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ypical Yersinia pseudotuberculosis serotype O:3 isolatedm hunted wild boars in Italy

. Magistrali *, L. Cucco, E. Manuali, C. Sebastiani, S. Farneti,Ercoli, G. Pezzotti

uto Zooprofilattico Sperimentale dell’Umbria e delle Marche, via Salvemini, 1, 06126 Perugia, Italy

ntroduction

Yersiniosis, caused by Yersinia enterocolitica and, lessmonly, Y. pseudotuberculosis, was the fourth mostuently reported zoonosis in the EU in 2011, with anrall notification rate of 1.63 cases per 100,000 populationSA, 2013). In Europe, Y. pseudotuberculosis infectionsically cause ileocolitis and mesenteric lymphadenitis,n mistaken for appendicitis, while in the Far East a

iety of systemic manifestations, such as scarlatiniformh and erythema nodosum, are also described (Galindol., 2011). Moreover, Y. pseudotuberculosis is an agent ofase in animals, causing enterocolitis, abortion, mastitis,

pneumonia and septicaemia in several domestic and wildspecies, such as birds, sheep, cattle, pigs, deer and hare(Shwimmer et al., 2007; Zhang et al., 2008; Wuthe et al.,1995). Y. pseudotuberculosis has been divided into 15 O-serotypes (O:1-O:15) and ten subtypes (O:1a-c; O:2a-c;O:4a-b and O:5a-b) on the basis of variable lipopolysac-charides O-side chains (Laukkanen-Ninios et al., 2011). Thedistribution and pathogenicity of Y. pseudotuberculosis

strains vary according to the serotype: in Europe, O1a andO1b are usually identified in cases of gastroenteritis inhumans, whereas in the Far East, serotypes O1b, 2b, 4b and5b are dominant in human cases. The pathogenicity of Y.

pseudotuberculosis is determined by several virulencefactors; invasion (Inv) and Y. pseudotuberculosis derivedmitogen (YPM) are chromosomial (Galindo et al., 2011;Yoshino et al., 1995). A plasmid, pYV, encodes Yersiniaadhesion (Yad A) and Yersinia outer proteins (Yops), and it isconsidered essential for virulence (Galindo et al., 2011).

T I C L E I N F O

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A B S T R A C T

Atypical Yersinia pseudotuberculosis serotype O:3 was isolated from rectal contents of two

wild boars hunted in Italy within a regional wildlife management program. No outbreak of

yersiniosis was reported in this area in the same period and no lesions were found by the

veterinarian at post-mortem inspection. Nevertheless, after histological examination,

granulomatous lesions were detected in submandibular lymph nodes of one of the two

wild boars. Microbiological and bio molecular characterization of the isolates revealed a

melibiose-negative, biotype 2, wbyK + O:3 genotype, carrying inv, yop (yopH and yopB),

virF, and R-HPI. Strains showing the same profile, matching to the criteria of genetic group

5, have been recently reported in fatal cases of yersiniosis in cynomolgus macaques and in

farmed deer and atypical O:3 serotype has been suggested as a pathogenic subtype of O:3.

This is the third report of an atypical O:3 Y. pseudotuberculosis strain, the first outside the

American continent and the first one not associated to fatal yersiniosis. Wild boars could

be a possible reservoir of this emerging pathogen.

� 2014 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +3907534333045; fax: +39075343289.

E-mail addresses: c.magistrali@izsum.it, chiaramagistrali@gmail.com

. Magistrali).

Contents lists available at ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

://dx.doi.org/10.1016/j.vetmic.2014.03.010

8-1135/� 2014 Elsevier B.V. All rights reserved.

C.F. Magistrali et al. / Veterinary Microbiology 171 (2014) 227–231228

Moreover, a chromosomal segment, designated as HighPathogenicity Island (HPI), is associated with highlypathogenic Y. pseudotuberculosis strains; HPI is only foundin strains of serotype O:1, or, in its truncated form (R-HPI), inisolates belonging to serotype O:3 (Galindo et al., 2011;Carniel, 1999). The different geographical distributiondescribed for serotypes has also been reported for virulencefactors (Fukushima et al., 2001; Yoshino et al., 1995). YPM-producing strains are isolated from systemic forms in the FarEast, while yersiniosis in Europe is usually associated withHPI + YPMa strains (Fukushima et al., 2001).

Recently, an atypical O:3 serotype, carrying an addi-tional gene, wbyK, has been reported in fatal cases ofyersiniosis in farmed deer and cynomolgus macaques inNorth America. This atypical O:3 serotype carried severalvirulence genes, in both cases inv, yopB, and yopH weredetected; ypmC, irp1, ybtP-ybtQ, yadA and lcrF were foundin isolates from cynomolgus macaques, which were alsoreported as melibiose-negative (Zhang et al., 2008; Zaoet al., 2013). To the best of our knowledge, this atypical O:3strain has never been reported in healthy animals oroutside North America.

Here we report the isolation of two atypical Y.

pseudotuberculosis serotype O:3 strains from rectal con-tents of hunted wild boars in Italy.

2. Materials and methods

The wild boars originated from two different areas ofthe Umbria region and were hunted in December, 2009,within a regional wildlife management program. Rectalcontents, submandibular lymph nodes and tonsils weresampled and sent to the laboratory within 24 h.

For histological examinations, representative portionsof submandibular lymph nodes and tonsils were fixed in10% neutral buffered formalin (pH 7) and then dehydratedand embedded in paraffin. Three to 4-m-thick consecutivesections were cut, stained with haematoxylin and eosin(HE). The image was digitalized using a video cameraconnected to a microscope (DMR Fluo HC, Leica, USA).

2.1. Microbiology

The two Y. pseudotuberculosis isolates were culturedfrom rectal contents following a cold enrichment proce-dure as described by Laukkanen et al. (2009). Briefly, 5 g ofrectal content were put in 45 ml phosphate-buffered salinewith 0 5% peptone, 1% mannitol and 0 15% bile salts (PMB),incubated at 4 8C for 7–14 days and then subcultured ontoa CIN agar plate and SSI Enteric Medium (34121, StatensSerum Institut of Copenhagen). Y. pseudotuberculosis

suspect colonies were presumptively identified usingoxidase tests, urea slants and indole. Oxidase-negative,urea-positive and indole negative isolates were thenconfirmed by ID 32 E (bioMerieux Italia Spa, Bagno aRipoli, FI, Italia), incubated at 28 8C as suggested byNeubauer et al. (1998).

The isolates were further characterized by serotyping, bythe slide agglutination method using commercial antiseraO:1-O:6 (Denka Seiken, Tokyo, Japan). Antimicrobialsusceptibility was assessed by the disc diffusion method,

following the CLSI M31-A2 (2002) procedure, except forincubation, which was performed at 30 8C for 24 h. Thefollowing commercially available antimicrobial discs(Oxoid Ltd., Cambridge, UK) were used: amoxycillin/clavulanic acid (30 mg), ampicillin (10 mg), cefotaxime(30 mg), ceftazidime (30 mg), cephalexin (30 mg), chlor-amphenicol (30 mg), ciprofloxacin (5 mg), erythromycin(15 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixicacid (30 mg), streptomycin (10 mg), sulphamethoxazole/trimethoprim (25 mg) and tetracycline (30 mg). Autoag-glutination test to detect the presence of pYV was carriedout in trypticase soy broth, incubated at 25 8C and 37 8C, asalready described (Fukushima et al., 2011). Finally, theisolates were biotyped as described by Tsubokura andAleksic (1995), using citrate utilization, and melibiose andrhaffinose fermentation.

2.2. Extraction of bacterial DNA

DNA was extracted from bacterial cells by the boilingmethod. Briefly, 2–3 colonies grown on Tripticase Soy Agarplates were suspended in 100 ml of DNase/RNase freewater, vortexed and boiled for 10 min. The tube was againvortexed and cooled on ice for five minutes before beingcentrifuged at 12.000 � g for 5 min at 4 8C. Five microlitersof the resulting supernatant were used as template for PCR.

2.3. PCR detection of virulence genes

Four sets of primers were used for detection of thevirulence genes inv, virF, yopH, yopB in a multiplex PCR(Thoerner et al., 2003; Zhang et al., 2008). The primers usedfor analysing YPMa, YPMb, YPMc and HPI genes were thesame as those described previously (Fukushima et al.,2001). Separation was performed using the AM320method (100 ng/ml sample was injected at voltage 5 kVfor 10 s, and separation voltage was 6 kV for 320 s). A DNAladder (100.0 bp–2.5 Kb) was used to estimate PCRproducts size in base pairs (bp).

2.4. O-antigen genotyping

A multiplex PCR based O-genotyping was performedusing HotStartTaq1 Master Mix Kit (Qiagen GmbH, D-40724 Hilden) and nine sets of primers targeting differentregions of the O-antigen gene cluster of Y. pseudotubercu-

losis. Primer sequence and concentration were the same asthose described previously (Bogdanovich et al., 2003).

Bacterial strains, ranging from O:1 to O:15, were kindlyprovided from Skurnik Laboratory (Haartman Institute,University of Helsinki) and included, as positive control,with each test run.

2.5. Sequencing

wbyK and ypm PCR products were purified using theQIAquick PCR purification kit following the recommendedprotocol (Qiagen). DNA sequencing was carried out usingBigDye Terminator Cycle Sequencing kit v1.1 on an ABIPRISM310 Genetic Analyzer (Applied Biosystems – LifeTechnologies Italia, Monza MB, Italy). The resulting

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C.F. Magistrali et al. / Veterinary Microbiology 171 (2014) 227–231 229

uences were aligned using the BioEdit sequencenment editor software (Hall, T.A. 1999. BioEdit: ar-friendly biological sequence alignment editor andlysis program for Windows 95/98/NT. Nucl. Acids.p. Ser. 41:95–98). Blast searches were performed to

firm PCR product identity.

esults

Granulomatous lesions were detected in submandibu-lymph nodes of one of the two wild boars. They wereracterized by a necrotic center surrounded by a thine of macrophages, epithelioid and multinucleate giants and marked perifollicular fibroplasia (Illustration 1).granulomatous lesions were found in tonsils.

Microbiology

The two Y. pseudotuberculosis isolates were character- as non- melibiose- fermenting, biotype 2 and sensitivell the tested antimicrobials. The strains reacted with

antisera against Y. pseudotuberculosis O:3 in theventional slide agglutination method.Finally the two isolates showed a positive reaction to

autoagglutination test performed at 37 8C as describedFukushima (2011), revealing the presence of pYV.

PCR detection of virulence genes

The two Y. pseudotuberculosis isolates analysed were positive for chromosomal genes inv and ypmA/ypmC.

sequence analysis of ypmA/ypmC gene confirmed thesence of ypmC in our strains. Virulence gene of HPI irp1

ybtP-yptQ were positive, however IS100, yptE and psn

the left hand part of HPI were PCR negative. Thelence plasmid-borne genes virF, yopB and yopH were all

positive in the strains tested.

O-antigen genotyping

The results of the PCR O-antigen-based genotypingboth isolates matched with those of an O:3 pattern:

gmd-flc+; ddhC-prt+; manB+; ddhA-B+ but an additionalgene, wbyK, was also detected. The sequence of the wbyK

gene, amplified by Ypf-13231 and Ypr-13538 primers,shared 86% of homology with the wbyK gene of Y.

pseudotuberculosis O:1b kindly provided by M. Skurnik(strain Pa3606 from human source; Fukushima-Carniel)and a 100% identity to Y. pseudotuberculosis O:3 wbyK geneisolated from the farmed deer (GenBank accession numberEU338401) (Zhang et al., 2008).

4. Discussion

The epidemiology of Y. pseudotuberculosis is poorlyunderstood: cases of this infection in humans are sporadic,and outbreaks of the disease are rarely described inEurope: recently, they were reported in Russia, France andFinland (EFSA, 2013; Vincent et al., 2008, Vasala et al.,2013). As far as we know, no cases of yersiniosis by Y.

pseudotuberculosis have been described in humans in Italyin the last ten years: on the other hand, no nationalsurveillance program has been conducted and this diseasecould have been underestimated, since the clinicaldiagnosis is not easily achieved and routine culture isnot sufficient to detect this organism (Wunderink et al.,2013).

Several cases have been reported in Italy in animals, inboth domestic and wild species: in particular, Y. pseudo-

tuberculosis is very commonly isolated in cases ofsepticaemia and enteritis in hares, where it is seldomconfused with tularaemia (Wuthe et al., 1995; Gyuraneczet al., 2010).

Despite its importance in both humans and animals,few data are available on the possible sources andreservoirs of Y. pseudotuberculosis. The disease is usuallysporadic, outbreaks rarely occur and this reduces thepossibility that a trace back investigation aimed at findingthe source of infection be carried out. In an outbreak inFinland in 2004, the disease was foodborne, and linked tothe consumption of fresh vegetables, in particular lettuce.The authors suggest that wild animals could havecontaminated lettuce directly by faeces or indirectly,contaminated trough irrigation water sources (Nuortiet al., 2004). In another outbreak, observed in France in2004, cases of yersiniosis in humans were related with anincreased prevalence of this pathogen in rodents, since thecases were reported in rural areas and had a five-yearoscillation pattern resembling the cyclical oscillation of therodent population (Vincent et al., 2008).

In this scenario, the better understanding of virulencedeterminants and serotype diversity of Y. pseudotubercu-

losis in animals living in the wild could give usefulinformation during epidemiological investigations on thesource of yersiniosis in man.

The two Y. pseudotuberculosis strains characterized inthis work were from a survey carried out in wild boarshunted in Umbria in 2009. At slaughter, no lesions wererecorded by the veterinarian who carried out the post-mortem inspection, but the histological examination onsubmandibular lymph nodes of one of the two animalsshowed a lesion attributable to Y. pseudotuberculosis

infection. Therefore, the pathogenicity of this organism1. Illustration 1: Light Micrograph of granulomatous lesions detected

ubmandibular lymph nodes stained with HE.

C.F. Magistrali et al. / Veterinary Microbiology 171 (2014) 227–231230

in wild boar could not be concluded. The two strainsshowed a specific profile: they belonged to an atypical Y.

pseudotuberculosis serotype, carrying a wbyK gene, firstdescribed in deer in North America in 2008 and then inmacaques in 2013. In both cases, the infection wasassociated with severe symptoms and death of theanimals. The source of the infection was not detected,but in both cases the animals had direct or indirect accessto the wild (Zhang et al., 2008; Zao et al., 2013).

The two strains isolated in this study had the samevirulence profile of the atypical Y. pseudotuberculosis isolatesfrom deer in North America in 2008 and from macaques inthe US in 2012: in particular, they were positive to inv, yop

(yopH and yopB) and virF, and carried a R-HPI: nevertheless,no outbreak of yersiniosis associated with mortality wasreported in the wild boar population in the same period.Since wild boars are generally considered an importantreservoir of pathogenic Yersinia species (Fredriksson-Ahomaa et al., 2009); it can be postulated that they couldalso act as a reservoir of this atypical serotype.

The characteristics of this atypical Y. pseudotuberculosis

strain match the criteria of genetic group 5, a European,non melibiose fermenting, low pathogenicity type. Thisgenetic group G5 is common among asymptomaticdomestic pigs in most European countries, and includesserotype O:3 strains harbouring pYV and inv gene(Fukushima et al., 2001; Laukkanen-Ninios et al., 2011).Y. pseudotuberculosis O:3 isolates belonging to G5 havenever been found in wild animals: therefore, the finding ofthe atypical Y. pseudotuberculosis in wild boars is ofparticular interest. It has been hypothesized that thisatypical strain could have originated from an O:3 strain,acquiring the wbyK gene from a recombination event withother Y. pseudotuberculosis serotypes (Zao et al., 2013).Unfortunately, we cannot support this hypothesis, since noother Y. pseudotuberculosis strain was isolated from wildboars during the survey.

In conclusion, in this work the presence of atypical Y.

pseudotuberculosis was reported in wild boars in Italy. Thisis the first time that a O:3 wbyK strain was not isolatedduring an outbreak of yersiniosis and outside the Americancontinent and it gives new insight on the pathogenicity andepidemiology of this emerging pathogen.

Acknowledgments

The authors acknowledge Prof. Mikael Skurnik forproviding us the reference strains used in this study. Thiswork was funded by the Italian Ministry of Health(Progetto di ricerca corrente 2010).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2014.03.010.

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