Campylobacter avium sp. nov., a hippurate-positive species isolated from poultry

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Campylobacter avium sp. nov., a hippurate-positive species isolated from poultry
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  Downloaded from www.microbiologyresearch.org byIP: 54.205.89.16On: Wed, 17 Aug 2016 11:37:14 Campylobacter avium  sp. nov., a hippurate-positivespecies isolated from poultry Mirko Rossi, 1 3  Lies Debruyne, 2 Renato Giulio Zanoni, 1 Gerardo Manfreda, 3 Joana Revez 1 and Peter Vandamme 2 Correspondence Mirko Rossimirko.rossi@helsinki.fi 1 Department of Veterinary Public Health and Animal Pathology, Alma Mater Studiorum, Universityof Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy 2 Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, Ghent University,K. L. Ledeganckstraat 35, B-9000 Gent, Belgium 3 Department of Food Science, Alma Mater Studiorum, University of Bologna, Via del Florio 2,40064 Ozzano Emilia, Bologna, Italy Three strains of an unusual hippurate-positive  Campylobacter   species were isolated at 37  6 Cfrom caecal contents of broiler chickens and a turkey. All strains were initially identified as Campylobacter   by means of genus-specific PCR, but none was further identified using specificPCRs for known thermophilic species. Phylogenetic analyses based on 16S rRNA,  rpoB  and groEL  gene sequences revealed that these strains formed a robust clade distinct from other Campylobacter   species. Amplified fragment length polymorphism analysis and whole-cell proteinelectrophoresis were subsequently carried out and confirmed the divergence between the avianstrains and other taxa. These data indicate that the unidentified  Campylobacter   strains belong to anovel taxon which could be distinguished from other campylobacters through its phenotypic andgenotypic characteristics. The name  Campylobacter avium  sp. nov., is proposed for the novelspecies, with the type strain 86/06 T (  5 LMG 24591 T 5 CCUG 56292 T ). The genus  Campylobacter   was proposed by Sebald & Ve´ron(1963) .  The genus has since been expanded, with speciessrcinating from mammals and birds, and now includes 19species and 6 subspecies (Foster  et al. , 2004; Vandamme et al. , 2005; Inglis  et al. , 2007; Zanoni  et al. , 2009). In thepresent study, we report a polyphasic taxonomic char-acterization of three unusual hippurate hydrolase-pro-ducing  Campylobacter   strains that were recovered from thecaecal contents of poultry.In the course of bacteriological investigations intended todefine the prevalence of   Helicobacter pullorum  in poultry,seven unusual hippurate-positive  Campylobacter   isolateswere recovered from caecal contents of six broiler chickensand one turkey srcinating from three different farms inItaly. Caeca were collected at the slaughterhouse betweenJuly and October 2006. Campylobacters were isolated after3–4 days of incubation at 37  u C in a microaerobicatmosphere with hydrogen on Brucella sheep blood agar [  Brucella broth (BBL) with 1.5% Bacto agar (Difco) and5% sheep blood ]  using a filter method (Zanoni  et al.  2007).The microaerobic atmosphere with hydrogen was obtainedby the gas replacement method using an anaerobic gasmixture (10% H 2 , 10% CO 2 , 80% N 2 ) as described by Bolton  et al.  (1992). After 3–4 days of incubation onBrucella sheep blood agar, growth appeared as a spreadinglayer on the agar medium. Single colonies were not seen.Pure cultures were obtained after dilution and repeatedsubculturing. Following subculturing after 48 h of incuba-tion at 37  u C, colonies appeared flat, greyish and finely granular with an irregular edge, and showed a tendency tospread along the direction of the streak and to swarm andcoalesce. Cells were Gram-negative, sigmoid to allantoid inshape, 1–3  m m long and 0.2–0.4  m m wide, when observedafter Gram staining, and appeared coccoid after 4–5 daysof incubation.For genotyping analysis, bacterial DNA was extracted by using a ChargeSwitch gDNA Mini Bacteria kit (InvitrogenLife Technologies). Abbreviation:  AFLP, amplified fragment length polymorphism. 3 Present address:  Department of Food and Environmental Hygiene,University of Helsinki, PO Box 66, Agnes Sjo¨bergin katu 2, FI-00014Helsinki, Finland.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, rpoB   and  groEL  gene sequences of strains 86/06 T , 24/06 and 87/06are respectively EU623473–EU623475 (16S rRNA gene), EU643476,EU643478 and EU643477 ( rpoB  ) and EU636014, EU636013 andEU636812 ( groEL ).Neighbour-joining dendrograms based on partial  rpoB   and  groEL sequences and a UPGMA dendrogram based on analysis of proteinprofiles of strains of  Campylobacter avium  sp. nov. and other Campylobacter   species are available as supplementary material withthe online version of this paper. International Journal of Systematic and Evolutionary Microbiology   (2009),  59,  2364–2369  DOI  10.1099/ijs.0.007419-02364 007419 G 2009 IUMS  Printed in Great Britain  Downloaded from www.microbiologyresearch.org byIP: 54.205.89.16On: Wed, 17 Aug 2016 11:37:14 Isolates were initially identified as  Campylobacter jejuni  because of their ability to hydrolyse hippurate. To confirmthe identification, isolates were further analysed by meansof a genus-specific PCR for the genus  Campylobacter  (Linton  et al. , 1996) and several species-specific PCRs fordifferent  Campylobacter   species (Denis  et al. , 1999; Lawson et al. , 1997; Linton  et al. , 1996). They were confirmed as Campylobacter  , but could not be identified at the specieslevel. The diversity among the unidentified  Campylobacter  isolates was assessed by visual analysis of whole-cell proteinprofiles in one-dimensional SDS-PAGE, carried out asdescribed by Zanoni  et al.  (2007). All seven  Campylobacter  isolates showed almost identical protein profiles (data notshown), suggesting that they belong to the same taxon.To obtain a more detailed identification, we selected threeisolates (86/06 T , 87/06 and 24/06) from different hosts(broiler chicken and turkey) and from different regions inItaly, thereby representing a geographically and epidemio-logically independent set of isolates.In order to define the taxonomic position of the avianisolates, a phylogenetic analysis based on 16S rRNA genesequences was carried out. The nearly complete 16S rRNAgene was amplified using universal primers p27f (5 9 -AGAGTTTGATCCTGGCTCAG-3 9 ) and p1492r (5 9 -TACGGCTACCTTGTTACGACT-5 9 ) and the PCR-amp-lified template was sequenced by primer walking (Primms.r.l.). Sequences were assembled with Vecton NTI software(Invitrogen) and then aligned in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) by   CLUSTAL W using  Campylobacter   reference sequences obtained fromGenBank. The alignment was adjusted visually removingintervening sequence regions and unknown bases. Finally,data were corrected for multiple base changes by themethod of Jukes & Cantor (1969). A phylogenetic tree wasconstructed in  MEGA 3 (http://www.megasoftware.net/)using the neighbour-joining method. Bootstrap analysiswas performed with 1000 resampled datasets.A fragment of approximately 1300 bp of the 16S rRNAgene was obtained. Analysis of the 16S rRNA genesequences using  MEGABLAST  (http://www.ncbi.nlm.nih.gov/BLAST/) indicated that the isolates were most closely related to taxa within the genus  Campylobacter  , confirmingtheir generic identification. Pairwise comparisons of 16SrRNA gene sequences showed that the three isolates weregenetically highly related, exhibiting 99.1–99.9% sequencesimilarity. Furthermore, the neighbour-joining dendro-gram (Fig. 1) indicated that they formed a robust clade(100% bootstrap support) which was clearly distinct fromother  Campylobacter   species. Pairwise sequence compar-isons of 86/06 T with type strains of the most closely relatedspecies revealed similarities of 96.6, 95.5, 94.9 and 94.2%with  Campylobacter upsaliensis  ,  C. helveticus  ,  C. cunicu-lorum  and  C. jejuni  , respectively.The phylogenetic relationships of these bacteria werefurther examined by   rpoB   (Korczak   et al.  2006) and groEL   (Ka¨renlampi  et al.  2004) sequence analysis.Sequences were processed as described above. Phylogenetictrees based on partial  rpoB   and  groEL   nucleotide sequencesare shown in Supplementary Figs S1 and S2 (available inIJSEM Online). In both trees, the unidentified strainsclustered together in a tight clade supported by a highbootstrap value (100%) and clearly separated from all Campylobacter   species. The  rpoB   sequences of the avianstrains were 99–100% similar, while similarity valuestowards those of other  Campylobacter   species varied from64 to 74.8%. Likewise, pairwise comparisons of the  groEL  sequences among the avian strains yielded similarity of 99.2–100%, while values towards other  Campylobacter   specieswere less than 82.4%. Like Korczak   et al.  (2006) andKa¨renlampi  et al.  (2004), we observed a good congruence Fig. 1.  Unrooted tree, based on 16S rRNAgene sequences, showing the phylogeneticrelationships of the three strains of  C. avium sp. nov. Bar, 0.02 nucleotide substitutions perbase. Numbers at nodes (  ¢ 88%) indicatesupport for the internal branches within thetree obtained by bootstrap analysis (percen-tages of 1000 bootstraps). Hippurate-positive  Campylobacter avium  sp. nov. from poultryhttp://ijs.sgmjournals.org 2365  Downloaded from www.microbiologyresearch.org byIP: 54.205.89.16On: Wed, 17 Aug 2016 11:37:14 between  rpoB  ,  groEL   and 16S rRNA gene sequence results,since each of the phylogenetic trees showed a similartopology. However, compared to the 16S rRNA gene,  rpoB  and  groEL   sequence analyses showed higher resolution as aresult of their lower interspecies similarity.Although all the sequence data demonstrated that the threeisolates represented a coherent taxon, amplified fragmentlength polymorphism (AFLP) analysis and whole-cellprotein electrophoresis were performed to further examinethe relationships between the strains.AFLP analysis was performed as described by Debruyne et al.  (2009). In brief, 1  m g genomic DNA was digested withthe  Hin  dIII– Hha  I restriction enzyme combination. Afterdigestion, site-specific adaptors were ligated to therestriction fragments and primers complementary to theadaptor and restriction site sequence were used insubsequent preselective and selective PCRs. The amplifiedand fluorescently labelled fragments were loaded on adenaturing polyacrylamide gel on an ABI Prism 377automated sequencer.  GENESCAN  version 3.1 (AppliedBiosystems) was used for data collection, and the generatedprofiles were imported, using the CrvConv filter, inBioNumerics version 4.61 (Applied Maths) for normaliza-tion and further analysis. Similarity between normalizedprofiles was determined by Pearson’s product–momentcorrelation coefficient and a UPGMA dendrogram wasconstructed. Numerical analysis of the AFLP profilesobtained (Fig. 2) differentiated the avian taxon from other Campylobacter   species. Moreover, the AFLP profiles of thethree strains were all different, thereby demonstrating thatthe isolates represent different clones.Whole-cell protein profile analysis was performed usingSDS-PAGE as described by Pot  et al.  (1994). For this analysis,strainsweregrown microaerobicallyonMueller–Hinton agar(Oxoid) supplement with 5% (v/v) defibrinated horse bloodand incubated microaerobically at 37  u C for 48 h. Whole-cellprotein profiles of   Campylobacter   reference strains wereavailable from previous studies (Vandamme  et al. , 1991).Densitometric analysis, normalization and interpolation of the protein profiles, and numerical analysis, were performedusing the GelCompar software package (version 4.2; AppliedMaths). For whole-cell protein SDS-PAGE analysis, similar-ity of the obtained normalized SDS-PAGE patterns wasdetermined by Pearson’s product–moment correlationcoefficient, after which clustering was performed by UPGMA. The dendrogram obtained by numerical analysisof the protein profiles of the three avian strains and of  Campylobacter   reference strains is shown in Supplementary Fig. S3. The three strains grouped in a single cluster above asimilarity level of 94% and were clearly distinct from theother  Campylobacter   species.For the determination of G + C content, DNA wasenzymically degraded into nucleosides as described by Mesbah & Whitman (1989). The nucleoside mixture wasseparated by HPLC using a Waters SymmetryShield C8column maintained at 37  u C. The solvent was 0.02 M(NH 4 )H 2 PO 4  (pH 4.0) with 1.5% acetonitrile. Non-methylated  l  phage DNA (Sigma) was used as thecalibration reference. The G + C content of the DNA of strain 86/06 T was 35 mol%. This value is within the rangeof 28–47 mol% reported for genus  Campylobacter  (Vandamme  et al. , 2005). Fig. 2.  Dendrogram of the three strains of  C. avium  sp. nov. based on UPGMA cluster analysis of AFLP profiles. M. Rossi and others2366  International Journal of Systematic and Evolutionary Microbiology   59  Downloaded from www.microbiologyresearch.org byIP: 54.205.89.16On: Wed, 17 Aug 2016 11:37:14 Table 1.  Phenotypic characteristics of  Campylobacter   species Taxa: 1,  C. avium  sp. nov.; 2,  C. canadensis  ; 3,  C. coli  ; 4,  C. concisus  ; 5,  C. cuniculorum ; 6,  C. curvus  ; 7,  C. fetus   subsp.  fetus  ; 8,  C. fetus   subsp.  venerealis  ; 9,  C. gracilis  ; 10,  C. helveticus  ; 11,  C. hominis  ;12,  C. hyointestinalis   subsp.  hyointestinalis  ; 13,  C. hyointestinalis   subsp.  lawsonii  ; 14,  C. insulanigrae  ; 15,  C. jejuni   subsp.  doylei  ; 16,  C. jejuni   subsp.  jejuni  ; 17,  C. lanienae  ; 18,  C. lari  ; 19,  C. mucosalis  ;20,  C. rectus  ; 21,  C. showae  ; 22,  C. sputorum ; 23,  C. upsaliensis.  Data for reference species were taken from On  et al.  (1996), Foster  et al.  (2004), Vandamme  et al.  (2005), Inglis  et al.  (2007) andZanoni  et al.  (2009). All taxa are negative for aerobic growth at 37  u C.  + , 90–100% of strains positive; 2 , 0–10% of strains positive; ( + ), 75–89% strains positive; ( 2 ), 11–25% of strains positive; V , 26–74% of strains positive;  W , weakly positive;  NA , no data available. CCDA, Charcoal cefoperazone deoxycholate agar (Oxoid); TTC, triphenyl tetrazolium chloride. Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a -Haemolysis  2 2  ( 2 ) ( 2 )  +  ( 2 )  2  V  2  +  NA V V NA  + + +  V  2  + + + + Oxidase  + + +  V  + + + +  2  + + + + + + + + + + +  V  + + Catalase  W V  +  2  +  2  +  ( + )  V  2 2  + + +  V  + + +  2  ( 2 )  +  V  2 Alkaline phosphatase  2 2 2  V  2  V  2 2 2 2 2 2  ( 2 )  NA  2 2  +  2  ( + )  2 2 2 2 c -Glutamyltranspeptidase  2  ( + )  2 2 2  NA  2  NA NA  2  NA  2 2  NA  2 2  NA  2  NA NA NA  2 2 Urease production  2  V  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  V  2 2 2  V *  2 Hippurate hydrolysis  +  2 2 2 2  ( 2 )  2 2 2 2 2 2 2 2  + +  2 2 2 2 2 2 2 Indoxyl acetate hydrolysis  +  2  +  2  +  V  2 2  V  +  2 2 2 2  + +  2 2 2  +  2 2  + Nitrate reduction  +  V  +  ( 2 )  + + + +  ( + )  +  2  + + +  2  + + +  2  + + + + Selenite reduction  2  NA V  ( 2 )  2 2  ( + )  2 2 2 2  + +  NA  2  + + +  2  + + + + TTC reduction  2  NA  +  2  V V  2 2 2 2  NA  2 2  NA V  +  NA  +  2 2 2 2  V Trace H 2 S on TSI agar  2  V  2 2 2  ( 2 )  2 2 2 2 2  + +  2 2 2 2 2  +  2  V  +  2 Growth at/in/on:25  u C (microaerobic)  2 2 2 2 2 2  + +  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 37  u C (microaerobic)  + + + + +  V  + +  2  + + + + + + + + + +  2  V  + + 42  u C (microaerobic)  + + +  ( + ) ( + )  V  ( + )  2  V  +  ( 2 )  + +  2 2  + + + +  ( 2 )  V  + + 37  u C (anaerobic)  2  +  2  +  2  +  ( 2 )  V  +  2  +  2  +  2 2 2  +  2  + + + +  2 Nutrient agar  2 2  +  ( 2 )  + + + + +  ( + )  NA  + +  NA  + +  NA  + +  ( 2 )  V  + + CCDA  2  + +  ( 2 ) ( + ) ( + )  + +  V  +  NA  + +  NA  + +  NA  + +  2  +  ( + )  + MacConkey agar  2  +  V  2 2  ( + ) ( + )  V  ( + )  2 2  V V NA  2 2  +  2  ( + )  2  +  V  2 1% Glycine  2  V  +  ( 2 )  2  + +  2  +  V  + +  V  +  ( 2 )  +  2  +  V  +  V  + + 2% NaCl  2  NA  2  ( 2 )  2  V  2 2  V  2  +  2 2 2 2 2 2  ( + )  +  V  + +  2 1% Bile  V NA  ( + )  2  V  2  + +  2  +  NA  +  ( + )  NA  + +  NA  + +  2 2  V  + Requirement for H 2  V  2 2  +  2  +  2 2  +  2  +  V V NA  2 2 2 2  + + +  2 2 Resistance to:Nalidixic acid  2  V  2  ( + )  V  + +  V V  2  V  + + +  2 2  +  V  ( + ) ( + )  2  ( + )  2 Cephalothin  +  2  +  2  ( + )  2 2 2 2 2 2  ( 2 )  2  +  2  + + +  2 2 2 2  ( 2 ) Hi      p  p ur   a t    e-  p o s i     t   i    v  e  C am  p  y l     o b   a c t    er   av i     um  s   p.n ov .f   r   om  p o ul     t   r    y h   t    t     p:  /  /  i      j     s . s   gm  j     o ur  n al     s . or    g2  3  6 7   Downloaded from www.microbiologyresearch.org byIP: 54.205.89.16On: Wed, 17 Aug 2016 11:37:14 The results of physiological characterization of the threeavian strains, determined using standard methods (On & Holmes, 1991a, b; 1992; Ursing  et al. , 1994; On  et al. ,1996), are presented in Table 1 and in the speciesdescription. These characteristics allowed differentiationof the poultry strains from established  Campylobacter  species. Among the hippurate-positive species, the poultry strains can be distinguished from  C. jejuni   subsp.  jejuni   by their inability to grow in the presence of 1% (w/v) glycineand to reduce selenite or triphenyl tetrazolium chlorideand from  C. jejuni   subsp.  doylei   by their ability to grow at42  u C and to reduce nitrate.In conclusion, the results of this polyphasic taxonomicstudy indicate that the three strains recovered from caecalcontents of poultry represent a unique  Campylobacter  species for which we propose the name  Campylobacter avium  sp. nov. Sequence analysis of   rpoB   and  groEL   genesand also AFLP and whole-cell protein profile analysis andtraditional biochemical analysis allow the novel species tobe distinguished from established species.Campylobacters are the most common bacterial cause of human enteric infections worldwide. Species identificationof human isolates is usually carried out in routine clinicalmicrobiology laboratories by means of phenotypic meth-ods. The ability of   C. jejuni   to hydrolyse hippurate ( N  -benzoylglycine) to benzoic acid and glycine is commonly used to distinguish it from other  Campylobacter   species,and only hippurate-negative strains are usually tested by molecular methods (Nakari  et al. , 2008; Wainø  et al. ,2003). Nakari  et al.  (2008) recently standardized thehippurate test by determining cell suspension turbidity limits using  C. jejuni   and  Campylobacter coli   referencestrains. All strains of   C. avium  sp. nov. were also able tohydrolyse hippurate when tested with the low suspensionturbidity described for  C. jejuni   by Nakari  et al.  (2008),thus confirming the previous results. Hippurate hydrolaseactivity in  C. jejuni   is due to the presence of an enzymeencoded by the  hipO   gene (Hani & Chan, 1995), anddifferent  hipO  -based species-specific PCR tests for  C. jejuni  have been described (Slater & Owen, 1997; Burnett  et al. ,2002; Bang  et al. , 2002). None of these PCRs amplified thehippurate hydrolase gene of   C. avium  sp. nov. (data notshown). These results suggest that hippurate-positivecampylobacters may erroneously be considered as  C. jejuni  if insufficient biochemical characterization or subsequentmolecular confirmation is performed. Description of  Campylobacter avium  sp. nov. Campylobacter avium  (a 9 vi.um. L. gen. pl. n.  avium  of birds).Cells are spiral, Gram-negative rods, motile, 0.2–0.4  m mwide and 1–3  m m long. On Brucella sheep blood agar at37  u C after 48 h under microaerobic conditions, coloniesappeared non- a -haemolytic, flat, greyish and finely granu-lar with an irregular edge and show a tendency to spreadalong the direction of the streak and to swarm andcoalesce. Strictly microaerobic. Able to grow at 37 and42  u C, but not at 25  u C or under anaerobic and aerobicconditions. Most strains do not require hydrogen to grow.Oxidase and weak catalase activity are observed, but noturease,  c -glutamyltranspeptidase or alkaline phosphatase.Strains hydrolyse hippurate and indoxyl acetate and reducenitrate but not selenite or triphenyl tetrazolium chloride.Strains do not produce H 2 S in TSI agar. Most strains grow in the presence of 1% (w/v) bile. No growth occurs onnutrient agar without blood, on MacConkey agar or in thepresence of 1% (w/v) glycine or 2% (w/v) NaCl. Growthon charcoal cefoperazone deoxycholate agar (CCDA;Oxoid) appears after 4–5 days of incubation, and growthon this medium is slightly restricted. Strains are susceptibleto nalidixic acid (30  m g) and resistant to cephalothin(30  m g) by disc diffusion tests. Pathogenicity is unknown.The type strain is 86/06 T ( 5 LMG 24591 T 5 CCUG56292 T ), which was isolated from a broiler chicken inItaly in 2006. Strains 24/06 ( 5 CCUG 56294) and 87/06( 5 LMG 24592  5 CCUG 56293) are also strains of thespecies. Strains have been recovered from poultry caecalcontents. Acknowledgements We thank Dr Jean Euze´by (Laboratoire de Bacte´riologie, E´coleNationale Ve´te´rinaire, Toulouse, France) for help with naming thenovel species and Professor Valeria Sanguinetti for all the supportgiven. References Bang, D. D., Wedderkopp, A., Pedersen, K. & Madsen, M. (2002). Rapid PCR using nested primers of the 16S rRNA and the hippuricase( hipO  ) genes to detect  Campylobacter jejuni   and  Campylobacter coli   inenvironmental samples.  Mol Cell Probes   16 , 359–369. Bolton, F. J., Wareing, D. R. A., Skirrow, M. B. & Hutchinson, D. N.(1992).  Identification and biotyping of campylobacters. In Identification Methods in Applied and Environmental Microbiology  ,pp. 151–161. Edited by R. G. Board, D. Jones & F. A Skinner. Oxford:Blackwell Scientific. Burnett, T. A., Hornitzky, A. M., Kuhnert, P. & Djordjevic, S. P. (2002). Speciating  Campylobacter jejuni   and  Campylobacter coli   isolates frompoultry and humans using six PCR-based assays.  FEMS Microbiol Lett  216 , 201–209. Debruyne, L., On, S. L. W., De Brandt, E. & Vandamme, P. (2009). Novel  Campylobacter lari  -like bacteria from humans and molluscs:description of   Campylobacter peloridis   sp. nov.,  Campylobacter lari  subsp.  concheus   subsp. nov. and  Campylobacter lari   subsp.  lari   subsp.nov.  Int J Syst Evol Microbiol   59 , 1126–1132. Denis, M., Soumet, C., Rivoal, K., Ermel, G., Blivet, D., Salvat, G. &Colin, P. (1999).  Development of a m-PCR assay for simultaneousidentification of   Campylobacter jejuni   and  C. coli  .  Lett Appl Microbiol  29 , 406–410. Foster, G., Holmes, B., Steigerwalt, A. G., Lawson, P. A., Thorne, P.,Byrer, D. E., Ross, H. M., Xerry, J., Thompson, P. M. & Collins, M. D.(2004).  Campylobacter insulaenigrae   sp. nov., isolated from marinemammals.  Int J Syst Evol Microbiol   54 , 2369–2373. M. Rossi and others2368  International Journal of Systematic and Evolutionary Microbiology   59
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