 Roquemore Rd, Athens, GA 30607 2BASF SE, ENS/LD, Chemiestrasse 22, 68623 Lampertheim, Germany

3BASF Corporation, 100 Campus Drive, Florham Park, NJ 07932 4Southern Poultry Research Group, Inc., 1061 Hale Rd, Watkinsville, GA 30677

Clostridium perfringens-induced necrotic enteritis (NE) has become a great concern to the poultry industry. A 28 day cage broiler chicken study consisting of 8 treatments and 8 replications examined the reduction of NE by feeding no additive (non-challenged (NM) and (challenged (NMI)), SILOhealth 104 (SH) a 1-monglycerides; 2.5 and 5.0 kg/mt, MIYA-GOLD (MG) a Clostridum butyricum probiotic, 0.5 and 1.0 kg/mt, Amasil NA (AN), sodium formate, 8.0 and 12.0 kg/mt, or BMD, antibiotic, 50 g/t. On D14, all broilers were orally inoculated with a low dose of E. maxima. On D19, 20, and 21, all birds, except NM, were orally dosed with C. perfringens. Birds and feed were weighed on D0, 14, 21, and 28. On D21, 3 birds per cage were NE lesion scored (0-3). The NE model was successful in producing a severe infection with 50% reduction in weight gain, 46 points higher feed conversion ratio (FCR), and 31% NE mortality in the NMI birds. Challenged birds fed either level of the MG, SH, or AN had significantly better weight gain (D0-28), lower NE lesion scores and % NE related mortality compared to NMI. FCR was significantly improved with the higher dose level of each feed additive and was not different from the BMD fed birds. The antibiotic BMD improved the performance (FCR and weight gain) and reduced NE lesions and NE mortality (0.9 and 6% respectively). These results emphasize the benefits of either feeding SILOhealth, MIYA-GOLD, Amasil NA, or BMD by reducing the negative impact of NE challenged broiler chickens.

Effect of replacing in-feed antibiotics with synergistic organic acids, with or without trace minerals and/or water acidification, on growth performance and health of broiler chickens

under a Clostridium perfringens type A challenge.

Gregory Page, Leslie McKnight, and Yanming Han

Trouw Nutrition R&D

The objective of this research was to examine the effects of an organic acid blend (OAB), organic acid water acidification (OAA) and/or hydroxy trace minerals (HTM) as alternative feed programs in broiler chickens under a Clostridium perfringens (Cp) challenge. A total of 2376 Ross 708 cockerels were allocated to one of 6 dietary treatments (9 replicates with 44 birds/pen) with a 3 phase feeding program (0-20 days; 21-28 days; and 29-35 days). Treatments included: 1) a Cp-challenged control (CpC), 2) CpC+BMD (55 ppm bacitracin), 3) CpC+OAB (1.5g/kg, 1.0 g/kg, and 0.5 g/kg by phase), 4) CpC+OAA (1L/1000L drinking water), 5) CpC+OAB+OAA, and 6) CpC+OAB+OAA+HTM (125 ppm Cu). Birds were subjected to an in feed Cp challenge (~108 cfu/ml Cp) on day 14 and sampled on day 17. Body weights (d35), and cumulative feed conversion ratio (FCR) and mortality (d0-35) were not significantly different between treatments. Prior to challenge (d14), birds fed OAB+OAA+HTM combination had higher body weight (BW) than CpC (+3.5%, p<0.01) and BMD (+2.2%, p=0.02)). OAB+OAA had significantly higher BW than CpC (+2.6%, p<0.05) and a tendency toward increased average daily gain (ADG) relative to CPC (+3.9%, p<0.05) pre-challenge (D0-14). All additive treatments and combinations numerically reduced ileal Cp counts relative to CpC, the most effective being OAB (-35%, p = 0.29) and OAB+OAA+HTM (-32%, p=0.35). The findings of this study support the use of OAB+OAA+HTM as an effective application in antibiotic-free feeding programs given the enhanced growth performance over BMD in the starter phase, and comparable growth to BMD after challenge.

Effect of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative status of broiler chickens under

Clostridium perfringens type A challenge

Yanming Han

Trouw Nutrition R&D

The objective of the study was to compare the effect of supplementing antibiotic (AGP) or synergistic organic acids (OA) in broiler chickens challenged with Clostridium perfringens (CP). 600 day-old male broiler chicks were allocated into 40 pens (15 birds each). There were five treatments, with 8 replicates each, in a two-phase feeding program (d0-21, d22-28). The treatments included: non-challenged control, CP- challenged control (CP), CP + AGP (50 ppm of Aureomycin chlortetracycline), CP + OA1 (0.2 or 0.1% in phases 1 & 2, comprising of butyrate, MCFAs, organic acids, and phenolics), and CP + OA1+OA2 (0.45 or 0.3% in phases 1 & 2, comprising of buffered short chain fatty acids). On d15, 16, and 17, birds in treatments 2-5 were orally gavaged with 0.5 mL culture of CP containing 2.0×108 CFU/mL. Before CP challenge, the birds were not affected by the treatments. CP challenge reduced body weight (BW) on d19 (P < 0.0001) and d22 (P < 0.0001), while AGP, OA1, and OA1+OA2 were able to maintain BW to that of the unchallenged controls over the same period. The intestinal lesions in the AGP, OA1, or OA1+OA2 group were numerically lower compared to CP-challenged control. The immune organ weight (spleen, liver, thymus, bursa of fabricius) was not impacted. OA1 or OA1+OA2 was able to reduce serum malondialdehyde (P < 0.002) and improved breast meat percentage (P<0.005) on d28, compared with the CP challenged group. The results indicated that OA1 or OA1+OA2 supported similar performance as AGP under a mild Clostridium perfringens challenge.

Evaluation of different doses of a novel Bacillus multi-strain DFM to control Clostridium perfringens induced necrotic enteritis (NE) in broiler chickens

Alfred Blanch1 and Charles Hofacre2

1Chr Hansen A/S, Bøge Allé 10-12, 2970 Hørsholm (Denmark)

2Southern Poultry Research Group, 1061 Hale Road, Watkinsville, GA 30677 (USA)

The aim of the study was to evaluate the efficacy of different doses of a Bacillus multi-strain DFM to control Clostridium perfringens induced necrotic enteritis in broilers from 0 to 42 days. 2500 male broilers (Ross 708) were used. Chickens were allocated to five treatment groups: T1 (challenged control); T2 (challenged + 250 g DFM/MT feed); T3 (challenged + 500 g DFM /MT feed); T4 (challenged + 1000 g DFM/MT feed) and T5 (challenged + 2000 g DFM/MT feed). Each group had 10 pens of 50 birds. The NE challenge model consisted of Eimeria vaccine (D0) and Clostridium perfringens (1x108 CFU in drinking water/pen/day, D18, D19 and D20). Zootechnical parameters (mortality, weight gain, feed consumption and feed conversion ratio –FCR-) were recorded on day 21, 35 and 42. No significant treatment effect by each individual DFM dose on zootechnical parameters was observed. DFM treatment reduced FCR numerically compared to the control group (T1: 2.000; T2: 1.890; T3: 1.865; T4: 1.910 and T5: 1.622; p=0.270). Mortality rate observed was higher than expected under the challenge model of this study (average 35%), without any significant effect by DFM treatments. However, when comparing T1 (challenged control) with all DFM-treatment groups together, regardless of the dose (data pool from T2, T3, T4 and T5), the addition of DFM in the diet significantly improved FCR by 6.4% (T1: 2.000 and DFM groups: 1.872; p=0.025). The results indicate that this novel DFM improves FCR in broiler chickens even under very severe NE conditions.

Comparative efficacy of a novel multi-strain Bacillus-based DFM and each one of its single strains for the control of necrotic enteritis caused by Clostridium perfringens in broiler

chickens

Dorthe Sandvang1, Alfred Blanch1 and Greg Mathis2

1Chr Hansen A/S, Bøge Allé 10-12, 2970 Hørsholm (Denmark) 2Southern Poultry Research Inc., 2011 Brock Road, Athens (Georgia, USA)

The objective of this study was to evaluate the effect of a multi-strain Bacillus-based DFM and its three single strains on performance of broilers challenged with a commercial Eimeria vaccine and a field isolate of Clostridium perfringens. 2,250 healthy male broiler chickens (Cobb) were allocated to five treatment groups: T1 (control group; challenged birds without DFM); T2 (DSM32324 strain at 8 X 105 CFU/g of feed); T3 (DSM32325 strain at 5 X 105 CFU/g of feed); T4 (DSM25840 strain at 3 X 105 CFU/g of feed) and T5 (the three strains together at 1.6 X 106 CFU/g of feed). There were nine 50 chicken-pens per treatment group. All birds were spray vaccinated with Eimeria vaccine on day of hatch. On Days 19, 20, and 21 all pens were challenged with a field isolated C. perfringens. Each pen received the same amount of C. perfringens (1.0 X 108 CFU/day). On Day 21, five birds per pen were sacrificed and examined for the degree of NE lesions. Zootechnical parameters were recorded on day 21, 35 and 42. DFMs significantly improved feed conversion ratio (FCR) compared to the control group, the multi-strain DFM showing the lowest value. Furthermore, DFM treatments significantly reduced lesion scores and mortality compared to the control group. In conclusion, the results of this study show the efficacy of a combination DFM and its single strains to minimize gut lesions and improve performance of broilers in a sub-clinical NE challenge.

Evaluating the performance of Bacillus subtilis DSM 32315 under different necrotic enteritis models in broiler chickens

A. Sokale1, A. Menconi1, E. Franco-Lara2, and K. Doranalli3

1Evonik Corporation, Kennesaw, GA, USA

2Evonik Nutrition & Care GmbH, Halle/Westfalen, Germany 3Evonik Nutrition & Care GmbH, Hanau, Germany

Reproducible Necrotic Enteritis (NE) infection models are necessary to understand the pathogenesis of NE and provide the basis of understanding the mechanisms of action of candidate alternative to antibiotics interventions. Many multiple methods of inducing the infection in broilers have been described. Experimental models that utilize toxin- producing Clostridium perfringens strains with co-infection with Eimeria or immunosuppressive pathogens, and dietary modifications, have been shown to increase the level of NE-related mortality. In general, the negative impact of NE on broiler chickens are exhibited by reduced body weight gain (BWG), presence of intestinal lesions, and high mortality. In five different NE broiler trials, the effect of Bacillus subtilis DSM 32315 (B. subtilis) was tested using NE model consisting of C. perfringens in co-infection with Eimeria. The differentials % change in BWG, feed conversion ratio (FCR), lesion scores (LSC), and mortality across all five trials was compared to the infected control (IC) using the following equation: Δ = ((B. subtilis/IC)*100)-100). Trial 1 (28 days cage trial) BWG, FCR, LSC, and mortality difference in comparison to IC were +21%, -1.3%, -43%, and -20%, respectively. In trials 2 to 5 in which birds were raised to either 35 or 42 days, the average differential BWG, FCR, LSC, and mortality in comparison to IC were +5%, -6%, -32%, and -4%, respectively. Overall, in all the NE models, supplementation of B. subtilis DSM 32315 significantly give (P ≤ 0.05) lowered intestinal lesions and mortality, and improved in the performance of broiler chickens, in a consistent manner.

Effect of dietary supplementation of Bacillus subtilis DSM 32315 on the performance and intestinal microbiota of broiler chickens under Clostridium perfringens challenge

C. Bortoluzzi1, JCP. Dorigam2, A. Menconi3, A. Sokale3, AM. Villegas1, TJ. Applegate1,

K. Doranalli2

1Department of Poultry Science, University of Georgia, Athens, GA, USA 2Evonik Nutrition & Care GmbH, Hanau, Germany.

3Evonik Corporation, Kennesaw, GA, USA The objective of this study was to evaluate the effects of the dietary supplementation of Bacillus subtilis DSM 32315 (probiotic) on the performance and intestinal microbiota of broiler chickens infected with Clostridium perfringens (CP). One-day-old broiler chickens were assigned to three treatments with 8 replicate pens (50 birds/pen). The treatments were: non-infected control; infected control; and infected supplemented with probiotic (1x106 CFU/g of feed). On day of hatch, all birds were sprayed with coccidia vaccine based on recommended dosage. On d18-20 the infected birds were inoculated with CP via feed. Necrotic enteritis (NE) lesion score was performed on d21. Digestive tract of 2 birds/pen was collected on d21 to analyze the ileal and cecal microbiota by 16S rRNA sequencing. Performance was evaluated on d 28 and 42. On d21, probiotic supplementation reduced (p<0.0001) NE related lesions vs. infected control birds. On d28, feed efficiency improved (p<0.0001) in birds supplemented with probiotic vs. infected control birds. On d42, body weight gain (BW) and feed conversion ratio (FCR) were higher (p<0.0001) in probiotic supplemented birds vs. infected control birds. The diversity of the intestinal microbiota was impaired in infected birds, but not in infected birds supplemented with probiotic. Infection with CP decreased the frequency of Ruminococcaceae and Ruminococcus (p=0.05 and 0.03, respectively) in the cecal microbiota, but the supplementation with probiotic partially restored its frequency. Therefore, dietary supplementation of Bacillus subtilis DSM 32315 was beneficial in attenuating the negative effects of CP challenge on performance and intestinal microbiota of broilers chickens.

Synbiotic (probiotic + prebiotic) as a potential alternative to ionophore to alleviate the effects of coccidiosis and necrotic enteritis in broiler chickens

G. Raj Murugesan1, Ashley Markazi2, Revathi Shanmugasundaram2, Chasity Pender1,

and Ramesh Selvaraj3

1Biomin America Inc., Overland Park, KS 2Department of Animal Sciences, Ohio Agricultural Research and Development Center,

Wooster, OH 3University of Georgia, Athens, GA

Effects of a synbiotic as a potential alternative to ionophore in broiler chickens challenged with coccidiosis and necrotic enteritis was studied. The synbiotic used is a combination of live probiotics isolated from adult broilers (Lactobacillus reuteri, Bifidobacterium animalis, Pediococcus acidilactici, and Enterococcus faecium) and prebiotic (fructooligosaccharide). 75 one-day-old broiler chicks were randomly distributed to control, salinomycin, and synbiotic groups in five replication pens per group and five birds per pen. Salinomycin was included at 0.05 g/kg and synbiotic at 0.5 g/kg of feed. At 14 days of age, all birds were orally inoculated with 1 X 104 Eimeria maxima oocysts. At 17 d of age, all birds were inoculated with 1 X 103 cfu/bird of Clostridium perfringens. At 35 days of age, birds supplemented with salinomycin or synbiotic had 8.2, 5.1% higher body weight gain (BWG) (P = 0.264) and 6.8, 5.1% better (P =0.10) feed conversion ratio (FCR) than the control group, respectively. Salinomycin or synbiotic supplementation increased illeal villi length 28.3, 20.4% (P = 0.002) and 26.8, 21.2% better villi:crypt ratio (P = 0.085) than the control group. Salinomycin or synbiotic lowered IL-1 mRNA by 0.31, 0.23 fold (P = 0.0009) and increased cecal tonsil IL-10 mRNA levels by 4.1, 5.6 fold (P = 0.028) than the control group. Overall supplementation of synbiotic increased BWG, improved FCR, and increased intestinal villi length in birds challenged with coccidiosis and necrotic enteritis by decreasing the disease severity as measured through decreased pro-inflammatory cytokine and increased anti- inflammatory cytokine mRNA expression at the site of infection.

Use of Alquermold Natural as an effective manner to control Clostridium perfringens induced necrotic enteritis in poultry

Julia Pi and Elvira Cano, Biovet, S.A.

An experiment was conducted to determine the effectivity of Alquermold Natural (AMN), a natural biocide based on cimenol ring (botanical origin), as a prevention and treatment for necrotic enteritis (NE) in broilers. Productive performance was evaluated, as well as mortality, uniformity, carcass yield and Clostridium perfringens (CP) intestinal colonies. 504 broilers were divided into four treatment groups: non-infected control (T1); infected control (T2); infected group receiving AMN as a prevention at 0.5 kg/Tm through feed, continuously during all the trial (T3); infected group receiving AMN as a treatment at 1 ml/L through water for one week, starting its administration the day after the infection (T4). Birds in T2, T3 and T4 were challenged with CP in drinking water on days 19, 20 and 21 of age with 108 cfu/bird. Results demonstrated that AMN as a preventive (T3) was effective in controlling NE (91.5 more grams/bird, 1.17% feed conversion reduction; compared to T2). AMN as a treatment (T4) also resulted effective in counteracting the negative effects of NE (65.34 more grams/bird, 0.58% feed conversion reduction; compared to T2), although less efficient in maintaining productive performance, carcass yield and controlling the proliferation of CP, compared to T3.

Effects of valeric acid glycerides on performance, intestinal morphology, and protective effects during necrotic enteritis challenge

Jacob Lum

Perstorp Feed and Food

Valeric acid is a C5 short-chain fatty acid (SCFA) that is naturally produced in the lower gastrointestinal tract of chickens by microbial fermentation. Unlike the more commonly known SCFAs, such as butyric, propionic, and acetic acid, valeric acid is produced in small quantities and its role in gastrointestinal health has not been extensively studied. In this study, valeric acid in the form of glyceride esters (valerins) were evaluated for their effect on growth performance and intestinal morphology and their protective effect during a necrotic enteritis (NE) challenge. In Trial 1, Ross 308 broilers were fed either a control diet or a diet containing valerins at an inclusion rate of 1.5 kg/MT for 37 days and treatments were evaluated on their effect on body weight gain and feed conversion. Treatment with valerins resulted in a 5 point improvement in feed conversion (p=0.021) compared to the control group during the trial. In Trial 2, intestinal morphology was evaluated and it was found that treatment with valerins significantly decreased crypt depth in the jejunum (p=0.0013) and increased villus height, though not significantly (p=0.09). In Trial 3, birds fed valerins had a significant increase in density of glucagon-like peptide-2 immunoreactive cells in the jejunum and ileum at 10 days of age (p<0.05). In trial 4, valerins were found to reduce the incidence of lesions and reduce lesion score in birds challenged in a NE model. Inclusion of valerins also significantly improved body weight gain during the NE challenge (69 g; p=0.0001).

Necrotic enteritis prevention and control in broilers: The effects of an animal-use only antibiotic compared to shared-class antibiotics

Jaime Ruiz, Sara Steinlage and Robert Evans

Elanco Animal Health, 2500 Innovation Way, Greenfield, IN 46140 USA

Avilamycin, an oligosaccharide antibiotic of the orthosomysin group, was recently introduced to the US broiler industry as an animal-use only antibiotic for prevention of mortality associated with Necrotic enteritis (NE) and the first to be introduced requiring veterinary oversight with the Veterinary Feed Directive (VFD). Studies assessing the impacts of avilamycin followed by an ionophore (animal-use only program) compared to antibiotics (shared-class) programs on NE prevention will be presented. We will review important factors impacting economics — such as the severity of Eimeria maxima lesions and feed conversion — and share additional comparisons from recent U.S. field trials.

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Nutritional modifications to decrease the risks of necrotic enteritis Todd J. Applegate and Cristiano Bortoluzzi*

Department of Poultry Science, University of Georgia, Athens, GA, USA

Even though the intestine represents a small proportion of body weight in broiler chickens, its requirements for energy and nutrients are high. A healthy broiler intestine has a well-coordinated immune system that must accommodate commensal microbiota while inhibiting the colonization and proliferation of harmful pathogens. Modern commercial intensive practices impose a high sanitary pressure that may exacerbate the progression of intestinal diseases such as coccidiosis and necrotic enteritis (NE). The incidence of these diseases may increase worldwide due to mounting pressure to limit the use of subtherapeutic antibiotics as growth promoters or ionophores for coccidia suppression/prevention in the diets of broilers. For this reason, dietary nutrients and gut health feed additives may be beneficial in modulating the intestinal physiology, immunology and microbiology of broiler chickens. For this presentation, modes of action of these in feed-gut modifiers will focus on alterations of the cecal microbiota. However, it should be noted that feed ingredient form, nutrient density, and feed additive modifiers are being investigated to more fully understand their physiologic and immunologic functions.

Studies have been conducted by our lab to determine the effects of sodium butyrate (SB), essential oils (EO), and probiotic supplementation in broilers under coccidia and Clostridium perfringens challenge. We have found that the supplementation of SB alone or in combination with EO had beneficial effects of the expression of tight junction (TJ) encoding genes and showed immunomodulatory effects mainly on the jejunum after Eimeria maxima challenge. Nevertheless, supplementation of SB and EO improved performance and differently modulated the establishment of the cecal microbiota, in terms of diversity and composition, and contributed to many unique changes in the predicted functions of the cecal microbiota over time, which was not observed in unsupplemented birds. Indeed, SB may directly modulate the intestinal microbiome, through its bactericidal effect, or indirectly by stimulating the growth of beneficial lactic acid bacteria. Essential oils, on the other hand, obtained from plant materials are known to possess antimicrobial, antioxidant, and anti-inflammatory properties and potentially reduce the negative impacts of NE, as showed by reduced gross lesions and beneficial effects on intestinal morphology.

Working with a Bacillus subtilis based probiotic supplemented to broiler chickens induced to NE, we observed that its supplementation reduced NE related lesions compared to infected control birds, and feed efficiency was partially restored in birds supplemented with probiotic compared to infected control birds on d 28 and 42. The diversity of the intestinal microbiota was impaired in infected birds, but not in infected birds supplemented with the probiotic. Induction to NE decreased the frequency of Ruminococcaceae and Ruminococcus in the cecal microbiota, but supplementation with probiotic partially restored its frequency. Therefore, we concluded that dietary supplementation of Bacillus subtilis was beneficial in attenuating the negative effects of NE on the performance and intestinal microbiota of broilers chickens.

Although the interactions between commensal bacteria, immune-system and nutrition in health and challenge situations are not fully understood, form of ingredient supply of dietary nutrients and feed additives are responsible for modulating the population of commensal and pathogenic microorganisms. As such, the understanding of these interactions, in both physiological and pathological situations, will allow the use of feed additives to promote a better growth during enteric pathogen challenges.

Antimicrobial effect of selected yeast fractions on Clostridium perfringens

Giuseppina Avantaggiato1, Donato Greco1, Virginie Marquis2, Ruth Raspoet2, Vito D’Ascanio1, and Elisa Santovito1

1National Research Council, Institute of Sciences of Food Production (CNR-ISPA), Bari, Italy

2Phileo - Lesaffre Animal Care, Marcq-en-Baroeul, France Saccharomyces cerevisiae derived products are used worldwide to improve the health and performance of animals. Their activity relies in part on their capacity to bind enteropathogenic bacteria, although the in vitro and in vivo mode of action is still not entirely understood. The inhibitory effect exerted by yeast fractions on bacteria seems to be limited to some gram-negative enteropathogens (Salmonella and Escherichia coli), although several in vivo studies report their anti-infectious activity on gram-positive pathogens like clostridia. The effectiveness of four yeast fractions in inhibiting the growth of Clostridium perfringens was quantitatively determined. The most effective fraction, at the minimum effective concentration of 1.25mg/mL, reduced the final cell count by 102 cfu/mL in 24hrs, increased by 3.6hrs the lag phase duration, and reduced by more than 50% the maximum growth rate with respect to the control. All products adsorbed C. perfringens in a dose- and time- dependent manner. The equilibrium adsorption isotherm approach was used to study the inhibition of C. perfringens as a consequence of adsorption, and to determine the maximum adsorption capacity and affinity. The selected yeast fraction sequestered up to 104 cfu of C. perfringens per mg of product. Results were confirmed on other strains, including a NetB toxin producing strain. Cell adsorption onto this product was fast, stable over time, and occurred with high affinity. To the best of our knowledge, this is the first report showing the in vitro efficacy of yeast fractions to inhibit the growth of C. perfringens, and to reduce the culturable cells by an adsorption process.

Efficacy of trace mineral-amino acid complexes in a necrotic enteritis challenge model

Charles Hofacre1, Greg Mathis1, Brett Lumpkins1, Marco Rebollo2 and John Smith3

1Southern Poultry Research, Inc., 96 Roquemore Rd, Athens, GA 30607

2Zinpro, Eden Prairie, MN 3Alectryon, Baldwin, GA

Zinc and copper are known to be important in maintaining intestinal integrity, especially under challenge situations, but there is little information regarding their role in ameliorating the effects of necrotic enteritis (NE). Two trials utilized the NE challenge model of Hofacre et al. 1998. Battery cages with six treatments and ten replicates were used in the first trial: NRC diet unchallenged; NRC diet challenged; 100 ppm each ZnSO4/MnSO4 challenged; 60 ppm ZnSO4/ 40 ppm Availa-Zn/ 100 ppm MnSO4 challenged; 60 ppm ZnSO4/ 60 ppm Availa-Zn/ 100 ppm MnSO4 challenged; 60 ppm ZnSO4/ 40 ppm Availa-Zn/ 60 ppm MnSO4/ 40 ppm Availa-Mn challenged. The second trial was a floor pen study with five treatments and ten replicates: 100 ppm ZnSO4/ 100 ppm MnSO4/ 20 ppm CuSO4 challenged; 60 ppm Availa-Zn/ 100 ppm MnSO4/ 20 ppm CuSO4/ 40 ppm ZnSO4 challenged; 40 ppm ZnSO4/ 100 ppm MnSO4/ 60 ppm Availa- Zn/10 ppm Availa-Cu challenged; 60 ppm ZnSO4/ 60 ppm MnSO4/ 20 ppm CuSO4/ 40 ppm Availa-Zn/ 40 ppm Availa-Mn challenged; 50 g/ton BMD challenged. Results: Battery NE mortality: 15%a challenge control, sulfate 2.5%b, Availa Zn low 0.0%b, Availa Zn high 2.5%b and Availa Zn/Mn 1.3%b. Results: Floor pen NE mortality: sulfate 12.7%a, Availa Zn 5.1%b, Availa Zn/Cu 8.6%ab, Availa Zn/Mn 3.9%b, BMD 7.6%ab. These studies demonstrated that replacing a portion of the dietary zinc, copper and manganese with metal-amino acid complexed minerals can be effective in reducing the effects of Eimeria maxima and Clostridium perfringens in an NE challenge model.

The use of in-feed mineral supplementation to control the impact of Clostridium perfringens on performance, lesion scores and necrotic enteritis associated mortality in

various necrotic enteritis challenge models in broilers

Daniel Moore, S. Davis S. Hendrix

Colorado Quality Research, Inc. Wellington, CO 80549

Necrotic enteritis (NE) has been a significant concern for the US poultry industry for many years and has typically been controlled through the use of antibiotics and ionophores. However, with an increase use of antibiotic-free diets in the US there is concern that NE will become an even bigger issue. In this new era of producing poultry in the US it is important to identify tools and antibiotic alternatives to assist in the control of NE in broilers. The use of minerals may be one tool that nutritionist and veterinarians can use to combat NE. Mineral supplementation is not a new concept in poultry production but in recent years alternative forms of minerals have become available that can be safely used at higher than traditional levels. Floor pen studies were conducted focusing on the supplementation of copper by itself or in combination with zinc during a NE challenge. Studies were initially conducted by challenging broilers in the feed with Clostridium perfringens to create NE followed up with a novel, 3 grow-out cycle model to induce a natural, mild NE outbreak on the third cycle. Results, discussion and conclusions from these studies will be presented.

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BIOLOGY OF Clostridium perfringens

Characterization of Clostridium perfringens netB+tpel+ type A strains isolated from necrotic enteritis-afflicted broiler chickens

Charles Li1*, Hyun S. Lillehoj1, Xianghe Yan2, Changqin Gu1,3, Zhifeng Sun1, YoungsSub

Lee1, Hongyan Zhao1,4, Zhezi Xianyu1,5, Gregory Siragusa6 and Arquette Grant1

1Animal Biosciences and Biotechnology Laboratory 2 Environment Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center,

Agricultural Research Service-US Department of Agriculture, Beltsville, MD, USA 3 HuaZhong Agricultural University, Wuhan, China;

4Yangzhou University, Yangzhou, China 5University of Delaware, Newark, DE, USA

6Poultry Microbiological Safety Research Unit, Agricultural Research Service-US Department of Agriculture, Athens, GA, USA.

Clostridium perfringens (CP) type A strains are the key etiological factor in induction of necrotic enteritis (NE), one of the important enteric diseases in poultry, responsible for the annual loss of $ 6 billions to worldwide poultry industry. Several CP toxin genes were found to be critical in the NE pathogenesis, but less information is available on CP lethal toxin tpel gene in chickens. In this report, 20 CP strains isolated from NE-afflicted birds were characterized microbiologically, molecularly, and 6 strains were tested for their virulence in chickens experimentally. Toxinotyping by PCR revealed that all the strains tested were alpha toxin-positive, but only five strains were netB+tpel+ (Tpel 13, Tpel 15, Tpel 17, Tpel 18, Tpel 19). The results from in vivo animal study by repeated CP infections with CP strains indicated that Tpel 17 was the most virulent inducing NE lesions in broiler chickens with least relative body weight gain. Quantitative PCR showed that 4 of CP tpel+ Strains contained the similar copy numbers of tpel genes among the bacteria strains normalized by 16S rRNA as a house-keeping gene marker. Availability of these virulent netB+tpel+ CP strains would greatly benefit the studies in CP pathogenesis and vaccine development as an inducible NE model.

Molecular characterization of type IV pilus operons in chicken Clostridium perfringens strains reveals a highly conserved organizational structure and a low level of gene sequence

conservation

Marie-Lou Gaucher1,4, Valeria R. Parreira2, Julie Arsenault3, Marie Archambault3, Martine Boulianne1 and John F. Prescott2

1 Chair in Poultry Research, Département de Sciences Cliniques, Faculté de médecine vétérinaire,

Université de Montréal, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6 2 Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 4Research Chair in Meat Safety, Département de pathologie et microbiologie, Faculté de

médecine vétérinaire, Université de Montréal, CP 5000, St- Hyacinthe, Québec, Canada J2S 7C6 Reduction in the use of antibiotic growth promoters in commercial broilers has generated a resurgence of necrotic enteritis (NE) and an increased interest in better understanding the role of its causative agent, Clostridium perfringens. Factors contributing to the colonization of the chicken gut by this bacterium are yet to be described. Among these, type IV pilus (T4P) may have an important role based on its functions include adhesion and colonization. There is little information in the scientific literature on T4P-encoding genes in C. perfringens strains isolated from broiler chickens. The aim of this study was to describe the T4P gene sequence and structural organization. T4P operon organization and gene sequences were compared among 15 publicly available genomes of C. perfringens reference strains. In parallel, 153 C. perfringens strains of chicken origin were assessed for the gene sequence conservation of these operons using an overlapping PCR approach. An in-depth comparison of the various T4P gene sequences identified was conducted through sequencing. Results obtained highlight a highly conserved organization within T4P operons, but a low level of sequence conservation within specific T4P-encoding genes among chicken C. perfringens. The T4P genes sequence variability was, however, significantly lower in C. perfringens netB- positive strains. This study is the first to describe the organization and variability of T4P- encoding genes in chicken C. perfringens strains. Data obtained suggest that the T4P gene sequence variability observed might influence the ability of C. perfringens strains to colonize the chicken intestine or to cause NE, and deserves further investigation.

PATHOGENESIS AND VIRULENCE OF Clostridium perfrigens

Viability of the Greater wax moth larvae (Galleria mellonella) as a model organism for the investigation of Clostridium perfringens associated disease (CPAD).

Sammy J. Kay1, Joseph R. Edwards1, Joseph C. S. Brown1, Emily P. Brandreth2, Danielle P.

Bramley2 and Ronald A. Dixon2

1Arden Biotechnology, Unit 1.11, Boole Technology Centre, Lincoln Science and Innovation Park, Lincoln, Lincolnshire, United Kingdom, LN6 7DJ

2University of Lincoln, Brayford Pool, Lincoln, Lincolnshire, United Kingdom, LN6 7TS Whole bird poultry models for the in vivo investigation of enteric diseases including necrotic enteritis can be constrained by ethical or financial issues. Greater wax moth larvae (Galleria mellonella) have become a well established alternative infection model for a variety of pathogens. The larval model is suited to study mammalian infections since it survives at 37°C, and requires no specialist equipment. Galleria mellonella produces basic immune responses and evidence suggests infection data correlates with that of mice. In this study, larvae were infected with varying doses of two distinct Clostridium perfringens strains associated with necrotic enteritis in poultry. Infections were reviewed for 72 hours using novel time-lapse methodology to track cuticle darkening caused by phenol-oxidase activation. Larval morbidity and mortality was assessed by melanisation scoring and histopathology. Antibiotic mono-therapy, using penicillin G, bacitracin, neomycin and tetracycline was administered and efficacy compared to in vitro assays. C. perfringens is pathogenic against G. mellonella although large doses were required to reduce larval survival. Antibiotic therapy increased larval survival however increased morbidity scores and correlated poorly to in vitro analysis. The G. mellonella model remains unexplored in the context of C. perfringens. Sub- lethal infections induce distinct melanisation in a dose dependent manner. The model will not replace mammalian equivalents for research but may be useful as a pre-screening assay for virulence of isolates during whole bird trials. The larvae presents a simple, cheap and rapid in vivo assay in the development of potential novel therapeutics.

EPIDEMIOLOGY OF NECROTIC ENTERITIS

Pulsed field gel electrophoresis and toxin type comparison of Clostridium perfringens isolates from birds with varied health statuses

Joseph C. S. Brown1,2, Sammy J. Kay1,2, Joseph R. Edwards1,2 and Ronald A. Dixon2

1Arden Biotechnology, Unit 1.11, Boole Technology Centre, Lincoln Science and Innovation Park,

Lincoln, Lincolnshire, United Kingdom, LN6 7DJ 2University of Lincoln, Brayford Pool, Lincoln, Lincolnshire, United Kingdom, LN6 7TS

Pulsed field gel electrophoresis (PFGE) is routinely used to investigate food poisoning episodes by linking specific isolates to outbreaks of human disease. In addition, it has proven helpful in the epidemiology of Clostridium perfringens in necrotic enteritis in poultry. In the present study from the UK, 75 birds that had been intensively reared commercially following postmortem, were shown to be either healthy (55) had subclinical necrotic enteritis (15) or had clinical necrotic enteritis (5). Microbiological and molecular analysis showed that all isolates in the study were identified and confirmed as C. perfringens and toxin gene typed to determine the presence of cpa, cpb, iA, etx, cpe (plasmid and chromosomal), cpb2 (atypical and consensus), netB, TpeL, becA, becB, pfoA and colA amongst the three categories of health or disease investigated. The isolates from necrotic enteritis affected birds showed that the netB gene was not identified, nor the TpeL gene, having only cpa, cpb2, pfoA and colA present. Birds with subclinical necrotic enteritis were found to have a much more diverse array of toxin genes, whilst isolates recovered from birds deemed healthy were found to have the least diverse array of toxin genes. Results of PFGE showed an overall similarity of 30% amongst isolates, with those recovered from cases of necrotic enteritis having a similarity of over 50%. This study would suggest that within the UK, isolates from necrotic enteritis shared more clonality than isolates recovered from healthy birds and that netB was not identified with the disease.

Clostridium perfringens populations in the gastrointestinal tracts of healthy broilers in Asia.

Evan Hutchison1, Jon Ferrel2, Tom Rehberger1

1Arm and Hammer, 2Elanco

Clostridium perfringens-associated necrotic enteritis (NE) is of great concern for the global poultry industry. Generally, NE incidence increases following the removal of antibiotic growth promoters (AGP) in commercial poultry systems. This trend was observed in Scandinavian countries and in the European Union at large after the banning of AGPs in 1995 and 1999, respectively. As regulations and consumer demand limit the use of AGP around the world, it is becoming increasingly important for poultry producers to understand the predisposing factors to NE, such as C. perfringens populations. Mapping the levels and genotype of C. perfringens type A in a particular region can help growers develop strategies to defend against NE. In an effort to understand the underlying C. perfringens challenges in various Asian regions, we sampled the gastrointestinal tracts of healthy (asymptomatic) broilers from five countries (Australia, Bangladesh, Japan, Thailand, and India) and plated for Clostridium species. Further molecular testing was done to determine if the cultures were C. perfringens (by possession of the alpha toxin) or non-perfringens Clostridium species. Our results show that average total clostridia across all countries was 4.4E+3 cfu/g, however, only broilers from Bangladesh harbored detectable levels of C. perfringens (average 1.3E+4 cfu/g). Interestingly, the genotypes of these C. perfringens isolates matched the observed genotypes in US C. perfringens. These results indicate that C. perfringens loads are highly specific to region, but that the genotypes are transcontinental.

Prevalence of Clostridium perfringens isolates containing the netB gene in UK environmental sources

Joseph R. Edwards1, Sammy J. Kay1, Joseph C. S. Brown1, Ellen O. Denness2, Emma L. Turnbull2

and Ronald A. Dixon2.

1Arden Biotechnology, Unit 1.11, Boole Technology Centre, Lincoln Science and Innovation Park, Lincoln, Lincolnshire, United Kingdom, LN6 7DJ.

2University of Lincoln, Brayford Pool, Lincoln, Lincolnshire, United Kingdom, LN6 7TS.

The netB toxin gene has been strongly linked to necrotic enteritis in poultry and is thought to be a critical virulence factor in the disease. Our study aims to determine the presence of this gene in the UK environment from isolates obtained from healthy poultry faeces, litter, soil, feed, and from retail chicken breasts or post mortem healthy bird gastrointestinal tract. Clostridium perfringens from environmental samples yielded a multitude of isolates con- taining the netB toxin gene from healthy chickens and their faeces, with soil yielding the most prevalent source (21.05%). Investigating other sources of environmental contamination, isolates of C. perfringens were recovered from retail packaged chicken breasts purchased from UK supermarkets. Sporadic results yielding netB suggest that no distinct correlation with either retail value or origin of the meat could be drawn. The presence of netB appears to be randomly distributed in our environmental samples since we were able to isolate netB from post mortem GIT of healthy broiler birds. It was completely absent from isolates retrieved from birds diagnosed with necrotic enteritis. In contrast 22.7% of isolates containing the netB gene were recovered from birds diagnosed with the subclinical variant of necrotic enteritis. Follow up studies investigating whether the food chain is a potential source of emerging C. perfringens infections in humans such as necrotising enterocolitis (NEC) in neonates asso- ciated are needed. Isolates originating from faeces of neonatal infants diagnosed with NEC have been found to carry the netB toxin and further research is required.

RISK FACTORS AND THEIR CONTROL

High body weight gain is a risk factor for the development and severity of necrotic enteritis lesions

Dierick E.*, Goossens E., Ducatelle R., Van Immerseel F.

Department of Pathology, Bacteriology and Avian diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

Over the past 50 years, intentional genetic selection within the broiler industry has led to major improvements in both body weight gain (BWG) and feed conversion efficiency. Undoubtedly, the use of antimicrobial growth promoters (AGP) has facilitated this process even more. Since the ban on AGP, alternative measures to maximize performance, such as optimising feed composition and using in-feed additives, are in high demand. In addition to its economic advantages, increasing BWG can decrease animal welfare and possibly affect intestinal health, hence performance. The aim of this study was to examine whether a higher BWG would predispose broilers to necrotic enteritis. In this study, 300 broilers were challenged with Clostridium perfringens using a well- established, previously described challenge model. It was found that birds with a higher BWG before challenge developed more severe lesions when compared to slower- growing birds. One possible explanation for the higher susceptibility of fast-growing birds is that increasing amounts of undigested material in the small intestine could be used as a substrate by Clostridium perfringens. After the challenge, the average BWG of the birds developing mild to severe lesions dropped significantly, negatively affecting animal welfare and performance. These results show a significant interplay between BWG and the development of necrotic enteritis. This raises the question whether there is a limit to broiler performance with respect to maintaining intestinal health, and whether decreasing BWG (at certain stages of the growth cycle) can be part of a plan to prevent intestinal pathology. *EvelienB.Dierick@ugent.be

Interaction of necrotic enteritis severity and live salmonella vaccination on salmonella prevalence and load in broilers

Manuel Da Costa1, Kalen Cookson1, Steve Davis2, Sam Hendrix2, Jon Schaeffer1,

John Dickson1

1Zoetis, U.S. Poultry 2Colorado Quality Research, Wellington, CO, USA

Necrotic enteritis (NE) and salmonella control are two major challenges for broiler integrators. Three broiler pen studies were performed to evaluate the interaction of NE severity and salmonella prevalence. The NE challenge methodology was common across studies and consisted of an oral gavage of Eimeria spp. at 14 days followed by a Clostridium perfringens plus Salmonella Heidelberg (SH) challenge at 18 days. The first study compared BMD fed birds with controls. A Poulvac®ST vaccination treatment was added to these two treatments for the second and third studies. Performance and SH prevalence/load (enumeration) were evaluated at 42 days. Across the three studies, BMD improved feed conversion ratio (FCR) (3 to 16 points), and reduced NE lesion scores and mortality (21 to 32% reduction). Regarding SH recovery results, in study 1 BMD fed birds had 20.2% and 43.0% less SH positives detection in spleen and carcass rinse samples, respectively. In addition, BMD resulted in significant SH load reduction in bootswabs (2.8x108

vs. 8.2x102 cfu/g). In both studies 2 and 3, BMD and Poulvac®ST reduced SH prevalence/load in cecae and bootswabs. Reductions of up to 20% in prevalence and 13 fold in SH loads were observed in BMD groups when compared to controls. Poulvac®ST significantly decreased SH prevalence (up to 30.5%) and loads (up to 130 fold) in relation to controls. In conclusion, NE control by BMD feeding can decrease salmonella incidence at the processing plant. Furthermore, it was shown that Poulvac®ST can significantly reduce salmonella detection even in the presence of a NE episode.

Effect of selected Eimeria maxima strains on pathology and subsequent development of necrotic enteritis in a model system

J. D. Latorre1*, G. Tellez1, R. Snyder2, J. R. Barta2, L. R. Bielke3 and B. M. Hargis1

1Department of Poultry Science, University of Arkansas, Fayetteville, AR USA

2Department of Pathobiology, University of Guelph, Guelph, ON, Canada 3Department of Animal Sciences, The Ohio State University, Columbus, OH, USA

Necrotic enteritis (NE) is a multifactorial disease that cause critical economical losses. Seemingly unrelated neonatal challenge with Salmonella Typhimurium at hatch has shown to increase the level of NE-related mortality using an Eimeria maxima (EM) and Clostridium perfringens (CP) challenge in published studies. When a more virulent EM (M6 Strain) was used in the model, NE related mortality reached 40% and 39% in the positive challenge groups (P < 0.05; Exp. 1 and 2). However, using a genetically and immunologically distinct EM (Guelph strain) in the same model, only modest lesion scores (Exp. 3: 2.0 vs. 0) and no impact on mortality (0%) were induced. Nevertheless, body weight gain (BWG) was reduced by 25% after EM-Guelph administration, and a further 86.8% following CP challenge, affecting feed conversion strain (1.949 vs. 1.586) in the positive control group (P < 0.05). Additionally, in Exp. 3, gut integrity was reduced in the challenged group showing an increased level of serum FITC-d and bacterial translocation to the liver compared to the control (P < 0.05). When this model was extended until 35 days of age using the EM- Guelph strain, BWG (2133 vs. 1925 g) was not recovered in Exp. 4. In the urgent search for antibiotic growth promoter (AGP) alternatives, some candidates may be more likely to promote recovery of the enteric epithelium whereas others may be more protective for the inflammation-induced shock and high acute mortality associated with the more virulent challenge. Consideration of appropriate models for different candidate AGP alternatives may be important in future studies.

Poultry feed linked with a clinical case of chicken necrotic enteritis in Quebec, Canada

Audrey Charlebois1,3, Eric Parent1,3, Daniel Venne2, Martine Boulianne1,3, and Marie Archambault3*

1Chaire en recherche avicole, Faculté de médecine vétérinaire, Université de Montréal, Saint-

Hyacinthe, Québec, Canada Centre de Recherche en Infectiologie Porcine et Aviaire (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada; 2Scott

Hatchery Ltd, Québec, Canada In April 2014, a visit to a broiler chicken farm with a sudden increase in mortality was made. Following examinations, necrotic enteritis was diagnosed on this farm. Representative birds and feed samples were sent for necropsy and Clostridium perfringens isolation. The LEPAQ laboratory report emitted a diagnosis of severe necrotic enteritis and moderate coccidiosis. As for bacteriology, one isolate of C. perfringens was recovered from caecum samples and six isolates from poultry feed. All were of type A and carried the netB toxin gene. Isolates had low susceptibility to neomycin, streptomycin, spectinomycin, tetracycline, lincomycin and bacitracin. At the genetic level, all seven isolates were found to be identical by PFGE. In the present report, we describe a clinical case of necrotic enteritis caused by a C. perfringens isolate found in contaminated poultry feed.

Presence of NetB positive isolates of Clostridium perfringens in gizzards of broiler chickens at slaughter.

Audrey Charlebois 1,2, Antoine Hayer 1,2, Samuel Lemire 1, Martine Boulianne 1,2 and Marie

Archambault 1*

1 Centre de Recherche en Infectiologie Porcine et Aviaire (CRIPA), Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada

and 2Chaire de recherche avicole, Département de sciences cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada

Clostridium perfringens, a commensal of the intestinal tract of many animal species, is also responsible for causing necrotic enteritis (NE), an economically significant poultry disease. NetB toxin is an important virulence factor associated with NE. C. perfringens has been isolated from the duodenum, jejunum, ileum and caecum of healthy chickens and more often, these strains were not carrying the NetB toxin. To date, there is no data available on the prevalence and netB carriage of C. perfringens in gizzards. The aim of this work was to evaluate this prevalence and to characterize these isolates. Gizzards and matching caeca were sampled from 40 different flocks at two abattoirs over a 7 month-period in Quebec, Canada. Out of 200 gizzards, 35 were positive for C. perfringens. Among positive gizzards, 86 isolates were recovered with 33.7 % carrying the netB toxin gene and 26 % the cpb2 toxin gene. Moreover, 79.3 % of these isolates were positive for the bacitracin resistance genes bcrABDR. The majority of the C. perfringens isolates were also able to produce moderate to strong biofilm (64%). A significant association (p = 0.0004) between C. perfringens recovery and the presence of feed in gizzards was observed. Genotyping analysis of isolates recovered from the gizzards and from the caeca revealed that both populations were different. This study demonstrates for the first time the presence of netB-positive C. perfringens in the gizzards of healthy broiler chickens.

DIAGNOSTIC OF NECROTIC ENTERITIS

Optimization of an in vitro hemolysis assay for the detection of Clostridium perfringens NetB toxin in culture supernatant

L. Van Damme*, E. Goossens, R. Ducatelle, F. Van Immerseel

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary

Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

Clostridium perfringens is a widespread pathogen that causes enteric diseases in a broad range of hosts, including necrotic enteritis (NE) in broilers. NetB is proven to be an essential virulence factor in NE, but so far no method has been described in detail to measure the production of this toxin by C. perfringens. Recently, purified NetB has been shown to cause hemolysis in different animal species, a characteristic which can be useful in the development of a detection assay. However, both C. perfringens alpha- toxin (α) and perfringolysin O (PFO) have hemolytic activity as well, which impedes the creation of such an assay. The aim of this study was to develop a quantitative NetB hemolysis assay to specifically evaluate NetB toxin production in C. perfringens culture supernatant fluids. Using the agar well diffusion method, we showed that red blood cells from chicken, goose or Muscovy duck allow to discriminate NetB-positive and NetB-negative C. perfringens strains by the presence or absence of an outer hemolysis zone. Based on these findings, a microplate hemolytic assay was developed using 1% avian red blood cells, which were pre-incubated with serum containing anti-alpha toxin and anti- perfringolysin O, to eliminate hemolytic effects of respectively α-toxin and PFO. The microplate NetB hemolysis assay showed to be a simple and inexpensive method, which can be easily used to evaluate NetB production in broth cultures. * lorevdam.vandamme@Ugent.be

Evaluation of media for the enumeration of Clostridium perfringens from poultry feces

M. Hustá*, E. Goossens, F. Van Immerseel

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Meiecine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

Enteric diseases caused by Clostridium perfringens are frequently associated with overgrowth of the organism in the intestinal tract. In subclinical disease or in healthy animals, numbers of C. perfringens in the gut are often low. Isolation of C. perfringens in feces of these animals can be quite challenging with the currently used media, due to other hemolytic strains and overgrowth of undesirable facultative anaerobes which interfere with the isolation. This study aimed to compare a range of selective media for the enumeration, and easy and fast detection of C. perfringens in fecal samples from poultry. The routinely used columbia blood agar (CBA), Shahidi-Ferguson-perfringens agar (SFP) and tryptose sulfite cycloserine agar (TSC), all selective by the addition of antibiotics, and a novel medium, CHROMagar, were tested. Fresh chicken feces were spiked with different concentrations of either pathogenic NetB-positive, or non-pathogenic NetB- negative C. perfringens strains. Ten-fold dilutions were plated on the media, after which the ease of C. perfringens identification, the detection limit of each medium and the growth of other bacteria was assessed. While on CBA a typical hemolysis zone appears around C. perfringens colonies, on SFP, the inclusion of egg yolk results in an opaque halo. CHROMagar shows pink C. perfringens colonies, whereas other strains are grey, making it easier to identify C. perfringens. When lower C. perfringens counts were present in the feces, detection was impaired due to overgrowth of other microbiota. * martina.husta@ugent.be

CONTROL OF NECROTIC ENTERITIS WITH ADDITIVES

Comparative efficacy of Alquermold Natural in a necrotic enteritis model as a treatment in drinking water and as a preventative in feed

Carlos Domenech1, Charles L. Hofacre2, Christa S. Hofacre2, Júlia Pié1, Anna Tesouro1

1Biovet S.A., Tarragona (Spain) 2Southern Poultry Research Group, GA (USA)

An experiment was conducted to determine the efficacy of Alquermold Natural (AMN), a natural biocide based on cimenol ring (botanical origin), as a treatment and preventative for necrotic enteritis (NE) in broilers. Productive performance, mortality and lesion score were evaluated. 2000 day-of-hatch male broilers were divided into four treatment groups. Treatments were: non-infected control (T1); infected control (T2); infected group receiving AMN as a preventative continuously in feed at 0.5 kg/ton (T3); and infected group receiving AMN as a treatment at 1 ml/L in drinking water for one week, starting its administration at the start of NE mortality (T4). To have a mild NE challenge similar to a broiler farm the birds were given 1 dose of a commercial coccidiosis vaccine at 1 day of age, then, T2, T3, and T4 were challenged with Clostridium perfringens in drinking water on days 14, 15 and 16. Results showed no significant differences in weight, FCR or lesion score, probably because the infection was not so severe. However, AMN in the feed (T3) had the numerically lowest FCR and heaviest body weights on day 42 (2% decrease and 4% increase, respectively, compared to T2). These broilers consistently performed as well or better than non-infected birds. Water treatment (T4) significantly reduced NE mortality, while feed treatment numerically reduced NE mortality. In conclusion, AMN as a preventative and as a treatment alleviated the negative consequences of NE due to C. perfringens and, overall, resulted in better performance. Therefore, the in-feed product could be used on all farms and in addition treat in the water those experiencing clinical NE.

In vitro evaluation of the potential of lactic acid bacteria isolated from chicken ceca to inhibit Clostridium perfringens

N. Vieco-Saiz1,2, R. Raspoet2, F. Gancel1, E. Auclair2 & D. Drider1*

1Institut Charles Viollette, EA7394-ICV, Université de Lille. F-59655 Villeneuve d’Ascq, France

2Phileo Lesaffre Animal Care, Marcq en Baroeul, France

Necrotic enteritis leads to heavy economic losses in the poultry industry. This disease is caused by type A Clostridium perfringens and is mostly treated with by antibiotics. As part of an international action plan to withdraw the use of antibiotics in livestock, global health organizations such as FAO/WHO and OIE currently promote the use of alternative strategies to fight this bacterium. Lactic acid bacteria (LAB) are of major interest because of their generally recognized as safe (GRAS) status and their abilities to secrete different antimicrobial compounds. In this work, strains were isolated from chicken ceca and identified as Lactobacillus reuteri or Lactobacillus salivarius. Their potential to inhibit C. perfringens in vitro was evaluated against 13 strains including the reference strain C. perfringens DSM756 and 12 strains isolated from animals with necrotic enteritis. LAB strains were grown in MRS agar for 24 hours, then slabs were cut and placed onto agar inoculated with C. perfringens. Plates were incubated for 24 hours in anaerobic conditions and then inspected for zone of inhibition formation. The results demonstrated that all lactobacilli tested could inhibit C. perfringens in a strain-dependent manner by lactic acid production. Overall, L. salivarius strains displayed significantly wider zones of inhibition with an average halo size of 1.43 cm in diameter, compared to L. reuteri strains, which had halos with an average of 1.18 cm. At this point, we can confirm the potential of these novel lactobacilli strains isolated from chicken ceca to inhibit the growth of several isolates of C. perfringens in vitro. * E-mail : djamel.drider@univ-lille.fr

Effects of NeutraPath™ with or without nicarbazin on the performance of broiler chickens during simulated field challenge conditions

H. Xue*, S. Johnston, S. Ching, E. DeBoer

Amlan International, Chicago, IL, USA

A 28-day study evaluated the effects of necrotic enteritis (NE) on growth performance of broiler chickens fed a diet containing a natural antimicrobial, NeutraPath™, and no commercial ionophore. On hatch day, 2,080 Ross 708 chicks were randomly assigned to treatments: non-challenged control (CON), challenged (CH) control, CH plus nicarbazin (0.01%), CH plus NeutraPath (1 kg/MT), and CH plus NeutraPath (1 kg/MT) and nicarbazin (0.01%). NeutraPath is a synergistic blend of antimicrobial fatty acids and essential oils that is optimized for pathogen control. NeutraPath was fed for 28 days; nicarbazin was fed through day 21. Except for CON, birds were placed on litter used during previous growouts. On day 7, litter containing C. perfringens and Eimeria was added to CH pens to simulate natural field exposure. Intestines were examined for lesions on day 28. Treatment differences were tested using one-way ANOVA. Compared to CON, NeutraPath/nicarbazin- or nicarbazin-treated birds exhibited no significant difference in 28-day mortality. However, compared to CH control, all treatments significantly decreased mortality (P<0.05). Although no statistical difference was found among treatments for overall average feed intake, NeutraPath/nicarbazin- and nicarbazin-treated birds had significantly greater weight gain and improved feed conversion ratio (FCR) compared to CH control birds (P<0.05). Birds in all treatments also had significantly lower lesion scores than CH control birds (P<0.05). NeutraPath-supplemented broilers exhibited significantly lower mortality and lesion scores compared to CH control (P<0.05). Adding nicarbazin resulted in further statistical improvement in these parameters. These data indicate NeutraPath can be used to manage C. perfringens-induced NE in broilers.

In vitro comparison of the growth inhibition concentration of two agents (essential oil blend and antibiotic) against 8 probiotics and Clostridium perfringens from monogastric gut flora.

Isabelle Deuve Riou1, Louise Mercier2, Claire Girard1, Thibaut Chabrillat1,

Sylvain Kerros1

1Phytosynthese, 57 avenue Jean Jaurès, 63 200 Mozac France 2Phyto-Plus, 7640 Pointe du Jour, J2R1H8 Sainte Hyacinthe, Canada

Plant extracts are used as alternatives to antibiotics in animal feed. The aim of this study is to evaluate the in vitro activity of an essential oil blend (EO) and salinomycin antibiotic, on eight probiotics (Lactobacillus acidophilus, L. casei, L. para casei, L. plantarum, Bacillus subtilis, B. longum, L. debrueckii, L. rhamnosus) and one pathogenic bacteria (Clostridium perfringens). Microdilution in liquid broth was used to determine the minimal growth inhibition concentration (GIC) of both agents. GIC corresponds to the lowest concentration of EO or antibiotic able to inhibit the bacterial growth for 18H at 37°C. EO shows a stronger selectivity on Clostridium than Lactobacilli spp compare to salinomycin; EO and antibiotic obtained respectively 150 µg.mL-1 and 0.2 µg.mL-1 on C. perfringens compare to 310 up to 2500 µg.mL-1 and <0.01 to 3.13 µg.mL-1 for Lactobacilli spp. This result confirms the bacterial growth inhibition capacity of EO and antibiotic is not comparable. On broilers, Salinomycin recommendation is 60 ppm and 30 to 75 ppm for EO. Previous in vivo results confirmed that EO was able to inhibit sulphite-reducing bacteria without change on Lactobacilli population (Juin et al., 2005); in opposite salinomicyn had a negative impact on lactobacilli population despite a strong activity on C. perfringens (Johansen et al., 2007). EO could reduce C. perfringens development without disturbing the beneficial flora populations. Such in vitro results probably involve other EO mode of actions. Further studies on broilers must be launched to better understand EO’s activity on gut populations.

Protective effect for broiler chickens of valerins and the antibiotic BMD in necrotic enteritis Challenge model

C. L. Hofacre1, G. F. Mathis 2, R. Sygall3 and J. Smith4

1Southern Poultry Research Group, Watkinsville, GA

2Southern Poultry Research, Athens, GA 3Perstorp BU Feed & Food, Waspik B.V., The Netherlands

4Alectryon, Baldwin, GA

Short chain fatty acids have been effective for both clinical and subclinical necrotic enteritis (NE). Valerins, glycerolesters of valeric acid, may provide benefits of triggering the physiological responses associated with improving intestinal health by the short chain fatty acid minus any negative effects associated with non-esterified short chain fatty acids. Battery cages 32, 8 replicates Cobb male broilers: No additive/No Clostridium perfringens (CP) challenge; No additive CP challenge; BMD 50g/ton/CP challenge; Valerins 1.5 kg/mt/CP challenge. The NE challenge model used was Eimeria maxima at 14 days, CP on days 19, 20, and 21, per Hofacre et al. 1998. Feed consumption and body weight (BW) were evaluated on days 14 and 28. Clinical NE results; the no additive/no CP had 0%c N. E. mortality and lesion scores (LS) were 0.0c; no additive/CP 43.8%a NE mortality and LS 1.3a; BMD NE mortality 7.8%c and LS 0.8b; Valerins NE mortality 17.2%b and LS 0.9b. Subclinical NE results Day 0-28 were no additives/no CP BW 1.015kga and mortality adjusted (adj.) feed conversion ratio (FCR) 1.526c; no additive/CP BW 0.845kgb and adj FCR 1.966a; BMD BW 1.044kga and adj FCR 1.548bc; Monovalerin BW 1.064kga and adj FCR 1.675b. In conclusion, this study demonstrated, as expected, that antibiotic BMD had the greatest impact in reducing effects of C. perfringens for both clinical and subclinical NE In a program without antibiotics. Valerins alone in feed significantly prevented both clinical and subclinical NE.

Evaluation of the in vitro and in vivo antibacterial effects of NeutraPath™

D. Wang*, C. Ching, H. Xue

Amlan International, Chicago, IL, USA

A formulated feed additive, NeutraPath consists of a synergistic blend of antimicrobial fatty acids, essential oils and a proprietary bacterial toxin adsorbent that is optimized for pathogen control in livestock production. An in vitro assay was used to determine minimal inhibitory concentrations (MICs) of NeutraPath™ against Clostridium perfringens, Escherichia coli O157, Salmonella ser. Heidelberg and Vibrio parahaemolyticus. With MICs of 3 to 5 mg/ml, NeutraPath exhibits strong antibacterial activity in vitro. Two studies using an experimental model of C. perfringens-induced necrotic enteritis (NE) evaluated the in vivo antibacterial effects of NeutraPath. Day-old male Cobb 500 broilers were randomly assigned to treatments: non-challenged control, challenged (CH) control, BMD (55 ppm bacitracin) and NeutraPath (0.17% active ingredients). Except for the non-challenged group, birds received ~108 CFU of C. perfringens on three consecutive days. In Study 1, NeutraPath, but not BMD, showed a trend in reducing ileal C. perfringens populations, as enumerated by cultivation-based MPN method (P=0.056). This reduced bacterial load may explain the observed improvement in mortality (27% vs. 54%, P<0.05) and NE-related lesion scores (0.6 vs. 1.2, P<0.05) between NeutraPath treatment and CH control. To further investigate the decreased bacterial load, Study 2 used an ELISA to quantify C. perfringens and its -toxin in cecal contents. Results showed NeutraPath reduced C. perfringens populations 7.7-fold (P<0.05) and -toxin levels 4-fold (P<0.05) compared to CH control. These reductions were associated with significantly improved mortality (26.6% vs. 4.7%, P<0.05). Collectively, these studies show NeutraPath exerts potent in vitro and in vivo antibacterial effects that reduce bacterial load and can be used to control C. perfringens-induced necrotic enteritis in broilers.

Evaluation of diet acidification with sodium bisulfate on broiler necrotic enteritis lesion scores, mortality, and performance in a Clostridium perfringens challenge model.

Josh Payne1, John Pitts1 and Michael Sims2.

1Jones-Hamilton Co. and 2Virginia Diversified Research Corporation.

Necrotic enteritis (NE) is a severe disease in chickens that is caused by Clostridium perfringens (CP). The disease is very costly to the poultry industry due to reduction in bird performance, carcass condemnation, treatment costs and mortality. A pen study using a CP challenge model was conducted to evaluate the impact of feed acidification with sodium bisulfate on controlling NE in broilers. Treatments consisted of (1) non challenged control; (2) challenged control; and (3) challenged sodium bisulfate basal diet inclusion. Treatments were assigned using a complete randomized block design with 10 replicates per treatment (30 pens total). The stocking density was 0.062 m2 (0.667 ft2) per bird with 30 broilers per pen. Birds were inoculated with CP via oral gavage on days 17 thru 19 and reared to 42 days on standard formulated corn, soybean meal and dried distillers grain based diets. Bird growth, feed conversion, mortality and intestinal lesion scores were measured at 21 days. Data were analyzed using the ANOVA procedure of SAS. Compared to results for the unchallenged control group, the challenged control group had significantly increased 21 day lesion scores, decreased 28 and 42 day body weights, and higher 28 day and 42 day feed conversion ratio (FCR). Sodium bisulfate diet inclusion to CP challenged broilers significantly lowered 42 day FCR (1.777 vs. 2.234) compared to results for control CP challenged birds. Sodium bisulfate diet inclusion can be a valuable alternative management strategy for improving FCR for necrotic enteritis challenged broilers.

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Devoted to success in gut health and preservationPerstorp Feed & Food is a global specialist in acid-based solutions withover 50 years of experience. We know the importance of gut health and feed preservation for the livestock industry. As Perstorp Feed & Food, we belong to the Swedish multinational Perstorp Group – a basic producer of organic acids such as propionic acid, formic acid and butyric acid. We have focused our skills, knowledge and resources on fi nding new and improved ways to improve gut health and preservation.

www.phytosynthese.com

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Like no one else.™vetagro.com

We are Vetagro ®.We aren’t from around here. We approach things differently.If you, too, challenge the norm and look at things from a unique perspective, then our conversation is just beginning.

For we have developed novel combinations of microencapsulated nutrients and additives to help maximize animal nutrition. The data is there. The performance is there. The proof is there. Now, we want you – a professional as distinct as us – to be there too.

For we are Vetagro®. And we, as you, are proudly like no one else.

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