Phenotypic and genotypic presence of the Yersinia virulence plasmid do not affect the production of enterotoxin YstA by Yersinia enterocolitica strains

The aim of the study was to determine whether the presence of the Yersinia virulence plasmid could affect the production of enterotoxin YstA by Y. enterocolitica strains isolated from pigs which are the main source of infection for humans. The phenotypic features characteristic for the Yersinia virulence plasmid were detected on CRMOX agar in 8 out of 12 strains producing enterotoxin YstA, in 5 out of 12 doubtful strains, and in 11 out of 12 strains not producing YstA. Autoagglutination ability was detected in all 12 Y. enterocolitica strains that were positive in the suckling mice bioassay, in 11 doubtful strains and 10 negative strains. CRMOX+ colonies were generally ystA, myfA, virF and yadA positive, while CRMOX- colonies were only ystA and myfA positive. The amplicons of yadA were not detected in 2 (8.3%) out of 24 CRMOX+ and virF positive strains. The results of this study indicate that the presence of pYV does not affect the enterotoxin-producing ability of Y. enterocolitica strains.     

Multiplex PCR method for differentiating highly pathogenic Yersinia enterocolitica and low pathogenic Yersinia enterocolitica,and Yersinia pseudotuberculosis

A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 10¹–10³ CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.     

鱼源小肠结肠炎耶尔森菌5种毒力基因的检测和分析

采用煮沸法和细菌基因组DNA试剂盒两种方法制备鱼源小肠结肠炎耶尔森菌DNA模板,运用聚合酶链反应(PCR)方法检测小肠结肠炎耶尔森菌的5种毒力基因(ail、yadA、virF、ystB和HPI-int),并对HPI-int基因进行测序分析.结果表明,小肠结肠炎耶尔森菌ail、virF和HPI-int 3种毒力基因被检测出,而另外两种毒力基因ystB和yadA未被检测到.HPI-int基因长度为722 bp,GenBank序列号为JX041513,与该数据库中的小肠结肠炎耶尔森菌强毒力岛基因的相似性达到99％,由此推断该菌株具有较强的致病性.这为深入研究小肠结肠炎耶尔森菌的毒力和危害积累了科学资料.     

Analysis of occurrence of virulence genes among Yersinia enterocolitica isolates belonging to different biotypes and serotypes

The 150 Y enterocolitica strains isolated from humans and from pigs belonged to biotypes 4 (68.7%), 1A (18.7%) and 2 (4%), or were biochemically untypeable (8.6%). Biotype 4 was comprised of Y. enterocolitica strains representing serotype O:3, within biotype 1A the strains either belonged to serotypes O:5 and O:6 or were untypeable, and biotype 2 was represented by the strains of serotype O:9. The strains which were biochemically untypeable belonged to serotypes O:5, O:6 and O:3. Among the strains tested there also were those of an unidentified biotype and serotype. Nearly all the strains of biotype 1A represented genotype ystB+myfA+, and few belonged to genotype ystB+. The presence of the ystB gene in the strains of biotype 1A and only occasional occurrence of the gene in the other biotypes makes ystB a distinguishing marker of biotype 1A. The strains of genotype ystA+ail+myfA+yadA+ predominated in biotype 4 (serotype O:3). The strains of biotype 2 (serotype O:9) represented genotype ystA+ail+myfA+, and the plasmid yadA gene was detected in some of them. Within the group of biochemically untypeable strains ystB- and myfA-specific PCR products were mainly obtained. The genotypes determined for the tested biotypes and serotypes of Y. enterocolitica, based upon the selected genes of virulence, can be applied as distinguishing markers and indicators of the potential virulence of Y. enterocolitica strains, excluding bioserotyping.     

The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains

The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.     

Enteritis in sheep and goats due to Yersinia enterocolitica infection

Yersinia enterocolitica biotype 5, serotype 02,3 was isolated from the intestine of 38 sheep and 8 goats submitted to the laboratory for disease diagnosis. Infected animals were usually young, had diarrhoea and were in poor condition or emaciated. A number were moribund or dead when submitted. Characteristic microabscesses were demonstrated in the intestine of 5 of 38 sheep and 3 of 8 goats and no alternative cause of morbidity or mortality was established in these animals. Of the 33 sheep and 5 goats infected with Y. enterocolitica in which microabscesses were not demonstrated, a number of other diagnoses were made, including internal parasitism (18), selenium deficiency or white muscle disease (6) and cobalt deficiency (2), so that morbidity and mortality were possibly unrelated to Y. enterocolitica infection. Five of 6 sheep exposed experimentally by mouth to Y. enterocolitica biotype 5, serotype 02,3 developed an intestinal infection. Although infected sheep showed no clinical evidence of disease and haematological and biochemical indices remained normal, multiple intestinal microabscesses typical of yersiniosis were demonstrated in 3 of 5 infected sheep. It is concluded that Y. enterocolitica biotype 5, serotype 02,3 is an enteropathogen of sheep and goats. Since sheep and goats may be the specific hosts of this bacterium, its virulence for these species is apparently low. Morbidity and mortality may, therefore, be unusual manifestations of infection.     

黄颡鱼感染小肠结肠炎耶尔森氏菌的组织病理学观察

为探讨小肠结肠炎耶尔森氏菌GM2402株对黄颡鱼的致病性,本试验采用人工回感试验测定其半数致死浓度(LC₅₀),同时采用组织病理学方法探讨该菌对黄颡鱼组织的影响.结果表明:回感18 h后,发病黄颡鱼腹部肿大,胸鳍、腹鳍基部出血,肛门红肿,其LC₅₀为3.0×10^6.2 CFU/mL.组织病理学观察肝细胞肿大、排列紊乱、广泛空泡变性;肾小球萎缩,肾小管上皮细胞肿大;脾脏组织结构疏松充满大量红细胞;鳃小片轻度水肿充血.超微结构观察结果显示肝脏和肠道细胞内线粒体均出现不同程度的肿胀,嵴断裂囊泡化.本试验结果表明小肠结肠炎耶尔森氏菌GM2402株对黄颡鱼有较强的致病性,可导致鱼体多个组织出现病变.     

Experimental mixed infection of rabbits with Yersinia enterocolitica and Listeria monocytogenes

Experimental mixed infection was reproduced in rabbits after per os infection with Yersinia enterocolitica serotype 0:3 cells. Four days later some of animals were re-infected orally with Listeria monocytogenes serotype 4b cells. A third group of healthy rabbits was also infected per os with Listeria monocytogenes. The infectious process was followed dynamically from days 1-28. The experimental animals were examined for clinical, paraclinical and morphological findings. Augmentation of body temperature and alveolar macrophage number, a decreased number of peritoneal macrophages, leucopenia as well as purulent meningoencephalitis, catarrhal pneumonia, lienitis, lymphadenitis and enteritis were detected after experimental mixed infection. Both types of macrophages demonstrated a weak bactericidal activity against Yersinia enterocolitica and a highly expressed killing effect against Listeria monocytogenes. Yersinia and Listeria cells were isolated from the viscera and brain. Both species of bacteria were established intracellularly in the macrophages by electron-microscopic examination. The data received showed that mixed Yersinia enterocolitica 0:3 and Listeria monocytogenes 4b infection of rabbits runs with transitory hyperthermia as a generalized infection and is similar to the Listeria mono-infection. The immunosuppressive effect induced by oral Yersinia enterocolitica infection of rabbits promotes the expression of listerious agents.     

Arthritis after experimental infection with Yersinia enterocolitica 0:3 in rabbits

Arthritis in rabbits was caused after experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, pYV+). Clinical and laboratory signs, bacterial dissemination to the viscera, immune response and morphological findings were studied from day 1 to day 40 post-infection (p.i.). Augmentation of body temperature and erythrocyte sedimentation rate occurred on day 1, and on day 8 p.i. was accompanied by leucopenia. The number of alveolar macrophages was increased up to the 15th day p.i., in contrast to peritoneal macrophage numbers. Extensive bacterial colonization of the internal organs was detected at necropsy until the end of the experiment. Analysis of the cell immune response revealed activation of B cells in peripheral blood, spleen and thymus as well as augmentation of T-cell number in the lymphoid organs examined on days 15, 28 and 40 p.i. Histological changes typical of a generalized infection, such as purulent meningoencephalitis, catarrhal pneumonia and lymphadenitis, were observed. Clinical and morphological manifestations of arthritis were also established. The results obtained show that Y. enterocolitica (serotype 0:3, pYV+) induces a generalized, non-lethal infection in Chinchilla rabbits, complicated by arthritis.     

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.     

Ovine abortion and stillbirth due to purulent placentitis caused by Yersinia pseudotuberculosis

Yersinia pseudotuberculosis was isolated from an aborted placenta and stillborn lamb from a sheep flock having multiple abortions. Given intravenously, it caused elevated body temperatures and purulent placentitis in eight of nine ewes. Two ewes died following infection at 2.5 months of gestation. Two ewes infected at 3.5 months gestation aborted; three infected at four months gave birth to weak, premature, or moribund lambs. One ewe infected at 4.5 months gave birth to a healthy lamb. One lamb which died minutes after birth had focal necrotizing hepatitis, a lesion observed in a stillborn lamb during the original disease outbreak. Y. pseudotuberculosis was reisolated from endometrial, placental, and fetal lesions of experimentally infected animals.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Experimental infection of newborn piglets with Yersinia enterocolitica: an animal model of enteritis

Newborn, colostrum-deprived Large White piglets infected with a human isolate of Yersinia enterocolitica biotype 4 serotype 0:3 were used as an animal model of yersiniosis. Within 3 hours of birth and before being fed, 14 piglets were inoculated orogastrically with 10 ml of bacterial suspension containing about 3 x 10(10) colony forming units of Y. enterocolitica, followed by 10 ml of 10% NaHCO3 solution. A further 14 litter mates acted as controls. The animals were reared on an artificial milk formula and humanely killed at 3 or 5 days after infection. Of the 14 infected piglets, 11 became anorexic, five vomited and 13 developed diarrhoea. Yersinia enterocolitica was isolated from their faeces and small intestinal contents. Body weight gains and the plasma glucose concentrations were significantly lower in the infected piglets than in the controls. Damage to the mucosa was observed in the whole gastro-intestinal tract, but was more severe in the small intestine and caecum. Micro-abscesses surrounding bacteria were present at the base of the villi in all parts of the small intestine, particularly in the distal ileum. Lesions were present in the small intestine in all infected piglets by day 3 and were more extensive by day 5. The liver was damaged by day 5, but not day 3. Similar lesions were seen in the mucosa of the stomach in one of six piglets at 3 days and in two of eight piglets at 5 days. It is hypothesised that the hypoacidity in the newborn stomach, as well as the administration of the NaHCO3 solution, may have produced favourable conditions for bacterial invasion.     

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.