Research ArticleMultilocus Sequencing of Corynebacterium pseudotuberculosisBiotype Ovis Strains

Boglaacuterka Sellyei1 Krisztiaacuten Baacutenyai1 Daacuteniel Bartha1 Istvaacuten Hajtoacutes2

Laacuteszloacute Fodor3 and Laacuteszloacute Makrai3

1CAR HAS Institute for Veterinary Medical Research PO Box 18 Budapest 1581 Hungary2Government Office for Borsod-Abauj-Zemplen County Vologda U 1 Miskolc 3525 Hungary3Department of Microbiology and Infectious Diseases University of Veterinary Medicine PO Box 22 Budapest 1581 Hungary

Correspondence should be addressed to Laszlo Makrai makrailaszlounivethu

Received 4 March 2017 Revised 26 July 2017 Accepted 8 August 2017 Published 11 October 2017

Academic Editor Chiu-Chung Young

Copyright copy 2017 Boglarka Sellyei et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Thirteen Corynebacterium pseudotuberculosis biotype ovis strains isolated from clinical cases of caseous lymphadenitis in Hungarywere characterised using multilocus sequencing and their phylogenetic comparison was carried out on the basis of fourhousekeeping genes (groEL1 infB dnaK and leuA) The in silico analysis of the 16 frequently studied housekeeping genesshowed that C pseudotuberculosis strains could be readily distinguished from C diphtheriae and C ulcerans strains howeversequences of the same genes in the two biotypes of the C pseudotuberculosis were highly similar the heterogeneity values werelow Genes dnaK infB groEL1 and leuA showed marked genetic variation within C pseudotuberculosis and strains of the twobiotypes of C pseudotuberculosis could be differentiated Analysis of the individual genes showed a fairly conservative nature of Cpseudotuberculosis biotype ovis strainsThe greatest genetic differentiation was seen in the dnaK and infB genes and concatenationsof these two genes were very useful in the genetic separation of the studied strains

1 Introduction

Corynebacterium pseudotuberculosis was isolated from asheep and described by Preisz [1] as a Gram-positive fac-ultative intracellular pathogen Two major biotypes can bedistinguished based on the ability of reduction of nitrate thenitrate negative biotype (C pseudotuberculosis biotype ovis)causes caseous lymphadenitis (CLA) in small ruminants [2ndash4] whereas the nitrate positive biotype (C pseudotuberculosisbiotype equi) is the causative agent of ulcerative lymphangitisin horses cows camels buffaloes and occasionally humans[5 6] CLA is a chronic contagious disease that is char-acterised by abscess formation in or near major peripherallymph nodes (external form) or within the internal organsand lymph nodes (internal form) CLA occurs worldwidehowever it is more frequent in the tropical areas and causesimportant economic losses in ovine and caprine herds byreducing wool meat and milk production [4 7ndash10]

Classification of C pseudotuberculosis was originallybased on cultural morphological and biochemical char-acteristics [11] In addition to nitrate reduction variousmolecular methods restriction fragment length polymor-phism (RFLP) of chromosomal DNA ribotyping and wholegenome sequence analysis were used for the differentiation ofC pseudotuberculosis biotypes [12ndash14] C pseudotuberculosisbiotype equi strains showed greater genetic divergence thanC pseudotuberculosis biotype ovis strains when examinedwith pulsed field gel electrophoresis (PFGE) BOX-PCRrandom amplified polymorphic DNA (RAPD) and amplifi-cation of DNA surrounding rare restriction sites (ADSRRS)[3 15 16]

Multilocus sequence typing has become popular in bac-terial genotyping technique over the past decade includinggenotyping Corynebacterium spp it allows the identificationof potential molecular genetic marker(s) in conservative

HindawiBioMed Research InternationalVolume 2017 Article ID 1762162 7 pageshttpsdoiorg10115520171762162

2 BioMed Research International

Table 1 The list and registered data of C pseudotuberculosis strains included in the examinations

ID Host Place of isolation Strains YearC1 Goat Szentistvan C pseudotuberculosis biotype ovis 1994C3 Sheep Vizsoly C pseudotuberculosis biotype ovis 2003C7 Goat Vizsoly C pseudotuberculosis biotype ovis 2003C11 Sheep Boldogkovaralja C pseudotuberculosis biotype ovis 2005C12 Goat Erd C pseudotuberculosis biotype ovis 2005C14 Goat Alsozsolca C pseudotuberculosis biotype ovis 2006C15 Goat Rudabanya C pseudotuberculosis biotype ovis 2006C19 Goat (DSM-7180) C pseudotuberculosis biotype ovis 1988C21 Horse (DSM-7177) C pseudotuberculosis biotype equi 1988C22 Sheep Mezocsat C pseudotuberculosis biotype ovis 2013C23 Goat Cseteny C pseudotuberculosis biotype ovis 2013C24 Sheep Petervasara C pseudotuberculosis biotype ovis 2009C25 Goat Szendro C pseudotuberculosis biotype ovis 2011C26 Goat Csongrad C pseudotuberculosis biotype ovis 2014C27 Goat Kisvasarhely C pseudotuberculosis biotype ovis 2014

genes for the differentiation of closely related bacterial strains[17 18]

The objective of this study was characterisation of Hun-garian field strains ofC pseudotuberculosis biotype ovis usingmultilocus sequencing and their phylogenetic comparison onthe basis of selected housekeeping genes

2 Materials and Methods

21 Bacterial Strains and Culture Condition Thirteen Cpseudotuberculosis biotype ovis field strains isolated between1994 and 2014 were randomly selected from the straincollection of the Department of Microbiology and Infec-tious Diseases Faculty of Veterinary Science Szent IstvanUniversity Budapest Hungary After identification usingstandard methods [20] they had been stored at minus80∘C till theexaminations Details about the strains used in this study aredescribed in Table 1

Reference strains of C pseudotuberculosis biotype ovis(DSM-7180) and C pseudotuberculosis biotype equi (DSM-7177) from the Leibniz Institute German Collection ofMicroorganisms and Cell Cultures (Braunschweig Ger-many) were also included in the examinations

The strainswere cultured onColumbia blood agar (LabMLancashire UK) with the addition of 5 (volvol) steriledefibrinated sheep blood and incubated at 37∘C for 48 h

22 In Silico Sequence Analysis and Primer Design In orderto distinguish C pseudotuberculosis biotypes from the closelyrelated species and to find potential markers the nucleotidesequences of 16 housekeeping genes of 15 C pseudotuber-culosis 13 C diphtheriae strains and one C ulcerans strain(Table 2) were comparedThe nucleotide sequences of 7 genesincluding the ATP synthase alpha chain (atpA) the DNApolymerase III alpha subunit (dnaE) the chaperone Hsp70(dnaK) the elongation factor G (fusA) the 2-isopropylmalatesynthase (leuA) the 2-oxoglutarate dehydrogenase E1 and

E2 components (odhI) and the DNA-directed RNA poly-merase beta chain (rpoB) were taken from [22 23] whilesequences of 9 genes involving ATP synthase beta chain(atpD) the heat shock proteins GroEL and GroES (groESgroEL1 and groEL2) the translation initiation factor IF2(infB) the DNA recombinational repair system (recA recN)the alpha subunit of DNA dependent RNA polymerase(rpoA) and the manganese-dependent superoxide dismu-tase (sodA) were downloaded from the NCBI database(httpwwwncbinlmnihgovgenbank) In silico analysisof the selected genes was performed with the ClustalWmultiple alignment algorithm [24] in MegAlign software ofthe Lasergene 7 program suite (DNAStarMadisonWIUSA)to find potentially useful markers for genetic differentiationand molecular epidemiological investigations

Four genes (groEL1 infB dnaK and leuA) were selectedfor primer design they are located at different genomicregions of bacteria (Figure 1) Primer sets were designed toamplify 580 bp 654 bp 641 bp and 647 bp long fragmentsfrom the respective genomic regions pending on the genesthe number of SNPs varied from 7 to 31 within the amplifiedfragments (Table 3) PCR primers were designed using OligoPrimer Analysis software 7 [25]

23 PCR and Sequencing A loopful of 48 h culture of Cpseudotuberculosis was suspended in 6 Chelex-solution(Bio-Rad Laboratories Hercules CA USA) and then heatedto 65∘C for 30min and to 100∘C for 8min afterwards it wascentrifuged The supernatant was collected in a new tubeand stored at minus20∘C until use Fragments of the selectedhousekeeping genes were amplified by PCR in an AppliedBiosystems 2720 Thermal Cycler (Thermo Fisher ScientificCarlsbad CAUSA)The 25 120583l amplification reactionmixturecontained 1x Dream Taq Buffer 200 nM dNTP-mix 1 120583Mof each primer 1 U Dream Taq (Thermo Fisher ScientificCarlsbad CA USA) template DNA and water The PCRprogram included 3min of initial denaturation at 95∘C and

BioMed Research International 3

Table 2 The source of Corynebacterium sp examined in the in silico sequence analysis

C pseudotuberculosi119904amp Biotype Host Origin C diphtheria119890sect Host OriginFRC41 Ovis Human France NCTC 13129 Human UK399-5 Ovis Sheep Scotland BH8 Human Rio de JaneiroP54B96 Ovis Antelope South Africa C7 Human Atlanta GA USA1002 Ovis Goat Brazil 241 Human Rio de JaneiroC231 Ovis Sheep Australia HC01 Human Rio de JaneiroI19 Ovis Cow Israel VA01 Human Rio de JaneiroPAT10 Ovis Sheep Argentina CDCE 8392 Human Bethesda MD USA4202-A Ovis Sheep Australia 31A Human Rio de Janeiro267 Ovis Llama USA HC03 Human Rio de Janeiro316 Equi Horse USA HC02 Human Rio de JaneiroCIP 5297 Equi Horse Kenya INCA 402 Human Rio de Janeiro106-A Equi Horse USA PW8 Human New York31 Equi Buffalo Egypt HC04 Human Rio de Janeiro258 Equi Horse Belgium C ulceran119904

Cp162 Equi Camel UK BR-AD22 Dog Rio de Janeiroamp[14] sect[19] [21]

gi|300857417|ref|NC_0143291| Corynebacterium pseudotuberculosis2337913 bp

groEL1fusA

rpoB

rpoAgroES

infBdnaK (2081520ndash2083352)

sodA

groEL2

leuA

atpAatpD

odhI

11m

1m

12m

13m

14m

15m

16m17m 18m

19m

2m

21m

22m

23m

100k

200k

300k

400k

500k600k700k

800k

900k

2337913

recN

recA

dnaE

(1395670ndash1398324)

(161250ndash163067)

(432546ndash434168)

Figure 1 The location of in silico studied 16 housekeeping genes atthe complete genome map of C pseudotuberculosis FRC41 [19] Thepartial regions of rimmed four genes in frames were amplified andsequenced in this study

then 30 cycles at 95∘C for 30 s at 60∘C for 30 and at 72∘Cfor 30 s and 7min of the final elongation PCR productswere purified for direct sequencing using the Geneaid PCRpurification Kit (Geneaid Biotech New Taipei Taiwan)Templates were sequenced with the PCR primers using theBigDye Terminator Ready Reaction Mix v31 Nucleotidesequences were run on an ABI Prism 3100 Genetic Analyzer(Applied Biosystems Foster City CA)

24 Phylogenetic Analysis Fragments of four protein-codinghighly diverse housekeeping genes (groEL1 infB dnaK andleuA) were sequenced for all strains

Sequences were aligned using the ClustalW algorithm intheMEGA6 software [26] and trimmedmanually at the sameposition before being used for comparison with sequences ofotherC pseudotuberculosis strains deposited in theGenBankThe same gene set from C diphtheriae HC02 was used as anoutgroup

After the single gene alignments the sequences werejoined to make a concatemer of loci at head-to-tail in-frame The phylogenetic trees were constructed by using theneighbour joining (NJ)methodThebootstrap technique [27]was employed to evaluate the reliability of tree topologies byresampling the sequence alignment 1000 times

25 Nucleotide Sequence Accession Numbers Thedeterminedpartial gene sequences were deposited in GenBank underaccession numbers from MF491615 to MF491629 (dnaK)from MF464070 to MF464084 (infB) MF476990 and fromMF476992 to MF477005 (groEL1) MF446654 and fromMF446656 to MF446669 (leuA)

3 Results

As the use of MLST protocol of Corynebacterium diphtheriae(httpspubmlstorgcdiphtheriae) proved to be inadequatefor analysis of C pseudotuberculosis strains developing ofnew scheme has been required

The in silico analysis of the 16 frequently studied house-keeping genes showed that C pseudotuberculosis strainscould be readily distinguished by sequence analysis from Cdiphtheriae and C ulcerans strains however sequences of thesame genes of the C pseudotuberculosis strains belonging tothe two biotypes were highly similar and the heterogeneityvalues were low (Table 4) Genes dnaK infB groEL1 andleuA showed marked genetic variation within C pseudotu-berculosis biotypes and they were selected for further genetic

4 BioMed Research International

Table3Prim

ersequ

encesu

sedfora

mplificatio

nandsequ

ence

analysis

Locus

Putativ

egenep

rodu

ctPrim

ername

Sequ

ence

Prod

uctsize(bp

)Po

sitionon

NC

0143291(FRC

41strain)

dnaK

Chaperon

eHsp70

Cpsd

naKF

51015840-TCC

TTAC

CAGTG

CCCT

TATC

C-31015840

580

2081944ndash

2081964

Cpsd

naKR

51015840-G

AGTT

CCAG

CGCA

TCAC

C-31015840

2082507ndash2082524

groE

L1Heatsho

ckprotein

Cpsg

roEL

1F51015840-ACC

TTCA

CCGGAT

CATT

G-31015840

654

1978987ndash197900

4Cp

sgroEL

1R51015840-TTG

GTG

ATCG

TCGTA

AAG

C-31015840

1979623ndash197964

1

infB

Transla

tioninitiationfactor

IF2

CpsinfBF

51015840-ATT

GCG

GGAC

TTGGAC

G-31015840

641

1397768ndash1397784

CpsinfBR

51015840-G

CATT

ATGCT

GCA

CAAG

ACG-31015840

1398390ndash

139840

9

leuA

2-iso

prop

ylmalates

ynthase

CpsleuAF

51015840-AGCT

CAGTG

CGCG

GTT

GAC

C-31015840

647

161252ndash161271

CpsleuAR

51015840-ATG

GCG

TCGCG

GGTT

CG-31015840

161915ndash161899

BioMed Research International 5

Antelope Cow

Human

Goat Sheep

Llama

Horse

Bualo

Camel

Biotype ovis (2081520ndash2083352)

005

Biotype equi

C7

C12C15C23C24C25C3C pseudotuberculosis PAT10

C pseudotuberculosis C231

C14C26C11C22C1C19 (DSM-7180)

C pseudotuberculosis 267

C pseudotuberculosis I19C pseudotuberculosis P54B96C pseudotuberculosis FRC41C pseudotuberculosis 1002

C27C pseudotuberculosis 399-5

C pseudotuberculosis Cp162C pseudotuberculosis 316C pseudotuberculosis 106-AC pseudotuberculosis 31

C21 (DSM-7177)C pseudotuberculosis 258C pseudotuberculosis CIP5297C diphtheriae HC02

10074

9691

94

9887

97

95

70

78

71

89

C pseudotuberculosis 4202-A

Figure 2 Phylogenetic reconstruction based on the concatenated partial sequence of dnaK (520 bp) infB (577 bp) groEL1 (588 bp) and leuA(597 bp) genes from the Hungarian field isolates and DSM type strains Analysis was conducted using the neighbour joining (NJ) methodThe 1000 bootstrap (BT) values are indicated at branching points Bar estimated nucleotide substitutions

differentiation andmolecular epidemiological investigationsThe sequence diversity in these regions exceeded 5

C pseudotuberculosis biotype ovis and C pseudotuber-culosis biotype equi field strains were differentiated withsequence analysis of the target gene fragments Analysis ofthe individual genes showed a fairly conservative nature of Cpseudotuberculosis biotype ovis strains although segregationof some Hungarian strains (eg C1 and C19 C3 C23 andC27) together with strains from Australia Israel USA SouthAfrica Scotland France and Brazil was also evident but theywere clearly located in the cluster of C pseudotuberculosisbiotype ovis The greatest genetic differentiation was seen inthe dnaK and infB genes and concatenations of these two

genes were very useful in the genetic separation of the studiedstrains (Figure 2)

4 Discussion

C pseudotuberculosis biotype ovis is the causative agent ofcaseous lymphadenitis a contagious chronic disease of sheepand goats [3]

Several previous studies showed high sequential homol-ogy of the C pseudotuberculosis biovar ovis strains confirm-ing their clonal origin however diversity of the clinical signsin sheep and goats and different efficacy of the vaccinessuggest that more than one pathogen clone can exist [14]

6 BioMed Research International

Table 4 Nucleic acid average sequence divergence of housekeeping genes in Corynebacterium species in the in silico study

Gene Full length(bp)

Average sequence divergenceC diphtheriae

versusC pseudotuberculosis

C ulceransversus

C pseudotuberculosis

C pseudotuberculosisbiotype ovis versus

equigroEL1 1641 629 279 11infB 2886 136 60 08dnaK 1836 91 50 06leuA 1818 140 81 05odhI 3687 125 497 04atpD 1446 62 32 04recN 1740 270 106 04dnaE 3561 582 71 03groEL2 1641 595 276 03recA 1110 141 73 03rpoA 1017 77 35 03fusA 2127 39 26 02groES 297 65 35 02rpoB 2974 80 33 02sodA 600 100 43 02atpA 1630 84 21 00

The analysis of sequence data of 16 frequently studiedhousekeeping genes (atpA dnaE dnaK fusA leuA odhIrpoB atpD groES groEL1 groEL2 infB recA recN rpoAand sodA) of 15 C pseudotuberculosis 13 C diphtheriaeand C ulcerans genomes from the GenBank proved to berelated and all studied genes could be used to differentiateC diphtheriae C ulcerans and C pseudotuberculosis strainsAll these genes allow the differentiation of biotype ovis andbiotype equi strains of C pseudotuberculosis but for detailedexamination of C pseudotuberculosis biovar ovis field isolatesfour genes (dnaK groEL1 infB and leuA) seemed to havehigher differentiation power The partial sequence analysisof adequate genes delineated phylogenetic trees with varioustopology and depth of branches The highest resolution andtopology of the tree were obtained with the dnaK sequences

The clusters could be explained by movement of breed-ing animals but no epidemiological connection was foundbetween the examined C pseudotuberculosis strains Thedistance between the geographical place of isolation and theyear of the isolation do not support any epidemiologicalconnection between the strains so circulation of severalclones of C pseudotuberculosis biotype ovis was verified

According to our data multilocus sequence typing candifferentiate C pseudotuberculosis and the most importantCorynebacterium species and examination of selected geneshelps to differentiate the two biotypes of C pseudotuberculo-sis and it can be used for epidemiological follow-up of thesestrains

Conflicts of Interest

None of the authors have any conflicts of interest to declare

Authorsrsquo Contributions

All authors have been involved in designing the studyanalysing the data and drafting of the manuscript

Acknowledgments

This project was supported by the Hungarian ScientificResearch Fund (OTKA PD 101091) and by the Janos BolyaiResearch Scholarship of the Hungarian Academy of Scienceto B Sellyei This research was supported by the HungarianMinistry of Human Capacities (12190-42017FEKUTSTRATGrant)

References

[1] H Preisz About a case of pseudotuberculosis in sheep and aboutpseudotuberculosis in general 1891

[2] E L Biberstein H D Knight and S Jang ldquoTwo biotypes ofCorynebacterium pseudotuberculosisrdquo Veterinary Record vol89 no 26 pp 691-692 1971

[3] KM ConnorMMQuirie G Baird andWDonachie ldquoChar-acterization of United Kingdom isolates of Corynebacteriumpseudotuberculosis using pulsed-field gel electrophoresisrdquo Jour-nal of Clinical Microbiology vol 38 no 7 pp 2633ndash2637 2000

[4] LHWilliamson ldquoCaseous lymphadenitis in small ruminantsrdquoThe Veterinary clinics of North America Food animal practicevol 17 no 2 pp 359ndash371 2001

[5] M M Peel G G Palmer A M Stacpoole and T G KerrldquoHuman lymphadenitis due to Corynebacterium pseudotuber-culosis report of ten cases from Australia and reviewrdquo ClinicalInfectious Diseases vol 24 no 2 pp 185ndash191 1997

BioMed Research International 7

[6] D T L Liu W-M Chan D S P Fan and D S C Lam ldquoAninfected hydrogel buckle with Corynebacterium pseudotuber-culosisrdquo British Journal of Ophthalmology vol 89 no 2 pp 245-246 2005

[7] I Hajtos G Malik and J Varga ldquoOccurrence of caseouslymphadenitis caused by Corynebacterium pseudotuberculosisin goats in Hungary (In Hungarian A kecskek Corynebacteri-umpseudotuuberculosis okozta sajtos nyirokcsomo-gyulla-dasanak elofordulasa hazankbanrdquo MagyarAllatorvosokLapjavol 40 no 1 pp 25ndash29 1985

[8] I Hajtos I Makkai L Fodor et al ldquoOccurrence of ovinecaseous lymphadenitis (pseudotuberculosis) in Northern Hun-gary (In Hungarian Sajtos nyirokcsomo-gyulladas (pseudo-tuberculosis) eszak-magyarorszagi juhallomanybanrdquo MagyarAllatorvosok Lapja vol 127 no 6 pp 339ndash347 2005

[9] F A Dorella L G C Pacheco S C Oliveira AMiyoshi and VAzevedo ldquoCorynebacterium pseudotuberculosis microbiologybiochemical properties pathogenesis and molecular studies ofvirulencerdquo Veterinary Research vol 37 no 2 pp 201ndash218 2006

[10] A de Sa Guimaraes F Borges do Carmo R Barbosa Pauletti etal ldquoReview veterinary microbiology Caseous lymphadenitisepidemiology diagnosis and controlrdquo The IIOAB Journal vol2 no 2 pp 33ndash43 2011

[11] C A Muckle and C L Gyles ldquoCharacterization of strainsof Corynebacterium pseudotuberculosisrdquo Canadian Journal ofComparative Medicine vol 46 no 2 pp 206ndash208 1982

[12] S S Sutherland R A Hart and N B Buller ldquoGeneticdifferences between nitrate-negative and nitrate-positive Cpseudotuberculosis strains using restriction fragment lengthpolymorphismsrdquo Veterinary Microbiology vol 49 no 1-2 pp1ndash9 1996

[13] J C Ruiz V DrsquoAfonseca A Silva et al ldquoEvidence for reductivegenome evolution and lateral acquisition of virulence functionsin two Corynebacterium pseudotuberculosis strainsrdquo PLoS ONEvol 6 no 4 Article ID e18551 2011

[14] S C Soares A Silva E Trost et al ldquoThe Pan-Genome ofthe Animal Pathogen Corynebacterium pseudotuberculosisReveals Differences in Genome Plasticity between the Biovarovis and equi Strainsrdquo PLoS ONE vol 8 no 1 Article ID e538182013

[15] K M Connor M C Fontaine K Rudge G J Baird and WDonache ldquoMolecular genotyping of multinational ovine andcaprine Corynebacterium pseudotuberculosis isolates usingpulsed-field gel electrophoresisrdquo Veterinary Research vol 38no 4 pp 613ndash623 2007

[16] I Stefanska M Rzewuska and M Binek ldquoEvaluation ofthree methods for DNA fingerprinting of Corynebacteriumpseudotuberculosis strains isolated fromgoats in PolandrdquoPolishJournal of Microbiology vol 57 no 2 pp 105ndash112 2008

[17] A Deshpande T Inkster K Hamilton et al ldquoColonisationwith toxigenic corynebacterium diphtheriae in a scottish burnspatient june 2015rdquo Eurosurveillance vol 20 no 49 pp 1ndash42015

[18] T Eisenberg N Mauder M Contzen et al ldquoOutbreak withclonally related isolates of Corynebacterium ulcerans in a groupof water ratsrdquo BMCMicrobiology vol 15 no 1 article 384 2015

[19] E Trost J Blom S C de Soares et al ldquoPangenomic studyof Corynebacterium diphtheriae that provides insights intothe genomic diversity of pathogenic isolates from cases ofclassical diphtheria endocarditis and pneumoniardquo Journal ofBacteriology vol 194 no 12 pp 3199ndash3215 2012

[20] B Markey F Leonard and M Archambault Clinical veterinarymicrobiology MosbyElsevier Ltd Edinburgh UK 2nd edition2013

[21] A A S O Dias F C Silva Jr G A Pereira et al ldquoCorynebac-terium ulcerans isolated from an asymptomatic dog kept inan animal shelter in the metropolitan area of Rio de JaneiroBrazilrdquo Vector borne and zoonotic diseases (Larchmont NY)vol 10 no 8 pp 743ndash748 2010

[22] A Khamis D Raoult and B La Scola ldquorpoB gene sequencingfor identification of Corynebacterium speciesrdquo Journal of Clin-ical Microbiology vol 42 no 9 pp 3925ndash3931 2004

[23] F BoltThe population structure of the Corynebacteriumdiphthe-riae group [PhD thesis] University of Warwick 2009

[24] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[25] W Rychlik ldquoOLIGO 7 Primer Analysis Softwarerdquo in PCRPrimer Design A A Yuryev Ed pp 35ndash59 Humana Press IncTotowa NJ USA 2007

[26] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[27] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

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Microbiology

BioMed Research International 5

Antelope Cow

Human

Goat Sheep

Llama

Horse

Bualo

Camel

Biotype ovis (2081520ndash2083352)

005

Biotype equi

C7

C12C15C23C24C25C3C pseudotuberculosis PAT10

C pseudotuberculosis C231

C14C26C11C22C1C19 (DSM-7180)

C pseudotuberculosis 267

C pseudotuberculosis I19C pseudotuberculosis P54B96C pseudotuberculosis FRC41C pseudotuberculosis 1002

C27C pseudotuberculosis 399-5

C pseudotuberculosis Cp162C pseudotuberculosis 316C pseudotuberculosis 106-AC pseudotuberculosis 31

C21 (DSM-7177)C pseudotuberculosis 258C pseudotuberculosis CIP5297C diphtheriae HC02

10074

9691

94

9887

97

95

70

78

71

89

C pseudotuberculosis 4202-A

Figure 2 Phylogenetic reconstruction based on the concatenated partial sequence of dnaK (520 bp) infB (577 bp) groEL1 (588 bp) and leuA(597 bp) genes from the Hungarian field isolates and DSM type strains Analysis was conducted using the neighbour joining (NJ) methodThe 1000 bootstrap (BT) values are indicated at branching points Bar estimated nucleotide substitutions

differentiation andmolecular epidemiological investigationsThe sequence diversity in these regions exceeded 5

C pseudotuberculosis biotype ovis and C pseudotuber-culosis biotype equi field strains were differentiated withsequence analysis of the target gene fragments Analysis ofthe individual genes showed a fairly conservative nature of Cpseudotuberculosis biotype ovis strains although segregationof some Hungarian strains (eg C1 and C19 C3 C23 andC27) together with strains from Australia Israel USA SouthAfrica Scotland France and Brazil was also evident but theywere clearly located in the cluster of C pseudotuberculosisbiotype ovis The greatest genetic differentiation was seen inthe dnaK and infB genes and concatenations of these two

genes were very useful in the genetic separation of the studiedstrains (Figure 2)

4 Discussion

C pseudotuberculosis biotype ovis is the causative agent ofcaseous lymphadenitis a contagious chronic disease of sheepand goats [3]

Several previous studies showed high sequential homol-ogy of the C pseudotuberculosis biovar ovis strains confirm-ing their clonal origin however diversity of the clinical signsin sheep and goats and different efficacy of the vaccinessuggest that more than one pathogen clone can exist [14]

6 BioMed Research International

Table 4 Nucleic acid average sequence divergence of housekeeping genes in Corynebacterium species in the in silico study

Gene Full length(bp)

Average sequence divergenceC diphtheriae

versusC pseudotuberculosis

C ulceransversus

C pseudotuberculosis

C pseudotuberculosisbiotype ovis versus

equigroEL1 1641 629 279 11infB 2886 136 60 08dnaK 1836 91 50 06leuA 1818 140 81 05odhI 3687 125 497 04atpD 1446 62 32 04recN 1740 270 106 04dnaE 3561 582 71 03groEL2 1641 595 276 03recA 1110 141 73 03rpoA 1017 77 35 03fusA 2127 39 26 02groES 297 65 35 02rpoB 2974 80 33 02sodA 600 100 43 02atpA 1630 84 21 00

The analysis of sequence data of 16 frequently studiedhousekeeping genes (atpA dnaE dnaK fusA leuA odhIrpoB atpD groES groEL1 groEL2 infB recA recN rpoAand sodA) of 15 C pseudotuberculosis 13 C diphtheriaeand C ulcerans genomes from the GenBank proved to berelated and all studied genes could be used to differentiateC diphtheriae C ulcerans and C pseudotuberculosis strainsAll these genes allow the differentiation of biotype ovis andbiotype equi strains of C pseudotuberculosis but for detailedexamination of C pseudotuberculosis biovar ovis field isolatesfour genes (dnaK groEL1 infB and leuA) seemed to havehigher differentiation power The partial sequence analysisof adequate genes delineated phylogenetic trees with varioustopology and depth of branches The highest resolution andtopology of the tree were obtained with the dnaK sequences

The clusters could be explained by movement of breed-ing animals but no epidemiological connection was foundbetween the examined C pseudotuberculosis strains Thedistance between the geographical place of isolation and theyear of the isolation do not support any epidemiologicalconnection between the strains so circulation of severalclones of C pseudotuberculosis biotype ovis was verified

According to our data multilocus sequence typing candifferentiate C pseudotuberculosis and the most importantCorynebacterium species and examination of selected geneshelps to differentiate the two biotypes of C pseudotuberculo-sis and it can be used for epidemiological follow-up of thesestrains

Conflicts of Interest

None of the authors have any conflicts of interest to declare

Authorsrsquo Contributions

All authors have been involved in designing the studyanalysing the data and drafting of the manuscript

Acknowledgments

This project was supported by the Hungarian ScientificResearch Fund (OTKA PD 101091) and by the Janos BolyaiResearch Scholarship of the Hungarian Academy of Scienceto B Sellyei This research was supported by the HungarianMinistry of Human Capacities (12190-42017FEKUTSTRATGrant)

References

[1] H Preisz About a case of pseudotuberculosis in sheep and aboutpseudotuberculosis in general 1891

[2] E L Biberstein H D Knight and S Jang ldquoTwo biotypes ofCorynebacterium pseudotuberculosisrdquo Veterinary Record vol89 no 26 pp 691-692 1971

[3] KM ConnorMMQuirie G Baird andWDonachie ldquoChar-acterization of United Kingdom isolates of Corynebacteriumpseudotuberculosis using pulsed-field gel electrophoresisrdquo Jour-nal of Clinical Microbiology vol 38 no 7 pp 2633ndash2637 2000

[4] LHWilliamson ldquoCaseous lymphadenitis in small ruminantsrdquoThe Veterinary clinics of North America Food animal practicevol 17 no 2 pp 359ndash371 2001

[5] M M Peel G G Palmer A M Stacpoole and T G KerrldquoHuman lymphadenitis due to Corynebacterium pseudotuber-culosis report of ten cases from Australia and reviewrdquo ClinicalInfectious Diseases vol 24 no 2 pp 185ndash191 1997

BioMed Research International 7

[6] D T L Liu W-M Chan D S P Fan and D S C Lam ldquoAninfected hydrogel buckle with Corynebacterium pseudotuber-culosisrdquo British Journal of Ophthalmology vol 89 no 2 pp 245-246 2005

[7] I Hajtos G Malik and J Varga ldquoOccurrence of caseouslymphadenitis caused by Corynebacterium pseudotuberculosisin goats in Hungary (In Hungarian A kecskek Corynebacteri-umpseudotuuberculosis okozta sajtos nyirokcsomo-gyulla-dasanak elofordulasa hazankbanrdquo MagyarAllatorvosokLapjavol 40 no 1 pp 25ndash29 1985

[8] I Hajtos I Makkai L Fodor et al ldquoOccurrence of ovinecaseous lymphadenitis (pseudotuberculosis) in Northern Hun-gary (In Hungarian Sajtos nyirokcsomo-gyulladas (pseudo-tuberculosis) eszak-magyarorszagi juhallomanybanrdquo MagyarAllatorvosok Lapja vol 127 no 6 pp 339ndash347 2005

[9] F A Dorella L G C Pacheco S C Oliveira AMiyoshi and VAzevedo ldquoCorynebacterium pseudotuberculosis microbiologybiochemical properties pathogenesis and molecular studies ofvirulencerdquo Veterinary Research vol 37 no 2 pp 201ndash218 2006

[10] A de Sa Guimaraes F Borges do Carmo R Barbosa Pauletti etal ldquoReview veterinary microbiology Caseous lymphadenitisepidemiology diagnosis and controlrdquo The IIOAB Journal vol2 no 2 pp 33ndash43 2011

[11] C A Muckle and C L Gyles ldquoCharacterization of strainsof Corynebacterium pseudotuberculosisrdquo Canadian Journal ofComparative Medicine vol 46 no 2 pp 206ndash208 1982

[12] S S Sutherland R A Hart and N B Buller ldquoGeneticdifferences between nitrate-negative and nitrate-positive Cpseudotuberculosis strains using restriction fragment lengthpolymorphismsrdquo Veterinary Microbiology vol 49 no 1-2 pp1ndash9 1996

[13] J C Ruiz V DrsquoAfonseca A Silva et al ldquoEvidence for reductivegenome evolution and lateral acquisition of virulence functionsin two Corynebacterium pseudotuberculosis strainsrdquo PLoS ONEvol 6 no 4 Article ID e18551 2011

[14] S C Soares A Silva E Trost et al ldquoThe Pan-Genome ofthe Animal Pathogen Corynebacterium pseudotuberculosisReveals Differences in Genome Plasticity between the Biovarovis and equi Strainsrdquo PLoS ONE vol 8 no 1 Article ID e538182013

[15] K M Connor M C Fontaine K Rudge G J Baird and WDonache ldquoMolecular genotyping of multinational ovine andcaprine Corynebacterium pseudotuberculosis isolates usingpulsed-field gel electrophoresisrdquo Veterinary Research vol 38no 4 pp 613ndash623 2007

[16] I Stefanska M Rzewuska and M Binek ldquoEvaluation ofthree methods for DNA fingerprinting of Corynebacteriumpseudotuberculosis strains isolated fromgoats in PolandrdquoPolishJournal of Microbiology vol 57 no 2 pp 105ndash112 2008

[17] A Deshpande T Inkster K Hamilton et al ldquoColonisationwith toxigenic corynebacterium diphtheriae in a scottish burnspatient june 2015rdquo Eurosurveillance vol 20 no 49 pp 1ndash42015

[18] T Eisenberg N Mauder M Contzen et al ldquoOutbreak withclonally related isolates of Corynebacterium ulcerans in a groupof water ratsrdquo BMCMicrobiology vol 15 no 1 article 384 2015

[19] E Trost J Blom S C de Soares et al ldquoPangenomic studyof Corynebacterium diphtheriae that provides insights intothe genomic diversity of pathogenic isolates from cases ofclassical diphtheria endocarditis and pneumoniardquo Journal ofBacteriology vol 194 no 12 pp 3199ndash3215 2012

[20] B Markey F Leonard and M Archambault Clinical veterinarymicrobiology MosbyElsevier Ltd Edinburgh UK 2nd edition2013

[21] A A S O Dias F C Silva Jr G A Pereira et al ldquoCorynebac-terium ulcerans isolated from an asymptomatic dog kept inan animal shelter in the metropolitan area of Rio de JaneiroBrazilrdquo Vector borne and zoonotic diseases (Larchmont NY)vol 10 no 8 pp 743ndash748 2010

[22] A Khamis D Raoult and B La Scola ldquorpoB gene sequencingfor identification of Corynebacterium speciesrdquo Journal of Clin-ical Microbiology vol 42 no 9 pp 3925ndash3931 2004

[23] F BoltThe population structure of the Corynebacteriumdiphthe-riae group [PhD thesis] University of Warwick 2009

[24] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[25] W Rychlik ldquoOLIGO 7 Primer Analysis Softwarerdquo in PCRPrimer Design A A Yuryev Ed pp 35ndash59 Humana Press IncTotowa NJ USA 2007

[26] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[27] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

Table 4 Nucleic acid average sequence divergence of housekeeping genes in Corynebacterium species in the in silico study

Gene Full length(bp)

Average sequence divergenceC diphtheriae

versusC pseudotuberculosis

C ulceransversus

C pseudotuberculosis

C pseudotuberculosisbiotype ovis versus

equigroEL1 1641 629 279 11infB 2886 136 60 08dnaK 1836 91 50 06leuA 1818 140 81 05odhI 3687 125 497 04atpD 1446 62 32 04recN 1740 270 106 04dnaE 3561 582 71 03groEL2 1641 595 276 03recA 1110 141 73 03rpoA 1017 77 35 03fusA 2127 39 26 02groES 297 65 35 02rpoB 2974 80 33 02sodA 600 100 43 02atpA 1630 84 21 00

The analysis of sequence data of 16 frequently studiedhousekeeping genes (atpA dnaE dnaK fusA leuA odhIrpoB atpD groES groEL1 groEL2 infB recA recN rpoAand sodA) of 15 C pseudotuberculosis 13 C diphtheriaeand C ulcerans genomes from the GenBank proved to berelated and all studied genes could be used to differentiateC diphtheriae C ulcerans and C pseudotuberculosis strainsAll these genes allow the differentiation of biotype ovis andbiotype equi strains of C pseudotuberculosis but for detailedexamination of C pseudotuberculosis biovar ovis field isolatesfour genes (dnaK groEL1 infB and leuA) seemed to havehigher differentiation power The partial sequence analysisof adequate genes delineated phylogenetic trees with varioustopology and depth of branches The highest resolution andtopology of the tree were obtained with the dnaK sequences

The clusters could be explained by movement of breed-ing animals but no epidemiological connection was foundbetween the examined C pseudotuberculosis strains Thedistance between the geographical place of isolation and theyear of the isolation do not support any epidemiologicalconnection between the strains so circulation of severalclones of C pseudotuberculosis biotype ovis was verified

According to our data multilocus sequence typing candifferentiate C pseudotuberculosis and the most importantCorynebacterium species and examination of selected geneshelps to differentiate the two biotypes of C pseudotuberculo-sis and it can be used for epidemiological follow-up of thesestrains

Conflicts of Interest

None of the authors have any conflicts of interest to declare

Authorsrsquo Contributions

All authors have been involved in designing the studyanalysing the data and drafting of the manuscript

Acknowledgments

This project was supported by the Hungarian ScientificResearch Fund (OTKA PD 101091) and by the Janos BolyaiResearch Scholarship of the Hungarian Academy of Scienceto B Sellyei This research was supported by the HungarianMinistry of Human Capacities (12190-42017FEKUTSTRATGrant)

References

[1] H Preisz About a case of pseudotuberculosis in sheep and aboutpseudotuberculosis in general 1891

[2] E L Biberstein H D Knight and S Jang ldquoTwo biotypes ofCorynebacterium pseudotuberculosisrdquo Veterinary Record vol89 no 26 pp 691-692 1971

[3] KM ConnorMMQuirie G Baird andWDonachie ldquoChar-acterization of United Kingdom isolates of Corynebacteriumpseudotuberculosis using pulsed-field gel electrophoresisrdquo Jour-nal of Clinical Microbiology vol 38 no 7 pp 2633ndash2637 2000

[4] LHWilliamson ldquoCaseous lymphadenitis in small ruminantsrdquoThe Veterinary clinics of North America Food animal practicevol 17 no 2 pp 359ndash371 2001

[5] M M Peel G G Palmer A M Stacpoole and T G KerrldquoHuman lymphadenitis due to Corynebacterium pseudotuber-culosis report of ten cases from Australia and reviewrdquo ClinicalInfectious Diseases vol 24 no 2 pp 185ndash191 1997

BioMed Research International 7

[6] D T L Liu W-M Chan D S P Fan and D S C Lam ldquoAninfected hydrogel buckle with Corynebacterium pseudotuber-culosisrdquo British Journal of Ophthalmology vol 89 no 2 pp 245-246 2005

[7] I Hajtos G Malik and J Varga ldquoOccurrence of caseouslymphadenitis caused by Corynebacterium pseudotuberculosisin goats in Hungary (In Hungarian A kecskek Corynebacteri-umpseudotuuberculosis okozta sajtos nyirokcsomo-gyulla-dasanak elofordulasa hazankbanrdquo MagyarAllatorvosokLapjavol 40 no 1 pp 25ndash29 1985

[8] I Hajtos I Makkai L Fodor et al ldquoOccurrence of ovinecaseous lymphadenitis (pseudotuberculosis) in Northern Hun-gary (In Hungarian Sajtos nyirokcsomo-gyulladas (pseudo-tuberculosis) eszak-magyarorszagi juhallomanybanrdquo MagyarAllatorvosok Lapja vol 127 no 6 pp 339ndash347 2005

[9] F A Dorella L G C Pacheco S C Oliveira AMiyoshi and VAzevedo ldquoCorynebacterium pseudotuberculosis microbiologybiochemical properties pathogenesis and molecular studies ofvirulencerdquo Veterinary Research vol 37 no 2 pp 201ndash218 2006

[10] A de Sa Guimaraes F Borges do Carmo R Barbosa Pauletti etal ldquoReview veterinary microbiology Caseous lymphadenitisepidemiology diagnosis and controlrdquo The IIOAB Journal vol2 no 2 pp 33ndash43 2011

[11] C A Muckle and C L Gyles ldquoCharacterization of strainsof Corynebacterium pseudotuberculosisrdquo Canadian Journal ofComparative Medicine vol 46 no 2 pp 206ndash208 1982

[12] S S Sutherland R A Hart and N B Buller ldquoGeneticdifferences between nitrate-negative and nitrate-positive Cpseudotuberculosis strains using restriction fragment lengthpolymorphismsrdquo Veterinary Microbiology vol 49 no 1-2 pp1ndash9 1996

[13] J C Ruiz V DrsquoAfonseca A Silva et al ldquoEvidence for reductivegenome evolution and lateral acquisition of virulence functionsin two Corynebacterium pseudotuberculosis strainsrdquo PLoS ONEvol 6 no 4 Article ID e18551 2011

[14] S C Soares A Silva E Trost et al ldquoThe Pan-Genome ofthe Animal Pathogen Corynebacterium pseudotuberculosisReveals Differences in Genome Plasticity between the Biovarovis and equi Strainsrdquo PLoS ONE vol 8 no 1 Article ID e538182013

[15] K M Connor M C Fontaine K Rudge G J Baird and WDonache ldquoMolecular genotyping of multinational ovine andcaprine Corynebacterium pseudotuberculosis isolates usingpulsed-field gel electrophoresisrdquo Veterinary Research vol 38no 4 pp 613ndash623 2007

[16] I Stefanska M Rzewuska and M Binek ldquoEvaluation ofthree methods for DNA fingerprinting of Corynebacteriumpseudotuberculosis strains isolated fromgoats in PolandrdquoPolishJournal of Microbiology vol 57 no 2 pp 105ndash112 2008

[17] A Deshpande T Inkster K Hamilton et al ldquoColonisationwith toxigenic corynebacterium diphtheriae in a scottish burnspatient june 2015rdquo Eurosurveillance vol 20 no 49 pp 1ndash42015

[18] T Eisenberg N Mauder M Contzen et al ldquoOutbreak withclonally related isolates of Corynebacterium ulcerans in a groupof water ratsrdquo BMCMicrobiology vol 15 no 1 article 384 2015

[19] E Trost J Blom S C de Soares et al ldquoPangenomic studyof Corynebacterium diphtheriae that provides insights intothe genomic diversity of pathogenic isolates from cases ofclassical diphtheria endocarditis and pneumoniardquo Journal ofBacteriology vol 194 no 12 pp 3199ndash3215 2012

[20] B Markey F Leonard and M Archambault Clinical veterinarymicrobiology MosbyElsevier Ltd Edinburgh UK 2nd edition2013

[21] A A S O Dias F C Silva Jr G A Pereira et al ldquoCorynebac-terium ulcerans isolated from an asymptomatic dog kept inan animal shelter in the metropolitan area of Rio de JaneiroBrazilrdquo Vector borne and zoonotic diseases (Larchmont NY)vol 10 no 8 pp 743ndash748 2010

[22] A Khamis D Raoult and B La Scola ldquorpoB gene sequencingfor identification of Corynebacterium speciesrdquo Journal of Clin-ical Microbiology vol 42 no 9 pp 3925ndash3931 2004

[23] F BoltThe population structure of the Corynebacteriumdiphthe-riae group [PhD thesis] University of Warwick 2009

[24] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[25] W Rychlik ldquoOLIGO 7 Primer Analysis Softwarerdquo in PCRPrimer Design A A Yuryev Ed pp 35ndash59 Humana Press IncTotowa NJ USA 2007

[26] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[27] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

[6] D T L Liu W-M Chan D S P Fan and D S C Lam ldquoAninfected hydrogel buckle with Corynebacterium pseudotuber-culosisrdquo British Journal of Ophthalmology vol 89 no 2 pp 245-246 2005

[7] I Hajtos G Malik and J Varga ldquoOccurrence of caseouslymphadenitis caused by Corynebacterium pseudotuberculosisin goats in Hungary (In Hungarian A kecskek Corynebacteri-umpseudotuuberculosis okozta sajtos nyirokcsomo-gyulla-dasanak elofordulasa hazankbanrdquo MagyarAllatorvosokLapjavol 40 no 1 pp 25ndash29 1985

[8] I Hajtos I Makkai L Fodor et al ldquoOccurrence of ovinecaseous lymphadenitis (pseudotuberculosis) in Northern Hun-gary (In Hungarian Sajtos nyirokcsomo-gyulladas (pseudo-tuberculosis) eszak-magyarorszagi juhallomanybanrdquo MagyarAllatorvosok Lapja vol 127 no 6 pp 339ndash347 2005

[9] F A Dorella L G C Pacheco S C Oliveira AMiyoshi and VAzevedo ldquoCorynebacterium pseudotuberculosis microbiologybiochemical properties pathogenesis and molecular studies ofvirulencerdquo Veterinary Research vol 37 no 2 pp 201ndash218 2006

[10] A de Sa Guimaraes F Borges do Carmo R Barbosa Pauletti etal ldquoReview veterinary microbiology Caseous lymphadenitisepidemiology diagnosis and controlrdquo The IIOAB Journal vol2 no 2 pp 33ndash43 2011

[11] C A Muckle and C L Gyles ldquoCharacterization of strainsof Corynebacterium pseudotuberculosisrdquo Canadian Journal ofComparative Medicine vol 46 no 2 pp 206ndash208 1982

[12] S S Sutherland R A Hart and N B Buller ldquoGeneticdifferences between nitrate-negative and nitrate-positive Cpseudotuberculosis strains using restriction fragment lengthpolymorphismsrdquo Veterinary Microbiology vol 49 no 1-2 pp1ndash9 1996

[13] J C Ruiz V DrsquoAfonseca A Silva et al ldquoEvidence for reductivegenome evolution and lateral acquisition of virulence functionsin two Corynebacterium pseudotuberculosis strainsrdquo PLoS ONEvol 6 no 4 Article ID e18551 2011

[14] S C Soares A Silva E Trost et al ldquoThe Pan-Genome ofthe Animal Pathogen Corynebacterium pseudotuberculosisReveals Differences in Genome Plasticity between the Biovarovis and equi Strainsrdquo PLoS ONE vol 8 no 1 Article ID e538182013

[15] K M Connor M C Fontaine K Rudge G J Baird and WDonache ldquoMolecular genotyping of multinational ovine andcaprine Corynebacterium pseudotuberculosis isolates usingpulsed-field gel electrophoresisrdquo Veterinary Research vol 38no 4 pp 613ndash623 2007

[16] I Stefanska M Rzewuska and M Binek ldquoEvaluation ofthree methods for DNA fingerprinting of Corynebacteriumpseudotuberculosis strains isolated fromgoats in PolandrdquoPolishJournal of Microbiology vol 57 no 2 pp 105ndash112 2008

[17] A Deshpande T Inkster K Hamilton et al ldquoColonisationwith toxigenic corynebacterium diphtheriae in a scottish burnspatient june 2015rdquo Eurosurveillance vol 20 no 49 pp 1ndash42015

[18] T Eisenberg N Mauder M Contzen et al ldquoOutbreak withclonally related isolates of Corynebacterium ulcerans in a groupof water ratsrdquo BMCMicrobiology vol 15 no 1 article 384 2015

[19] E Trost J Blom S C de Soares et al ldquoPangenomic studyof Corynebacterium diphtheriae that provides insights intothe genomic diversity of pathogenic isolates from cases ofclassical diphtheria endocarditis and pneumoniardquo Journal ofBacteriology vol 194 no 12 pp 3199ndash3215 2012

[20] B Markey F Leonard and M Archambault Clinical veterinarymicrobiology MosbyElsevier Ltd Edinburgh UK 2nd edition2013

[21] A A S O Dias F C Silva Jr G A Pereira et al ldquoCorynebac-terium ulcerans isolated from an asymptomatic dog kept inan animal shelter in the metropolitan area of Rio de JaneiroBrazilrdquo Vector borne and zoonotic diseases (Larchmont NY)vol 10 no 8 pp 743ndash748 2010

[22] A Khamis D Raoult and B La Scola ldquorpoB gene sequencingfor identification of Corynebacterium speciesrdquo Journal of Clin-ical Microbiology vol 42 no 9 pp 3925ndash3931 2004

[23] F BoltThe population structure of the Corynebacteriumdiphthe-riae group [PhD thesis] University of Warwick 2009

[24] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[25] W Rychlik ldquoOLIGO 7 Primer Analysis Softwarerdquo in PCRPrimer Design A A Yuryev Ed pp 35ndash59 Humana Press IncTotowa NJ USA 2007

[26] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[27] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology