Isolation of pathogenic yersiniae from wild animals in Bulgaria

Pathogenic Yersinia strains were isolated between December 1998 and April 1999 from 37 wild animals: rabbit (Lepus europeus), boar (Sus scrofa scrofa), asiatic jackal (Canis aureus), red fox (Vulpes vulpes), mouflon (Ovis musimon), european river otter (Lutra lutra), beech marten (Martes foina), polecat (Musleta putorius) and wild cat (Felis silvestris). It was established that among the wild animals Y. enterocolitica strains of serotype 0:3 predominated, accompanied by Y. pseudotuberculosis strains of serotype 0:3. In one sample from asiatic jackal and one sample from rabbit, Y. enterocolitica serotype 0:8 was isolated. Yersinia enterocolitica and Y. pseudotuberculosis strains were isolated from tonsils and tongues as well as from the viscera--lung, liver, heart, spleen, kidney and lymph nodes, mainly in young animals (1-2 years of age). The results showed that wild animals are a possible natural reservoir for pathogenic Y. enterocolitica and Y. pseudotuberculosis and are included in the epidemiological chain of yersinioses.     

Diagnosis o Yersinia enterocolitica infections: a review on classical identification techniques and new molecular biological methods

Only three of the eleven species of the genus Yersinia are associated with disease. Y. pestis is the causative agent of plague, Y. pseudotuberculosis and several pathogenic bio/serovars of the species Y. enterocolitica cause yersiniosis. New Y. enterocolitica subspecies with diagnostic relevance have been proposed allowing the differentiation of European and American isolates. The ISO-standard (ISO 102739) summarizes the knowledge gained from enrichment and isolation of Y. enterocolitica from food and feed samples. The final biochemical identification must be carried out by classical tube testing, as commercially available test-systems are not sensitive and specific. For the assessment of the presumptive pathogenicity of a Y. enterocolitica isolate empiric virulence markers can be replaced by PCR assays targeting plasmoidal or chromosomal genes. Their evaluation in terms of routine diagnostic procedures is still missing. The definite identification of Y. enterocolitica isolates can also be achieved by sequencing the 16S rRNA gene. Immunoblot based on plasmoidal encoded Yersinia proteins enables the serological determination of animal and human infections. The development of simple, sensitive and specific rapid identification systems applicable for the direct and indirect diagnosis for veterinary use is a challenge for the future.     

Yersinia species isolated from sheep with enterocolitis

In 40 submissions to the Regional Veterinary Laboratory (RVL) Wagga Wagga from sheep in southern New South Wales from 1981 to 1989, 53 isolates of Yersinia sp were recovered from 45 sheep in 37 flocks. Of 53 isolates, 26 were identified as Y. pseudotuberculosis, 20 as Y. enterocolitica, 5 as Y. intermedia and 2 as Y. frederiksenii. Twelve isolates of Y. pseudotuberculosis tested in the slide agglutination test all belonged to serotype III. The 20 Y. enterocolitica isolates were categorised biochemically as biotype 5 strains and, of 6 isolates serotyped, all belonged to serogroup 2,3. Outbreaks of yersiniosis were most common in late winter and early spring and affected flocks often had experienced a change in husbandry. Infection with Yersinia sp was associated with diarrhoea, illthrift and mortality. At necropsy, congestion and occasionally thickening of the intestinal mucosa were observed in affected sheep. Gastrointestinal nematodiasis and coccidiosis often were concurrent findings. The characteristic histological lesion in sheep infected with Y. pseudotuberculosis was acute segmental suppurative erosive enterocolitis. There were no lesions consistently associated with Y. enterocolitica, Y. intermedia or Y. frederiksenii.     

The isolation of Yersinia sp. from feral and farmed deer faeces

Faecal samples from clinically normal farmed red deer, wapiti, fallow deer; and feral red deer and white tail deer were examined for members of the genus Yersinia. From 922 samples 176 strains of Y.enterocolitica, 56 strains of Y.frederiksenii, 29 strains of Y.kristensenii, eight strains of Y.intermedia, and seven strains of Y.pseudotuberculosis were isolated. High isolation rates of Yersinia sp. were recorded from some farms. Two herds had isolation rates of 33.3% and 36.8%. Sixteen strains of Yersinia sp. in addition to strains of Y.psuedotuberculosis were found to be Hela cell invasive. The majority of these strains were confined to a single herd and represented Y.enterocolitica biotypes I, II and III, Y.intermedia, Y. fredericksenii, and Y.kristensenii.     

Yersiniosis in deer from the Otago-Southland region of New Zealand

Samples from 350 routine deer cases submitted to Invermay Animal Health Laboratory for diagnosis during 1979-1982 were examined specifically for the presence of Yersinia sp. An analysis of 57 cases of yersiniosis due to Yersinia pseudotuberculosis is made and two cases due to Yersinia enterocolitica are described. The occurrence of cases appeared strongly correlated with periods of stress, predominantly in winter when cool wet conditions and lack of grazing combined to precipitate the disease. Animals up to one year old were most commonly affected (64% of cases). Of the 61 strains of Yersinia pseudotuberculosis isolated 35 were of Serotype I, 17 of Serotype II and nine of Serotype III. All strains of Yersinia pseudotuberculosis and the strains of Yersinia enterocolitica isolated from the two cases described were Hela cell invasive and gave a positive autoagglutination test for virulence.     

A cohort study of Yersinia infection in goats

OBJECTIVE: To determine the temporal pattern of Yersinia infections in three goat flocks and examine the influence of management and seasonal factors on the incidence of those infections over a 1-year period. METHODS: A longitudinal study involving monthly culture of faeces for Yersinia spp. from age groups of randomly selected goats on three farms in the Manawatu region of New Zealand. RESULTS: The incidence of excretion of potentially pathogenic Yersinia (Yersinia pseudotuberculosis and Y enterocolitica biotypes 2, 3 and 5) peaked in winter and fell in summer. In contrast, environmental Yersinia (Y enterocolitica biotype 1A, Y frederiksenii, Y intermedia and Y rohdei) showed no clear pattern of seasonal variation. Pathogenic Yersinia were more prevalent in young animals than in adults, while environmental Yersinia were more prevalent in adults. The same type was isolated from the same animal in two or more successive months in about 20 to 25% of cases, and in the remaining cases there was a gap of at least one month between successive isolations, with many animals yielding a particular type on only a single occasion. A notable difference was that with the potentially pathogenic types, no animal had more than one period of time when it was found to be excreting a particular type, suggesting that immunity develops following exposure. In contrast, it was common for environmental types to be isolated from the same animal throughout the study period. Two goats were suspected to have developed clinical yersiniosis but all remaining infected animals showed no clinical signs of infection. CONCLUSIONS: Asymptomatic Yersinia carriage was common in goats in New Zealand, with a clear seasonal and age group pattern of infection with potentially pathogenic types. There was evidence that immunity developed to potentially pathogenic types. This is the first time that Y rohdei has been isolated from goats.     

Prevalence of anti-Yersinia antibodies in European brown hares in North-Rhine Westphalia, Germany

Yersinia (Y.) pseudotuberculosis infections may lead to significant lethality in European brown hare (Lepus europaeus, Pallas) populations especially during the cold and wet seasons. In recent decades, also Y. enterocolitica was isolated from hares found dead. Consequently, a Western-blot technique proved to be valuable for the detection of antibodies against all pathogenic Yersinia isolates was applied to monitor the prevalence of antibodies in hare populations in North-Rhine Westphalia, Germany. A total of 89.6% of the 230 animals tested was seropositive. Further investigations should be performed to elucidate the role of subclinical yersiniosis in the decline of European brown hare populations in Germany.     

Prevalence of gastrointestinal bacterial pathogens in a population of zoo animals

Faecal prevalence of gastrointestinal bacterial pathogens, including Campylobacter, Escherichia coli O157:H7, Salmonella, Shigella, Yersinia, as well as Arcobacter, were examined in 317 faecal specimens from 44 animal species in Belfast Zoological Gardens, during July-September 2006. Thermophilic campylobacters including Campylobacter jejuni, Campylobacter coli and Campylobacter lari, were the most frequently isolated pathogens, where members of this genus were isolated from 11 animal species (11 of 44; 25%). Yersinia spp. were isolated from seven animal species (seven of 44; 15.9%) and included, Yersinia enterocolitica (five of seven isolates; 71.4%) and one isolate each of Yersinia frederiksenii and Yersinia kristensenii. Only one isolate of Salmonella was obtained throughout the entire study, which was an isolate of Salmonella dublin (O 1,9,12: H g, p), originating from tiger faeces after enrichment. None of the animal species found in public contact areas of the zoo were positive for any gastrointestinal bacterial pathogens. Also, water from the lake in the centre of the grounds, was examined for the same bacterial pathogens and was found to contain C. jejuni. This study is the first report on the isolation of a number of important bacterial pathogens from a variety of novel host species, C. jejuni from the red kangaroo (Macropus rufus), C. lari from a maned wolf (Chrysocyon brachyurus), Y. kristensenii from a vicugna (Vicugna vicugna) and Y. enterocolitica from a maned wolf and red panda (Ailurus fulgens). In conclusion, this study demonstrated that the faeces of animals in public contact areas of the zoo were not positive for the bacterial gastrointestinal pathogens examined. This is reassuring for the public health of visitors, particularly children, who enjoy this educational and recreational resource.     

The application of PCR to the identification of selected virulence markers of Yersinia genus

The subject of this study was thirty nine strains of Yersinia enterocolitica, isolated from faeces of humans who showed symptoms typical of intestinal yersiniosis, and seventy strains of Y enterocolitica, four strains of Y. pseudotuberculosis, and one strain of Y. kristensenii from healthy pigs. In the population tested the following serogroups appeared: O3, O9, O2, O5. A PCR was used to detect the presence of pathogenic chromosomal markers, such as myfA and inv genes of the tested Yersinia species. Among Y. enterocolitica strains isolated from humans and belonging to serogroup O3 (thirty four strains) and serogroup O9 (five strains) thirty three Y. enterocolitica O3 strains and four Y. enterocolifica O9 strains, gave a positive reaction to the nmyfA gene, yielding a fragment of 280 base pairs (bp). Among seventy Y. enterocolitica strains isolated from pigs forty strains belonging to serogroup O3 and fifteen strains belonging to serogroup O9 gave a positive reaction to the myfA gene. The presence of 390 bp amplified products, corresponding to the inv gene fragment, was detected in PCR products of three Y pseudotlluberculosis strains from pigs and only in one Y. enterocolitica O3 strain from humans, which had no myfA gene. The results obtained show that the myfA gene is only present in the strains that belong to pathogenic serotypes of Y. enterocolitica. The myfA gene prevailed in the Y. enterocolitica O3 and O9 strains from humans but was less common in the Y. enterocolitica O3 and O9 strains from pigs.     

Mastitis caused by Yersinia pseudotuberculosis in Israeli dairy cattle and public health implications

Yersinia pseudotuberculosis was isolated from mastitis cases in nine dairy cows in Israel. Six cases occurred on one farm (three instances of two cows, 2 months apart) and three cases on one farm each. Seven cows suffered from clinical and two from subclinical mastitis. All but two of the cows were culled. The literature describing cases of bovine mastitis caused by Y. pseudotuberculosis is reviewed and the human public health implications are discussed.     

Immuno-magnetic separation and agar layer methods for the isolation of freeze-injured Yersinia enterocolitica O:8 from water

To develop an effective method to isolate an injured pathogenic Yersinia enterocolitica O:8 organism from environmental samples, we compared the isolation of freeze-injured and non-injured Y. enterocolitica O:8 and found that the isolation was more successful when immuno-magnetic separation (IMS) with anti-Y. enterocolitica O:8 antibody was used. Plating onto cefsulodin-irgasan-novobiocin (CIN) agar and Virulent Yersinia enterocolitica (VYE) agar by means of the agar layer method was found to be effective in isolating the injured cells. The alkali treatment which is generally used for selective detection of Yersinia organism failed to isolate freeze-injured pathogenic Y. enterocolitica O:8 cells. Recovery methods without using the alkali treatment were superior for detecting freeze-injured Y. enterocolitica O:8. Our results demonstrate that the IMS and the agar layer methods should be used to isolate injured pathogenic Yersinia organisms from environmental samples such as water.     

Enteritis in sheep, goats and pigs due to Yersinia pseudotuberculosis infection

The features of naturally occurring Yersinia pseudotuberculosis serotype III infections in 16 sheep, one goat and 3 pigs, and Y. pseudotuberculosis serotype I infections in 3 goats, are described. Affected animals usually had diarrhoea and were in poor condition or emaciated. A number were moribund or dead when submitted for necropsy. Thickening of the caecal and colonic mucosa was the only gross lesion attributable to Y. pseudotuberculosis infection, with liver or other visceral abscesses not being seen. Characteristic microabscesses were demonstrated in the intestinal mucosa of 10 sheep, one goat and one pig infected with Y. pseudotuberculosis serotype III and one goat infected with Y. pseudotuberculosis serotype I. Sheep, goats and pigs dosed orally with Y. pseudotuberculosis serotype III, the serotype isolated most commonly from these species, developed intestinal infection. In sheep and pigs, infection was accompanied by diarrhoea. Haematological changes and specific antibodies were elicited in all 3 species in response to infection. Microabscesses were seen in the intestinal mucosa of all experimentally exposed animals. The occurrence of field cases and the results of experimental exposure confirm that Y. pseudotuberculosis serotype III is an enteropathogen of sheep, goats and pigs. The association of Y. pseudotuberculosis serotype I with lesions in a goat, indicates that this bacterium may also be a pathogen of this species. It is concluded that Y. pseudotuberculosis serotype III is an enteric pathogen of a wide range of ungulate species including cattle, buffalo, deer, antelopes, sheep, goats and pigs. Serotypes I and II, while having a more restricted host range, are probably also pathogens of ungulates and, in particular, deer, antelopes and goats.     

Enterocolitis in cattle associated with Yersinia pseudotuberculosis infection

A syndrome in cattle of diarrhoea and death associated with enteric Yersinia pseudotuberculosis infection is described. Outbreaks occurred during winter and early spring in adult cattle grazing pastures waterlogged by recent flooding or persistent heavy rain. Antibiotic therapy was effective early in the course of the syndrome. At necropsy there was severe acute enterocolitis, and bacteria consistent with Y. pseudotuberculosis were observed in the lesions. This organism could usually be isolated from the intestines of affected animals but was recovered less often from other organs. Representative isolates were identified as Y. pseudotuberculosis serotype III. The association of this syndrome with waterlogged pastures and low temperatures suggests that these conditions favour transmission of Y. pseudotuberculosis infection in cattle. The role of Y. pseudotuberculosis as primary pathogen requires confirmation.     

Recovery of Yersinia pseudotuberculosis from the faeces of healthy cattle

Attempts were made to recover and serologically identify Yersinia pseudotuberculosis from the faeces of groups of nine to twenty clinically healthy cattle eight to thirteen months old on each of 50 farms in the northern part of New Zealand. Yersinia pseudotuberculosis was recovered from 134 (26.3%) of 509 faeces samples from cattle on 42 (84%) farms and from nine of ten samples on two of these farms. Serotypes I, II, and III were identified, of which serotype III was by far the most common and accounted for 125 (93.2%) of the 134 isolates. Because of the common occurrence and widespread distribution of Y. pseudotuberculosis it is suggested that the clinical significance of isolation of this micro-organism from faeces samples or intestinal contents should be treated with caution.     

The activity of chosen bacteriophages on Yersinia enterocolitica strains

The aim of the present study was to evaluate the lytic activity of three bacteriophages on Yersinia enterocolitica strains isolated from humans and pigs. The Y. enterocolitica strains tested belonged to 0:3, 0:9 and 0:2 serogroups. The ZD5 phage was obtained from a water sample, but remaining phages were obtained from the lysogenic Y. frederiksenii 7291 and Y. enterocolitica 8684 strains. All the Y. enterocolitica strains tested which belonged to 0:9 serogroup did not show any susceptibility to the bacteriophages used. The bacteriophages tested showed different lytic activity on the Y. enterocolitica 0:3 strains investigated. The phage susceptibility of Y. enterocolitica 0:3 strains revealed 9 different phage patterns. ZD5 phage showed the highest lytic activity, because it produced confluent lysis of the most Y. enterocolitica 0:3 strains tested. The Y. enterocolitica 0:2 strains isolated from pigs showed the similar phage susceptibility. The Y. kristensenii and Y. pseudotuberculosis strains tested were not sensitive to the bacteriophages used.     

The role of wild birds and the environment in the epidemiology of Yersiniae in New Zealand

A survey was carried out to determine the prevalence of Yersiniae in wild passerines in the lower half of the North island of New Zealand over a period of 12 months. Samples of soil, water and foliage were also collected. Out of a total of 1370 avian samples, only two strains of Y. pseudotuberculosis were isolated and a total of 98 strains of environmental yersiniae were identified, including Y. enterocolitica biotype 1a, Y. frederiksenii, Y. kristensenii and Y. intermedia. No strains of Y. pseudotuberculosis were isolated from 1032 non-avian samples collected, which included 100 samples taken from wild mammals. From the non-avian samples, 51 strains of environmental Yersiniae were identified, of which the relative prevalence of Yersinia enterocolitica, biotype 1a, Y. frederiksenii, Y. kristensenii and Y. intermedia was similar to that in the rural passerines. The prevalence of Yersiniae in soil samples was greater in rural areas than in urban areas of the survey region. In both rural and urban passerine populations, the prevalence of Yersiniae was greater in the winter and early summer than at other times of the year.     

A combination of different polymerase chain reaction (PCR) assays for the presumptive identification of Yersinia pestis

A combination of four polymerase chain reaction (PCR) assays targeting the Yersinia pestis-specific plasmoidal genes of the fraction 1 capsular antigen and plasminogen activator/coagulase, the gene of the V antigen of the Yersinia virulence plasmid, and the chromosomal 16S rRNA gene was evaluated for the identification of Y. pestis isolates. All four assays were subjected to the same sample preparation technique, reagents and cycling conditions. Eighteen Y. pestis, 66 Y. pseudotuberculosis, 40 Y. enterocolitica strains, the type strains of the other Yersinia species, and 20 other pathogenic bacterial strains were investigated. By using the proposed combination of PCR assays all Y. pestis strains were identified correctly. The applicability of this combination of PCR assays was demonstrated by the detection of Y. pestis DNA in spiked tissues from Rattus norwegicus and fleas (Xenopsylla cheopis and Ctenocephalides spp.). As little as 60 genome equivalents were detected. This system is applicable for monitoring Y. pestis and its vectors in enzootic natural foci and in the diagnosis of plague in humans and animals.     

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.     

Faecal survey of deer for Yersinia pseudotuberculosis and Salmonella sp

The results of a national survey of faeces from clinically normal deer for Salmonella sp. and Yersinia pseudotuberculosis are reported. Five isolates of Y. pseudotuberculosis and none of Salmonella sp. were made from 3810 faeces representing 122 farms.     

Identification of Yersinia enterocolitica in minced meat: a comparative analysis of API 20E, Yersinia identification kit and a 16S rRNA-based PCR method

The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin-irgasan-novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous 'typical'Yersinia-like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia-like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nürtingen, Germany), the Yersinia identification kit (Merlin, Bornheim-Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.