Fecal immunoglobulin A (IgA) antibodies to Yersinia enterocolitica in mice orally vaccinated and infected

Fecal immunoglobulin-A (IgA) antibodies to Yersinia enterocolitica serovar O3 strain were detected in the mice orally immunized with formalin-killed organisms. Y. enterocolitica O3 organisms were inhibited to colonize in the intestines of the mice producing fecal IgA. The fecal IgA antibodies were detected in the mice orally infected with the bacteria. When IgA was produced in the mice infected, they ceased shedding the organisms in their feces.     

Ecological studies of Yersinia enterocolitica. III. Cross-protection against fecal excretion between Y. enterocolotica serovars 3 and 5.27 in pigs

Yersinia enterocolitica biovar 4 serovar 3 and biovar 2 serovar 5.27 failed to establish in the intestines of pigs when challenged with the homologous or heterologous strains. After inoculation, the serum O-agglutinin titers were 1/10 or less and were not boosted by challenge with either serovar. Y. enterocolitica were not recovered in any specimens at the time of slaughter.     

Introduction and reisolation of selected gram-negative bacteria from fermented edible wastes

A Lactobacillus fermentation process, using edible food wastes, was tested for its ability to eliminate selected bacterial pathogens. This fermentation process converts food wastes into a feed ingredient for animal consumption. Six gram-negative bacterial pathogens of potential zoonotic importance were tested. These experimental organisms were: Salmonella enteritidis serovar typhimurium, S enteritidis serovar anatum, S cholerae-suis, Yersinia enterocolitica, Y pseudotuberculosis, and Pasteurella multocida. Each organism was introduced into ground waste that had been previously inoculated with L acidophilus, and was mixed. This mixture was divided among 8 containers, and was incubated in duplicate at 5 C, 10 C, 20 C, and 30 C for 96 hours. The temperature of the reactant containers, reduction-oxidation potential, and pH were monitored. Waste samples were obtained initially and subsequently at 24-hour periods for 96 hours. Qualitative and quantitative recovery attempts from each sample were made for the introduced gram-negative bacteria. Pasteurella multocida and the S enteritidis serovars typhimurium and anatum survived the fermentation at 5 C and 10 C, but were killed after 48 hours at 20 C and 30 C. Salmonella cholerae-suis survived at 5 C, but was destroyed by 72 hours at the remaining temperatures. Yersinia enterocolitica was viable through 70 hours, but was killed by 96 hours. Yersinia pseudotuberculosis was not reisolated at any temperature.     

Development of an ELISA procedure to detect swine carriers of pathogenic Yersinia enterocolitica

An enzyme immunoassay (ELISA) was developed to detect antibodies in pigs against the lipopolysaccharidic antigen of the three serotypes of Yersinia enterocolitica mostly associated with human infections. Recent epidemiological evidence has demonstrated that pigs and pork are important sources of yersiniosis in humans. The purpose of this study was to clarify the use of an ELISA to detect swine carriers of this enteroinvasive bacteria by examining seroconversion and tissue distribution of Y. enterocolitica following experimental infection and then screening pigs at a slaughterhouse by bacterial culture and ELISA. It was observed that seroconversion occurred in animals experimentally inoculated with Y. enterocolitica but not with other enterobacteria. It was also found that 27% of swine at a slaughterhouse carried the bacterium in their tonsils and/or intestinal tract, whereas 66% showed serological evidence of previous infection. About 6% of swine at slaughter were culture-positive, but seronegative. Although, similar numbers of swine showed serological evidence of previous infection by each of the three Y. enterocolitica serotypes tested, virtually all culture isolates belonged to serotype O:3. This ELISA appears as a valuable control tool that can be used, in conjunction with culture, to identify pigs or herds infected by strains of Y. enterocolitica associated with human infections.     

Studies of the efficacy of Enterocoliticin, a phage-tail like bacteriocin, as antimicrobial agent against Yersinia enterocolitica serotype O3 in a cell culture system and in mice

The efficacy of enterocoliticin, a phage tail-like bacteriocin, as antimicrobial compound against infections with pathogenic Yersinia enterocolitica serotype O3 strains was assessed. In cell cultures, which were infected with the Y. enterocolitica strains 13 169 or 6471/76, bactericidal activity of enterocoliticin was found for bacteria adhering to the surface of eukaryotic cells, whereas bacteria, which had invaded the eukaryotic cells, were not accessible to the bacteriocin. The interaction of enterocoliticin with Y. enterocolitica was further examined in animals. Female BALB/c mice were experimentally infected with the two Y. enterocolitica strains and enterocoliticin was applied as antimicrobial compound by the oral route. Experimental variations concerning the infectious doses of the Y. enterocolitica strains and the time points of application of the bacteriocin were investigated. The increase of the Yersinia CFU titre in animals was retarded at time points shortly after the application of enterocoliticin indicating that the particles were effective on recently introduced Yersinia. The repeated application of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Yersinia.     

Prevalence of Yersinia enterocolitica in goat herds from Northern Germany

The prevalence of human pathogenic Yersinia enterocolitica isolates in livestock farming is of paramount interest. Raw goat milk has been proposed as a source of human yersiniosis; however, no data on the prevalence of human strains of Y. enterocolitica in goat herds are available. Therefore, fecal samples (n = 575) were collected from 24 goat herds from Lower Saxony, northern Germany. Pre-enrichment in peptone, sorbitol and bile salts broth was followed by plating on cefsuloidin irgasan novobiocin agar. Yersinia enterocolitica was isolated from 17 (3%) samples of five (21%) goat herds. All isolates were biovar 1A, but represented various serovars. PCR assays targeting Yersinia adhesin (yad) gene and the yopT gene, both associated with pathogenicity, produced no amplification products. Therefore, the isolates can be regarded as opportunistic apathogenic bacteria. Consequently, milk, cheese or meat from goats should not be considered as an important source for human yersiniosis.     

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.     

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.     

Pretreatment of guinea-pigs with iron or desferal influences the course of Yersinia enterocolitica infections

The effects of iron excess and desferrioxamine in pretreated guinea-pigs on the immune response (production of Yops) and on the histological changes in infections with Yersinia enterocolitica 0:3 and Y. enterocolitica 0:8 were investigated. The prior overload of the guinea pigs with Dextrofer or treatment with Desferal increased the pathogenic activity of Y. entercolitica 0:3 and led to a generalized infection. Immunoblot analysis showed that in conditions of iron overload the expression of outer membrane proteins (Yops) of Y. enterocolitica 0:8 was blocked. This was accompanied by weak changes in the tissues. The iron limited conditions stimulated production of a low molecular weight protein (17 kDa) on day 6 and easier proliferation of the bacterium. This in vivo study intends to show that in Y. enterocolitica infections a leading role is played not only by iron itself but also by the bacterial strain.     

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.     

Findings of Yersinia in slaughter pigs, synantropic animals and workers in meat products in a defined region

In 15 selected stocks in the Strakonice district, 507 slaughter pigs, 708 small terrestric mammals and 110 free-living birds were examined in a two-year period (1986-1987) to study the occurrence of carriers of yersiniae and their elimination. Rectal smears from 243 persons working in livestock production were examined in the same way. Standard bacteriological methods, recent examination procedures (Aulisio et al., 1980; Aldová, 1981) and a diagnostic antiserum (03 IMUNA Sarisské Michalany) were used for the examination. The following results were obtained: In pigs: 1. yersiniae were detected in 65 cases (12.8%); of this, in 31 cases they occurred in the tonsils, in 35 cases in ileum, and twice in the ileocaecal lymph nodes. 2. Epidemiologically significant Y. enterocolitica 4;03 was detected in 28 cases (5.5%); of this, 22 times in the tonsils, 7 times in ileum, and once in the ileocaecal lymph nodes. 3. The seasonal nature of the occurrence of yersiniae was confirmed in 1986, with maxima in winter-spring, but in 1987 their occurrence declined substantially to less than a quarter. In the small mammals, yersiniae were detected 28 times (4%); of this, 7 times in common field mouse, 11 times in common vole, 5 times in house mouse, twice in shrew, once in Apodemus flavicollis, and once in Apodemus sp. 2. Y. enterocolitica 4;03 was detected twice (0.26%), both cases in the house mouse. Other results: 1. In all the 110 free-living birds the examination for yersiniae had a negative result; 2. in the rectal smears of 243 persons employed in livestock production, yersiniae were identified twice (0.8%)--in one case Y. enterocolitica 1, in the other Y. enterocolitica biovars 1 and 2.     

Ecological studies of Yersinia enterocolitica I. Dissemination of Y. enterocolitica in pigs

Pigs were examined on five farms for carrier status of Yersinia enterocolitica. Yersinia enterocolitica biovar 4, serovar 3, phagovar VIII was isolated consistently from the feces of fattening pigs on one farm and sporadically from those of similar pigs on the other farms and of sows on all five farms, during a one-year period of weekly surveys. Seasonal variation was not a feature of fattening pigs on a highly contaminated farm. In other pigs, however, the organisms were not isolated during the summer months. On a highly contaminated farm, the organisms were excreted in the feces of 8 to 15-week-old pigs within 1–3 weeks of entering pens which were thought to be contaminated with the organisms. On a detailed observation of natural infection of Y. enterocolitica in eight pigs, the organism appeared in the pigs' feces within 2–7 weeks of them being moved to a pen which had been washed thoroughly after becoming contaminated, by a previous group of pigs, with feces containing 10⁵ viable organisms per g. Thus, Y. enterocolitica is apparently transmitted from infected feces or picked up from the floor of a contaminated pen, and the regular schedule of pig movement among the pens is an important factor in the spread of Y. enterocolitica within a piggery. Intestinal colonization continues for a long time and does not occur by re-infection. The organisms were not isolated from eight pigs at the time of slaughter, and their serum O-agglutinin titers were $\text{1}\text{40}$ or less. Thus, circulating antibody may not inhibit intestinal colonization by Y. enterocolitica.     

Morphology of intestinal colonization of Yersinia enterocolitica serovar O3 in mice

The present study was made to know the morphology of the initial invasion and lesions involved in the intestinal colonization of Yersinia enterocolitica serovar O3 in the epithelium of Peyer's patches of mice. Microfold (M) cells formed a specific structure like a pseudopodium and the bacteria were observed on the surface of the pseudopodium-like structure 4 hr after oral administration of serovar O3. The colonies of serovar O3 were observed in the epithelium and the lamina propria of the Peyer's patches dome region, and the bacteria grown in the Peyer's patches were in direct contact with the lumen without covered with the host tissue 24 hr after the administration.     

Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.     

Uptake of killed Yersinia enterocolitica by pseudopodia of M cells in the Peyers' patches of the murine small intestines

To understand the mechanisms of uptake of killed bacteria of Yersinia enterocolitica serovar O3 into the epithelium of Peyer's patches, the killed bacteria were perorally inoculated into mice and observation was carried out by scanning and transmission electron microscopy. Microfold (M) cells formed a specific pseudopodium-like structure and the bacteria were observed on the surface and the interior of the pseudopodium-like structure 8 hr after oral administration of killed bacteria of serovar O3.     

Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7

Sera from cattle naturally infected with Brucella abortus (n = 160), vaccinated with B. abortus S19 (n = 88) or immunized with Yersinia enterocolitica O:9 (n = 25) or Escherichia coli O157:H7 (n = 80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera (n = 523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA(Br)), CELISA and FPA (FPA(Br)) and the Y. enterocolitica IELISA (IELISA(Ye)) while the Y. enterocolitica FPA (FPA(Ye)) detected antibody in 93.8% and the E. coli IELISA (IELISA(Ec)) 86.9% and the E. coli FPA (FPA(Ec)) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA(Ec), 21.6% in the FPA(Br) and 5.7% in the FPA(Ye). Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA(Ec) and FPA(Ec) although surprisingly 87.5% reacted in the IELISA(Ye) and only 3.8% in the IELISA(Br). No reactions were observed with these sera in the FPA(Br) and FPA(Ye) but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA(Br) and IELISA(Ye) and 80% in the IELISA(Ec). In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA(Br), 28% in the FPA(Ye) and 12% in the FPA(Ec). Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA.     

Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A, serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD). METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5 x 10(9) colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR). RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non-pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain. CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

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.     

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.     

Suppression of heat-stable enterotoxin production by Yersinia spp. in milk

One of 16 raw milk isolates of Yersinia enterocolitica and Y. intermedia produced heat-stable enterotoxin (ST) in milk at 25 degrees C but not at 4 degrees C after 21 days of incubation. A catabolite repression of ST synthesis by the lactose-fermenting strain of Y. enterocolitica was observed when 4.6% lactose was added to trypticase soy broth. However, the lactose-fermenting strain was killed by acid produced by lactose fermentation in milk and did not produce ST in milk with the pH adjusted to neutrality. This study suggested that lactose and fat in milk are not the fundamental inhibitors of ST synthesis by Y. enterocolitica and that repression of ST synthesis may be related to other components.