Vet Microbiol. 2016 Dec, 197, 78-82 

Sheep carrying pathogenic Yersinia enterocolitica bioserotypes 2/O:9 and 5/O:3 in the feces at slaughter

Suvi Joutsen, Kirsi-Maria Eklund, Riikka-Laukkanen-Ninios, Roger Stephan, and Maria Fredriksson-Ahomaa

Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland

Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Switzerland

 

Abstract 

Yersinia enterocolitica is a heterogeneous species including non-pathogenic strains belonging to biotype 1A and pathogenic strains belonging to biotypes 1B and 2 to 5. Pathogenic strains of biotypes 2 to 4 carrying the ail virulence gene have frequently been isolated from domestic pigs at slaughter. In sheep, mostly non-pathogenic biotype 1A strains have been reported. In our study, the prevalence of ail-positive Y. enterocolitica was studied by PCR and culturing in 406 young sheep (<1 year of age) and 139 older sheep at slaughter in Finland. When using PCR, the detection rate was 11% (45/406) in young sheep originating from 11 (18%) farms. Surprisingly, Y. enterocolitica belonging to bio/serotypes 2/O:9 and 5/O:3, carrying both chromosomal and plasmid-borne virulence genes, were isolated from the fecal samples of 10 (2%) and 23 (4%) sheep, respectively. All isolates of bio/serotypes 2/O:9 (19 isolates) and 5/O:3 (53 isolates) carried the chromosomal virulence genes ail, inv, ystA, and myfA, and almost all isolates (71/72) also carried the virulence genes virF and yadA located on the virulence plasmid. The isolates showed high susceptibility to tested antimicrobials and low genetic diversity by PFGE. Y. enterocolitica bio/serotype 5/O:3 is a very rare bio/serotype, and has earlier only sporadically been reported in European wildlife and in sheep in Australia and New Zealand. Bio/serotype 2/O:9 is a common bio/serotype found in humans with yersiniosis, and has sporadically been isolated in wild and domestic animals.

KEY WORDS: Yersinia enterocolitica; biotype; sheep; feces

 

Supplement: 

Yersinia enterocolitica is one of the three Yersinia species causing a disease to humans and animals called yersiniosis. Y. enterocolitica causes mainly an enteric infection in humans obtained fecal orally through contaminated food or water or through direct contact with infected animals. Y. enterocolitica is the third most frequently reported gastrointestinal pathogen in humans in the European Union (EU) [1]. In 2015, the number of reported confirmed cases of human yersiniosis was 7,202 with a notification rate of 2.2 cases per 100,000 population, which was 6,8% higher than in 2014. Notification is not mandatory in all European countries. The most common serotype reported in humans in EU is O:3 followed by O:9 and O:5,27. Serotype O:3, which is the most common type found in human yersiniosis, is commonly isolated from pigs.

Y. enterocolitica is a very heterogeneous group of bacteria which can be divided into 6 biotypes and several serotypes. Five biotypes (1B, 2-5) are considered pathogenic and biotype 1A non-pathogenic. Some biotype and serotype combinations have been associated with yersiniosis and different animal reservoirs. Pigs are an important reservoir of bio/serotype 4/O:3 found most frequently in the tonsils of fattening pigs in Europe.

Bio/serotype 2/O:9 is associated with cattle and bio/serotype 5/O:2,3 is associated with wild hares. Wild boars have been shown carry bio/serotypes 2/O:5,27, 2/O:9 and 4/O:3 in the tonsils [2]. Biotype 1B, which is a high-pathogenic type and very rare in Europe, is associated with wild life. However, animal reservoirs and transmission routes of pathogenic Y. enterocolitica remain unclear. So far only contaminated pork has shown to be an important infection source of human yersiniosis linked bio/serotype 4/O:3 [3].

Isolation and identification of pathogenic Y. enterocolitica is very challenging especially from non-human sources [4]. Number of pathogenic strains in asymptomatic animals, food and environmental samples is typically very low and they are easily over grown even on selective agar plates by other bacteria in the samples. So far all available culture methods have a quite low sensitivity and specificity and none of the methods are optimal for all bio/serotypes. This can be overcome by screening the samples with PCR targeting important virulence genes before isolation and identification.

 

ail-positive Y. enterocolitica isolates found in sheep belonged to three different biotypes

Pathogenic Y. enterocolitica strains carry several important virulence genes in the chromosome and on the virulence plasmid, which is essential for the bacteria to survive and multiply in the lymphoid tissue. One of the important chromosomal virulence genes is the ail which is detected in pathogenic and not in non-pathogenic strains. In our study, ail-positive Y. enterocolitica was surprisingly isolated from 35 young sheep originating from 8 farms. Very few studies have been conducted on sheep and mainly non-pathogenic strains have been isolated from them before. The ail was detected in 97 isolates belonging to biotypes 1A, 2 and 5. Surprisingly, ail-positive Y. enterocolitica isolates of biotype 1A was found in 3 young sheep which shows that detection of ail gene alone is not enough to assess the pathogenicity. The ail was detected in all Y. enterocolitca isolates belonging to biotypes 2 and 5 which also carry two virulence genes virF and yadA located on the virulence plasmid.

 

Identification of Y. enterocolitica belonging to biotype O:5 is challenging

In total, 53 ail-positive isolates from 23 young sheep originating from 3 herds were identified as biotype 5. They grow as very tiny colonies on CIN which resembled the colonies of Y. kristensenii (Fig 1). The obtained API20E code (1015323) identified them as Pantoea. Furthermore, the biotyping was not clear because all isolates were rapidly xylose positive. All isolates agglutinated clearly with O:3 antiserum but only some with O:1,2. However, all isolates displayed a very similar genotype. Our results indicate that these isolates probably belong to bio/serotype 5/O:2,3 which is also called hare type found in wild hares in Europe.

 

 

Fig. 1. Yersinia colonies on CIN agar plates (A=Y. enterocolitica biotype 1A, B=Y. enterocolitica biotype 2, C=Yersinia enterocolitica biotype 5, Y. kristensenii)

 

Sheep is a reservoir for bio/serotypes 2/O:9 and 5/O:(1,2)3

The reservoirs of bio/serotypes 2/O:9 and 5/O:3 are so far not clear. However, bio/serotype 2/O:9 is associated with cattle and bio/serotype 5/O:2,3 with wild hares. Our results show that young sheep under one year old can excrete pathogenic Y. enterocolitica belonging to bio/serotypes 2/O:9 and 5/O:3 indicating that young sheep can be a reservoirs of these bio/serotypes (Fig. 2). All sheep were studied at the slaughterhouse. During slaughter the carcasses can easily be contaminated with Yersinia-positive feces. Yersinia may also contaminate raw milk during milking. As a psychrotrophic bacteria even small number of Yersinia can multiply during cold storage on the carcasses/meat and in the milk causing public health problems to humans, which handle/eat raw sheep meat and drink raw milk.

 

 

 

Future plans:

(1)    We are going to study the ail gene in Yersinia strains isolated from different sources in more detail

(2)    The ail-positive isolates from sheep will be sequenced to get more information about epidemiology

(3)     We are going to study what impact the wild life has on the presence of Y. enterocolitica in sheep

(4)    We are starting a project to study the occurrence of Yersinia on sheep/goat carcasses and in sheep/goat raw milk

 

Acknowledgements:

This work was supported by research funding from the Ministry of Agriculture and Forestry, Finland (999/311/2013) and the Walter Ehrström Foundation. Maria Stark and Anu Seppänen are acknowledged for technical assistance.

 

Contact:

Maria Fredriksson-Ahomaa, DVM

Professor

Department of Food Hygiene and Environmental Heath, Faculty of Veterinary Medicine, University of Helsinki, Finland

E- mail: maria.fredriksson-ahomaa@helsinki.fi

 

References:

  1. EFSA 2016. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA Journal, 2016, 14, 4634.
  2. Fredriksson-Ahomaa M, Wacheck S, Bonke R, Stephan R. Foodborne Pathogens and Diseases, 2011, 8, 733-737.
  3. Fredriksson-Ahomaa M, Stolle A, Korkeala H. Molecular epidemiology of Yersinia enterocolitica infections. FEMS Immunology and Medical Microbiology, 2006, 47, 315-329.
  4. Fredriksson-Ahomaa M, Korkeala H. Low occurrence of pathogenic Yersinia enterocolitica in clinical, food and environmental samples: a methodological problem. Clinical Microbiology Review, 2003, 16, 220-229.