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Research ArticleTrueperella pyogenes and Brucella abortusCoinfection in a Dog and a Cat on a Dairy Farm in Egypt withRecurrent Cases of Mastitis and Abortion

GamalWareth 12 Mohamed El-Diasty3 Falk Melzer1 Jayaseelan Murugaiyan4

Amir Abdulmawjood 5 Lisa D Sprague1 and Heinrich Neubauer1

1Friedrich-Loeffler-Institut Federal Research Institute for Animal Health Institute of Bacterial Infections and ZoonosesNaumburger Str 96a 07743 Jena Germany2Department of Pathology Faculty of Veterinary Medicine Benha University Moshtohor Toukh 13736 Egypt3Animal Health Research Institute-Mansoura Provincial Laboratory Mansoura Egypt4Institute of Animal Hygiene and Environmental Health Centre for Infectious Medicine Freie Universitat BerlinRobert-von-Ostertag-Str 7-13 14163 Berlin Germany5Institute of Food Quality and Food Safety Research Center for Emerging Infections and ZoonosesUniversity of Veterinary Medicine Hannover Bunteweg 17 30559 Hannover Germany

Correspondence should be addressed to Gamal Wareth gamalwarethhotmailcom

Received 19 September 2017 Accepted 11 February 2018 Published 20 March 2018

Academic Editor Giuliano Bettini

Copyright copy 2018 Gamal Wareth et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Trueperella pyogeneswas isolated from a dog and a cat with amixed infection with Brucella abortus Both lived on a dairy cattle farmwith a history of regular cases of abortion and mastitis Identification of the bacteria was done by means of MALDI-TOFMS loop-mediated isothermal amplification (LAMP) based on cpn60 partial 16S rRNA sequencing and growth on Loeffler SerumMediumIsolation of Trueperella pyogenes on the dairy farm highlights its neglected role in reproduction failure and draws attention to itseffects in the dairy industry in Egypt Diagnosis and control of abortion in Egypt should include Trueperella pyogenes as one ofpossible causes of abortion

1 Introduction

Trueperella (T) pyogenes is Gram positive haemolytic non-motile non-spore-forming facultative anaerobic coccobacil-lus The species was originally known as Corynebacteriumpyogenes and Actinomyces pyogenes and then as Arcanobac-terium pyogenes Based on the 16S rRNA signature nucleotidecomparisons and menaquinone and phospholipid compo-sitions the genus was divided into Arcanobacterium andTrueperella gen nov in honour of the German microbi-ologist Hans G Truper Currently the genus Trueperellaencompasses five different species which are T pyogenes Tabortisuis T bonsai T bernardiae and T bialowiezensis [1]

Trueperella pyogenes is a commensal and an opportunisticpathogen which can cause a variety of suppurative infectionsin livestock and wildlife and can elicit sporadic cases of

abortion due to endometritis and placentitis at any stage ofgestation [2] The bacteria can affect a wide range of animalspecies including cattle camel horse swine antelope bisonchicken pigeon turkey deer elephant gazelle macaw andreindeer [3] as well as companion animals such as dogsand cats [4] T pyogenes has recently been isolated fromlung abscesses of slaughtered one-humped camels in Cairopresenting with clinical and subclinical pulmonary infection[5] Different suppurative T pyogenes infections in livestockand companion animals are associated with a variety ofvirulence factors particularly exotoxin pyolysin and adhesins(fimbriae neuraminidases and collagen-binding protein)[6]

The present study describes the isolation of T pyogenesin the uterine discharge of a bitch after abortion and from acat presenting with pyometra Both animals lived on a dairy

HindawiVeterinary Medicine InternationalVolume 2018 Article ID 2056436 4 pageshttpsdoiorg10115520182056436

2 Veterinary Medicine International

farm with recurrent cases of abortion and mastitis due toBrucella (B) abortus biovar (bv) 1 infections [7] The aim ofthe current study was to identify and highlight a neglectedpathogen in Egypt which can circulate in pet animals incontact with livestock and cause reproductive failure withinthe dairy industry

2 Material and Methods

21 Bacteriology Uterine discharges were collected from thebitch and the cat under sterile conditions after positiveBrucella serology using Rose Bengal Test and ComplementFixation Test and sent to the OIE reference laboratoryof brucellosis at the Friedrich-Loeffler-Institut for Brucellaidentification and biotyping During cultivation on bloodagar for Brucella biotyping two types of bacterial colonieswere found Identification and biotyping of the putativeBrucella isolates were done by assessing colony morphologybiochemical reactions CO

2requirement production of H

2S

growth in the presence of the dyes thionine and fuchsinereaction with monospecific antisera (A M and R) andphage lysis (Wb Tb and F25) [8] Putative Trueperellastrains were additionally grown on Loeffler Serum Medium(Himeda Mumbai India) and tested for catalase activ-ity

22 MALDI Based Species Identification The microbialspecies identification was carried out using matrix-assistedlaser desorptionionization (MALDI-TOF MS) as describedelsewhere [9] In brief bacteria from single colonies wereinactivated by the addition of 300 120583L of HPLC grade waterand 900120583L of absolute ethanol For protein extractionthe suspensions were centrifuged at 11290119892 for 2min thesupernatants discarded and the resulting cell pellets air-driedto remove traces of ethanol Each pellet was reconstitutedin 50120583L of 70 formic acid and 50 120583L of acetonitrile Thesamples were then sonicated (100 amplitude and 10 dutycycle) for 1min on ice using a sonicator (UP100H HielscherUltrasound Technology Teltow Germany) Next sampleswere centrifuged at 11290119892 for 5min at room temperature andthe clear supernatant was collected One 120583L of each super-natant was spotted onto the MALDI target (MSP 96 targetpolished steel (MicroScout Target) plate Bruker DaltonikBremen Germany) air-dried and overlaid with 10 120583L ofsaturated 120572-cyano-4-hydroxycinnamic acid matrix solution(in 50 acetonitrile and 025 trifluoroacetic acid) TheMALDI measurements were carried out using a MicroflexLT (Bruker Daltonics Bremen Germany) instrument andMBT Compass Explorer 41 software (Bruker DaltonicsBremen Germany) The MALDI Biotyper manufacturerrsquosrecommendation on log score value of 0ndash3 for speciesidentification was followed Score values between 23 and30 indicate ldquohighly probable species identificationrdquo valuesbetween 20 and 229 indicate a ldquosecure genus identifica-tion and probable species identificationrdquo values between17 and 199 indicate ldquoprobable genus identificationrdquo andvalues between 0 and 169 indicate ldquono reliable identifica-tionrdquo

23 DNA Extraction AMOS-PCR and Partial 16S rRNASequencing Genomic DNA was extracted from heat inac-tivated individual bacterial colonies using the High PurePCR template preparation kit (Roche Applied SciencesMannheim Germany) according to the manufacturerrsquosinstructions AMOS-PCR for B abortus B melitensis Bovis and B suis was done as previously described [7 10]Partial 16S rRNAgenes of the bacterial isolates were amplifiedby PCR with the 16SUNI-L (51015840-AGA GTT TGA TCA TGGCTC AG-31015840) and 16SUNI-R (51015840-GTG TGA CGG GCG GTGTGT AC-31015840) primer pair (Jena Bioscience GmbH JenaGermany) to generate amplicons of approx 1400-bp [11]PCR products were analyzed by agarose gel electrophoresisbands were cut out and DNA was purified using a GelExtraction Kit (Qiagen Hilden Germany) according tothe manufacturerrsquos recommendations Cycle sequencing ofthe partial 16S rRNA genes was done in both directionsby using forward and reverse amplification primers witha BigDye Terminator Version 11 Cycle Sequencing Kit(Applied Biosystems Darmstadt Germany) according to therecommendations of the manufacturer Sequencing productswere analyzed with an ABI Prism 3130 Genetic Analyzer(Applied Biosystems) Identification of isolates was done bya BLAST search (httpswwwncbinlmnihgovblast) using16S rRNA gene sequences

24 Loop-Mediated Isothermal Amplification (LAMP) Anewly designed loop-mediated isothermal amplification(LAMP) assay based on cpn60 (encoding for chaperonin 60)was carried out in a total volume of 25 120583L containing 05 120583Leach of pho-F3 and Pyo-B3 primer (25 pmol120583L) equivalentto 05 120583M final concentration 1120583L each of Pyo-LoopF andPyo-LoopB primer (25 pmol120583L) equivalent to 10120583M finalconcentration 2120583L each of Pyo-FIP and Pyo-BIP primer(25 pmol120583L) equivalent to 20120583M final concentration and15 120583L Isothermal Master Mix Iso-001 (Optigene HorshamUK) Subsequently 3120583L DNA was added as a template TheLAMP assay was run at 70∘C for 30min with a meltingcurve analysis step (annealing curve 98∘C to 80∘C ramping at005∘C per s) in a portable real-time fluorometer (Genie IIOptigene West Sussex UK) according to the manufacturerrsquosinstructions as previously described [12]

3 Results and Discussion

In addition to B abortus bv1 a Gram positive catalasenegative aerobic nonmotile szlig-haemolytic bacterium wasrecovered from a bitch who had recently aborted and froma cat suffering from an open pyometra Both animals livedon a dairy farm with regular cases of abortion and mastitis[7] These recovered bacteria produced H

2S and were able

to grow under aerobic conditions with and without CO2

They did not react with the tested monospecific antisera andphages specific for Brucella and produced no amplicon in theAMOS-PCR MALDI-TOF MS identified these bacteria asT pyogenes with a log score of 218 (dog isolate) and a logscore of 202 (cat isolate) respectively These log score valuesconfirm the identification at the species level MALDI-TOF

Veterinary Medicine International 3

Table 1 Results of LAMP including detection time and annealing temperature of each isolate and positive and negative control

Number Isolate number 1 Isolate number 2 Positive control Negative controlSample ID 15RB7429H 15RB7430H T pyogenes DSM 20594 HPLC water and Master mixResult +ve +ve +ve minusveDetection time 1000 730 1730 00Annealing 897 896 896 00

000

500

001

000

001

500

002

000

003

000

002

500

Time (hhmmss)

FLI number 65FLI number 66

MM minusve controlDSM 20594 +ve T pyogenes [12]

minus20000

0

20000

40000

60000

80000

100000

Fluo

resc

ence

Figure 1 Loop amplification signal of the LAMPproducts of the twoT pyogenes isolates the reference strainT pyogenesDSM20594 andthe negative control

MS for Trueperella differentiation with a log score around2 shows comparable discriminating power with molecularmethods it is a rapid and accurate tool for T pyogenesdiagnosis [13] Partial 16S rRNA sequencing confirmed theisolates to be T pyogenes and cultivation on Loeffler serumrevealed the typical pitting of the serum slope The specieswas also identified using the cpn60 LAMP assay A loopamplification signal of the LAMP products was observedfor both T pyogenes isolates and for the reference strainT pyogenes DSM 20594 and none in the negative controlcontaining water and LAMP-Mastermix (Figure 1)Themeanof the annealing temperature of the amplicons was 896∘C sdplusmn 005 s (Figure 2) The cpn60 LAMP assay allowed a reliablerapid and low cost identification of T pyogenes (Table 1)

The chaperonin 60 encoding gene has been previouslyused for the identification of various Gram positive bacteriaand a chaperonin sequence database containing a largecollections of sequences including gene cpn60 of T pyogenesis available This was comparable to the previously describedLAMP-mediated identification ofA pluranimalium using plagene [14]

Trueperella pyogenes is a ubiquitous occurring organismand is frequently found as a commensal in the oropharynxupper respiratory tract and gastrointestinal tract of livestock[2] However underlying chronic illness innate immunitypoor animal husbandry and production methods appear to

Anneal derivative



minus2000000

000

2000000

4000000

6000000

Fluo

resc

ence

der

ivat

ive

8400 8900 94007900Temperature (∘C)

Figure 2 Annealing reaction curves of the respective amplicons

influence the virulence of the agent [15ndash18] It can readily betransmitted by biting flies and contaminated farm and dairyequipment [6]

In Egypt the dairy industry suffers from large financiallosses due to reduced fertility andmilk yield as a consequenceof uterine infections and mastitis caused not only by brucel-losis [5] but also by other agents such as E coli Fusobacteriumnecrophorum and T pyogenes [19 20] Although T pyogenesis a well-known agent causing reproductive disorders inmale and female livestock species [4] its role is neglected asprimary cause of abortion in cattle as diagnosis of abortionis focusing only on predominately classical agents such asBrucella species Circulating of the bacterium in dog and catkept in dairy farm with history of abortion and mastitis isalarming and representing a potential reservoir of infectionfor livestock Thus screening for this agent should be consid-ered in cases of seronegativity to Brucella On the other handeradication of pet animals has to be implemented and takeninto account in control measures to reduce dissemination ofpathogens T pyogenesis is not a sufficiently known pathogenin animals and humans because of inadequate identificationof this bacterium that should be better known to clinicalmicrobiologists Diagnosis of abortion in cattle and controlhas to include T pyogenesis especially in farm suffering frommastitis and sporadic abortion

Disclosure

This manuscript has not been submitted for publicationelsewhere and has been approved by all coauthors


Conflicts of Interest

The authors declare that they do not have any conflicts ofinterest

Acknowledgments

The authors would like to acknowledge the Academy of Sci-entific Research and Technology (ASRT) Egypt for financialsupport This work belongs to BrucMedNet project (ID 698)funded by the ARIMNet2

References

[1] A F Yassin H Hupfer C Siering and P Schumann ldquoCompar-ative chemotaxonomic and phylogenetic studies on the genusArcanobacterium Collins et al 1982 emend Lehnen et al 2006proposal for Trueperella gen nov and emended description ofthe genus Arcanobacteriumrdquo International Journal of Systematicand Evolutionary Microbiology vol 61 no 6 pp 1265ndash12742011

[2] B H Jost and S J Billington ldquoArcanobacterium pyogenesmolecular pathogenesis of an animal opportunistrdquo Antonie vanLeeuwenhoek vol 88 no 2 pp 87ndash102 2005

[3] S J Billington K W Post and B H Jost ldquoIsolation ofArcanobacterium (Actinomyces) pyogenes from cases of felineotitis externa and canine cystitisrdquo Journal of Veterinary Diag-nostic Investigation vol 14 no 2 pp 159ndash162 2002

[4] M G Ribeiro R M Risseti C A D Bolanos et al ldquoTrueperellapyogenes multispecies infections in domestic animals a retro-spective study of 144 cases (2002 to 2012)rdquoVeterinary Quarterlyvol 35 no 2 pp 82ndash87 2015

[5] G Wareth J Murugaiyan D F Khater and S A MoustafaldquoSubclinical pulmonary pathogenic infection in camels slaugh-tered in Cairo Egyptrdquo The Journal of Infection in DevelopingCountries vol 8 no 7 pp 909ndash913 2014

[6] R M Risseti E Zastempowska M Twaruzek et al ldquoVirulencemarkers associated with Trueperella pyogenes infections in live-stock and companion animalsrdquo Letters in Applied Microbiologyvol 65 no 2 pp 125ndash132 2017

[7] G Wareth F Melzer M El-Diasty et al ldquoIsolation of Brucellaabortus from a dog and a cat confirms their biological role inre-emergence and dissemination of bovine brucellosis on dairyfarmsrdquo Transboundary and Emerging Diseases vol 64 no 5 ppe27ndashe30 2017

[8] G G Alton L M Jones R D Angus and J M VergerTechniques for the Brucellosis Laboratory Instituttional de laRecherche Agronomique Paris France 1988

[9] J Murugaiyan B Walther I Stamm et al ldquoSpecies differen-tiation within the Staphylococcus intermedius group using arefined MALDI-TOF MS databaserdquo Clinical Microbiology andInfection vol 20 no 10 pp 1007ndash1015 2014

[10] B J Bricker and S M Halling ldquoDifferentiation of Brucellaabortus bv 1 2 and 4 Brucella melitensis Brucella ovis andBrucella suis bv 1 by PCRrdquo Journal of Clinical Microbiology vol32 no 11 pp 2660ndash2666 1994

[11] P Kuhnert S E Capaul J Nicolet and J Frey ldquoPhylogeneticpositions of Clostridium chauvoei and Clostridium septicumbased on 16S rRNA gene sequencesrdquo International Journal ofSystematic Bacteriology vol 46 no 4 pp 1174ndash1176 1996

[12] A Abdulmawjood J Wickhorst O Hashim et al ldquoApplicationof a loop-mediated isothermal amplification (LAMP) assay formolecular identification of Trueperella pyogenes isolated fromvarious originsrdquo Molecular and Cellular Probes vol 30 no 4pp 205ndash210 2016

[13] L Z Moreno C E C Matajira B L P da Costa et al ldquoChar-acterization of porcine Trueperella pyogenes by matrix-assistedlaser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS) molecular typing and antimicrobial sus-ceptibility profiling in Sao Paulo Staterdquo Comparative Immunol-ogy Microbiology amp Infectious Diseases vol 51 pp 49ndash53 2017

[14] A Abdulmawjood J Wickhorst O Sammra et al ldquoDevel-opment of a loop-mediated isothermal amplification (LAMP)assay for rapid and sensitive identification of ArcanobacteriumpluranimaliumrdquoMolecular andCellular Probes vol 29 no 6 pp468ndash472 2015

[15] N Dyer K B Register D Miskimins and T Newell ldquoNecroticpharyngitis associated with Mycoplasma bovisinfections inAmerican bison (Bison bison)rdquo Journal of Veterinary DiagnosticInvestigation vol 25 no 2 pp 301ndash303 2013

[16] J S Agerholm T K Jensen J F Agger M Y Engelsma andH I J Roest ldquoPresence of Coxiella burnetii DNA in inflamedbovine cardiac valvesrdquo BMC Veterinary Research vol 13 no 1article 69 2017

[17] L C Carneiro J G Cronin and I M Sheldon ldquoMechanismslinking bacterial infections of the bovine endometrium todisease and infertilityrdquo Reproductive Biology vol 16 no 1 pp1ndash7 2016

[18] P H Kvist E S Jensen B Aalbaek and H E Jensen ldquoEvalu-ation of the pathology pathogenesis and aetiology of auricularelephantiasis in slaughter pigsrdquo Journal of Veterinary MedicineSeries A Physiology Pathology Clinical Medicine vol 49 no 10pp 517ndash522 2002

[19] H El-Khadrawy W Ahmed M Zaabal and E Hanafi ldquoStrate-gies for diagnosis and treatment of uterine infection in bovinesrdquoGlobal Veterinaria vol 15 no 5 pp 98ndash105 2015

[20] M L S Bicalho F S Lima V S Machado et al ldquoAssociationsamong Trueperella pyogenes endometritis diagnosis and preg-nancy outcomes in dairy cowsrdquo Theriogenology vol 85 no 2pp 267ndash274 2016

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farm with recurrent cases of abortion and mastitis due toBrucella (B) abortus biovar (bv) 1 infections [7] The aim ofthe current study was to identify and highlight a neglectedpathogen in Egypt which can circulate in pet animals incontact with livestock and cause reproductive failure withinthe dairy industry

2 Material and Methods

21 Bacteriology Uterine discharges were collected from thebitch and the cat under sterile conditions after positiveBrucella serology using Rose Bengal Test and ComplementFixation Test and sent to the OIE reference laboratoryof brucellosis at the Friedrich-Loeffler-Institut for Brucellaidentification and biotyping During cultivation on bloodagar for Brucella biotyping two types of bacterial colonieswere found Identification and biotyping of the putativeBrucella isolates were done by assessing colony morphologybiochemical reactions CO

2requirement production of H

2S

growth in the presence of the dyes thionine and fuchsinereaction with monospecific antisera (A M and R) andphage lysis (Wb Tb and F25) [8] Putative Trueperellastrains were additionally grown on Loeffler Serum Medium(Himeda Mumbai India) and tested for catalase activ-ity

22 MALDI Based Species Identification The microbialspecies identification was carried out using matrix-assistedlaser desorptionionization (MALDI-TOF MS) as describedelsewhere [9] In brief bacteria from single colonies wereinactivated by the addition of 300 120583L of HPLC grade waterand 900120583L of absolute ethanol For protein extractionthe suspensions were centrifuged at 11290119892 for 2min thesupernatants discarded and the resulting cell pellets air-driedto remove traces of ethanol Each pellet was reconstitutedin 50120583L of 70 formic acid and 50 120583L of acetonitrile Thesamples were then sonicated (100 amplitude and 10 dutycycle) for 1min on ice using a sonicator (UP100H HielscherUltrasound Technology Teltow Germany) Next sampleswere centrifuged at 11290119892 for 5min at room temperature andthe clear supernatant was collected One 120583L of each super-natant was spotted onto the MALDI target (MSP 96 targetpolished steel (MicroScout Target) plate Bruker DaltonikBremen Germany) air-dried and overlaid with 10 120583L ofsaturated 120572-cyano-4-hydroxycinnamic acid matrix solution(in 50 acetonitrile and 025 trifluoroacetic acid) TheMALDI measurements were carried out using a MicroflexLT (Bruker Daltonics Bremen Germany) instrument andMBT Compass Explorer 41 software (Bruker DaltonicsBremen Germany) The MALDI Biotyper manufacturerrsquosrecommendation on log score value of 0ndash3 for speciesidentification was followed Score values between 23 and30 indicate ldquohighly probable species identificationrdquo valuesbetween 20 and 229 indicate a ldquosecure genus identifica-tion and probable species identificationrdquo values between17 and 199 indicate ldquoprobable genus identificationrdquo andvalues between 0 and 169 indicate ldquono reliable identifica-tionrdquo

23 DNA Extraction AMOS-PCR and Partial 16S rRNASequencing Genomic DNA was extracted from heat inac-tivated individual bacterial colonies using the High PurePCR template preparation kit (Roche Applied SciencesMannheim Germany) according to the manufacturerrsquosinstructions AMOS-PCR for B abortus B melitensis Bovis and B suis was done as previously described [7 10]Partial 16S rRNAgenes of the bacterial isolates were amplifiedby PCR with the 16SUNI-L (51015840-AGA GTT TGA TCA TGGCTC AG-31015840) and 16SUNI-R (51015840-GTG TGA CGG GCG GTGTGT AC-31015840) primer pair (Jena Bioscience GmbH JenaGermany) to generate amplicons of approx 1400-bp [11]PCR products were analyzed by agarose gel electrophoresisbands were cut out and DNA was purified using a GelExtraction Kit (Qiagen Hilden Germany) according tothe manufacturerrsquos recommendations Cycle sequencing ofthe partial 16S rRNA genes was done in both directionsby using forward and reverse amplification primers witha BigDye Terminator Version 11 Cycle Sequencing Kit(Applied Biosystems Darmstadt Germany) according to therecommendations of the manufacturer Sequencing productswere analyzed with an ABI Prism 3130 Genetic Analyzer(Applied Biosystems) Identification of isolates was done bya BLAST search (httpswwwncbinlmnihgovblast) using16S rRNA gene sequences

24 Loop-Mediated Isothermal Amplification (LAMP) Anewly designed loop-mediated isothermal amplification(LAMP) assay based on cpn60 (encoding for chaperonin 60)was carried out in a total volume of 25 120583L containing 05 120583Leach of pho-F3 and Pyo-B3 primer (25 pmol120583L) equivalentto 05 120583M final concentration 1120583L each of Pyo-LoopF andPyo-LoopB primer (25 pmol120583L) equivalent to 10120583M finalconcentration 2120583L each of Pyo-FIP and Pyo-BIP primer(25 pmol120583L) equivalent to 20120583M final concentration and15 120583L Isothermal Master Mix Iso-001 (Optigene HorshamUK) Subsequently 3120583L DNA was added as a template TheLAMP assay was run at 70∘C for 30min with a meltingcurve analysis step (annealing curve 98∘C to 80∘C ramping at005∘C per s) in a portable real-time fluorometer (Genie IIOptigene West Sussex UK) according to the manufacturerrsquosinstructions as previously described [12]

3 Results and Discussion

In addition to B abortus bv1 a Gram positive catalasenegative aerobic nonmotile szlig-haemolytic bacterium wasrecovered from a bitch who had recently aborted and froma cat suffering from an open pyometra Both animals livedon a dairy farm with regular cases of abortion and mastitis[7] These recovered bacteria produced H

2S and were able

to grow under aerobic conditions with and without CO2

They did not react with the tested monospecific antisera andphages specific for Brucella and produced no amplicon in theAMOS-PCR MALDI-TOF MS identified these bacteria asT pyogenes with a log score of 218 (dog isolate) and a logscore of 202 (cat isolate) respectively These log score valuesconfirm the identification at the species level MALDI-TOF




000

500

001

000

001

500

002

000

003

000

002

500

Time (hhmmss)



minus20000

0

20000

40000

60000

80000

100000

Fluo

resc

ence





Anneal derivative



minus2000000

000

2000000

4000000

6000000

Fluo

resc

ence

der

ivat

ive

8400 8900 94007900Temperature (∘C)




Disclosure





Acknowledgments


References

























Microbiology












Volume 2018



Agronomy







Volume 2018

Zoology






Volume 2018


Advances in

Virolog y










000

500

001

000

001

500

002

000

003

000

002

500

Time (hhmmss)



minus20000

0

20000

40000

60000

80000

100000

Fluo

resc

ence





Anneal derivative



minus2000000

000

2000000

4000000

6000000

Fluo

resc

ence

der

ivat

ive

8400 8900 94007900Temperature (∘C)




Disclosure





Acknowledgments


References

























Microbiology












Volume 2018



Agronomy







Volume 2018

Zoology






Volume 2018


Advances in

Virolog y










Acknowledgments


References

























Microbiology












Volume 2018



Agronomy







Volume 2018

Zoology






Volume 2018


Advances in

Virolog y











Microbiology












Volume 2018



Agronomy







Volume 2018

Zoology






Volume 2018


Advances in

Virolog y





