Yersinia enterolitica infections: 2. Impact on human health

The clinical picture of yersiniosis in humans and its prevalence in the human population is described in detail. Mass production of animals, development of meat factories based on sophisticated chains of cold storage units and international trade of meat products and animals are believed to be the reasons for the increasing prevalence of yersiniosis in humans. In Germany, anti-Yersinia antibodies are found in up to 40% of the average population. The financial losses for the national economy cannot be judged. Of special interest for industrial medicine are sequelae-like reactive arthritis in exposed occupational groups such as veterinarians or butchers. However, the lack of national and international data makes the assessment of the potential of yersiniosis as a zoonosis difficult. Therefore, intensive and interdisciplinary research is needed to close the gaps described. Already proven and proposed countermeasures at the different stages of mass production of animals and reglementations for international trade of meat products and animals are introduced. The need for development not only of cheap and rapid diagnostic tools but also for countermeasures and treatment strategies is discussed.     

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.     

Experimental Yersinia enterocolitica placentitis in sheep.

A strain of Yersinia enterocolitica of O serogroup 6,30 isolated from the liver of an aborted ovine fetus was inoculated intravenously into a group of pregnant ewes at about 90 days gestation and produced placentitis with abortion or delivery of infected lambs about 50 days later. Y. enterocolitica of the same serogroup was recovered from the necrotic placental cotyledons and most other fetal tissues and could be isolated from vaginal discharges of the ewes for a least 2 weeks after abortion. Histological changes were consistent with an acute bacterial necrotizing placentitis and systemic infection of the fetus. Subsequent pregnancies in the ewes proceeded to term without evidence of infection.     

Yersinia enterocolitica. Experimental pathogenicity for chinchilla.

An intragastric inoculation of approx. 2 × 10¹⁰ Yersinia enterocolitica cells killed chinchillas in three days in the case of four strains out of six tested. Because of the sensitivity of chinchillas to this bacterium, the test is useful for the evaluation of the virulence and invasiveness of Y. enterocolitica isolates. This animal model could also be used for studies on the mechanism of the infection.     

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.     

The pathogenicity of Yersinia enterocolitica for piglets.

The pathogenicity of Yersinia enterocolitica, a bacterium that has been isolated frequently from healthy swine, was studied in piglets by oral challenge of two litters, one derived by cesarean section and deprived of colostrum, and the other delivered at full-term. Eight cesarean-derived piglets were divided into groups of two and challenged with four serotypes of Y. enterocolitica (O:8, O:21, O:3, O:13). Two deaths occurred and two piglets were killed because of severe illness before termination of the experiment eight days after challenge. Surviving piglets showed no clinical signs of illness. Rectal cultures were consistently positive and all cesarean-derived piglets were colonized in the small intestine and throat at necropsy. Full-term piglets were allowed access for 36 hours to sow colostrum containing low levels of antibody against the challenge strains. Six full-term piglets challenged with three serotypes of Y. enterocolitica (O:8, O:21, O:13) survived for 15 days without any signs of illness. These piglets had fewer positive rectal cultures and showed less extensive colonization of internal organs at necropsy than did cesarean-derived piglets. It is uncertain whether this increased resistance to infection with Y. enterocolitica resulted from colostrum-derived antibody, intestinal colonization with other bacteria, or an improved physical condition which accompanied full-term development. Nevertheless, the results of this challenge experiment suggest that piglets are capable of restricting colonization by Y. enterocolitica to the throat and intestinal tract without development of serious illness.     

Survival of Yersinia enterocolitica in soil and water.

Yersinia enterocolitica O3, O4, O5A, O5B, O6, O6, 30, O9 and O13 and Yersinia intermedia were examined to clarify their survival in natural soil, river water and well water. The O3 strain disappeared most rapidly from soil at both temperature of 4 degrees C and 20 degrees C and from river water at 20 degrees C. Although the O5B and O9 strains disappeared before the O3 strain did from river water at 4 degrees C, the O9 strain survived as long as non-virulent strains did in soil at 4 degrees C. The O3, O5B and O9 strains survived longer at 4 degrees C than at 20 degrees C in soil and river water. Non-virulent strains of O4, O5A and O6 survived longer in well water than in soil and river water. Although the O3 and O5A strains disappeared from supernatant filtered with 100 microns and 5 microns pore size filters, they maintained their viable cell numbers in supernatant of soil filtered with 0.22 microns pore size filter and in the autoclaved supernatant.     

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.     

Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs

Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype O:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week post-inoculation observation period. In contrast, IFN-gamma responses in two B. suis inoculated pigs without bacteriological or serological evidence of infection were below a cut-point of 25pg/ml at all samplings. IFN-gamma responses in repeated samplings from 5 uninfected control pigs and 18 pigs experimentally infected with YeO:9 were all negative, except for solitary false positives in 3.7% of the samples from both the experimentally YeO:9 infected pigs and control pigs. Skin tests using the same commercial Brucella antigen confirmed the ability of cell-mediated immune responses to differentiate between the two infections. In addition, a field evaluation of the diagnostic use of cell-mediated immune responses by IFN-gamma assay and skin test to resolve serological suspicions of Brucella was conducted in an YeO:9 infected pig herd. Following a screening of 200 pigs 39 pigs were identified with false positive serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non-LPS Brucella antigens were specific and sensitive in discriminating subclinical experimental infections with B. suis from both natural and experimental infections with YeO:9.     

Experimental studies on Yersinia enterocolitica infection in chickens exposed at 1-day old

1. Seventy 1-d-old broiler chicks were experimentally inoculated orally with Yersinia enterocolitica serotype 0:3 (1.4 x 10(11) cells/chick), 0:8 (1.6 x 10(11) cells) and 0:9 (8.0 x 10(10) cells) with or without sodium bicarbonate solution (10 g/l). 2. None of the chicks showed any overt clinical signs or pathological lesions although the organism was demonstrated in the ileum and shedding was observed up to 13 d after exposure. 3. The serotype, dose of Y. enterocolitica and administration of NaHCO3 solution had no significant effect on the weight gain of exposed broiler chicks. 4. Y. enterocolitica was isolated from the liver, spleen, heart and gall bladder of infected chicks 70 d after exposure. 5. Although broiler chicks appear resistant to high doses of Y. enterocolitica by the oral route, detection of the organism in the organs of infected chickens is of public health significance.     

Impact of Yersinia enterocolitica enteritis on disaccharidase activity and small intestinal morphology in colostrum-deprived newborn piglets

The effect of enteritis on the development of the small intestine was examined in newborn, colostrum-deprived piglets infected with a human isolate of Y. enterocolitica (serotype 0:3, biotype 4) soon after birth. The piglets were killed 3 days (n = 6) or 5 days (n = 8) after infection, or antibiotic therapy was commenced on day 5 and the animals killed on day 14 (n = 5). Compared with the non-infected controls, infected animals had reduced mucosal lactase and sucrase, but not maltase activity, while after antibiotic therapy, previously infected piglets had a lower lactase and a higher maltase and sucrase activity. Lactase activity was significantly reduced in the duodenum and jejunum, and mean values were lower in the ileum, but the difference did not reach significance; maltase activity was greater at all ages from the distal jejunum to the mid-ileum; sucrase activity was reduced in all segments up to day 5 but after antibiotic therapy was increased in the jejunum and appeared early in the ileum. Enzyme profiles were more mature along the crypt-villus axis in some segments of the intestine in previously infected piglets. Sodium-potassium-ATPase activity was unchanged. There was a reduced villus height:crypt depth ratio, crypt hyperplasia and increased crypt cell proliferation. Morphological maturation, indicated by loss of vacuoles and location of the nucleus at the base of the enterocyte, proceeded distally from the duodenum to ileum from 3 to 14 days of age when only the ileum remained immature. In infected piglets, there was reduced vacuolation and earlier location of the nucleus at the base of the cell in the distal intestine. Accelerated maturity of specific disaccharidases and enterocyte morphology in infected piglets appears to be due to physical damage to the mucosa resulting in faster proliferation of crypt cells and migration of enterocytes. It is suggested that this may reduce macromolecular internalisation and impair the ability to utilise dietary carbohydrate and may have long-term effects on growth and immunological responses of the gut.     

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.     

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 5x10⁹ 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.     

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.     

鱼源小肠结肠炎耶尔森氏菌的耐药性研究

本试验采用聚合酶链反应(PCR)方法检测鱼源小肠结肠炎耶尔森氏菌的四环素类耐药基因(tetA、tetC、tetM)、磺胺类耐药基因(sul1、sul2、sul3)和氨基糖苷类耐药基因(aph(3')-Ⅲa、aac(6')-Ⅰb、ant(3')-Ⅰa、aac(3)-Ⅱa),并采用κ-B法检测该菌对14种抗生素的耐药表型。结果表明,小肠结肠炎耶尔森氏菌8种耐药基因(tetC、tetM、sul1、sul2,aph(3')-Ⅱa、aac(6')-Ⅰb、ant(3')-Ⅰa、aac(3)-Ⅱa)被检测出,而tetA、sul3基因未被检测出。该菌株对复方新诺明、磺胺异恶唑、红霉素、利福平、洁霉素和头孢噻吩6种抗生素耐药,对氟哌酸、氟苯尼考和头孢曲松等8种抗生素敏感。这为治疗小肠结肠炎耶尔森氏菌引起的鱼病积累了资料。     

Yersinia enterocolitica in sheep - a high frequency of biotype 1A

Background

Pigs are regarded as the main reservoir for human pathogenic Yersinia enterocolitica, which is dominated by bioserotype 4/O:3. Other animals, including sheep, have occasionally been reported as carriers of pathogenic strains of Y. enterocolitica. To our knowledge, this is the first study performed in the Nordic countries in which the presence of Y. enterocolitica in sheep is investigated.

Methods

Tonsils and faecal samples collected from sheep slaughtered on the island Gotland (Sweden) from September 2010 through January 2011 were analysed for presence of Y. enterocolitica. In an attempt to maximize recovery, several cultural strategies were applied. Various non-selective media were used and different temperatures and durations of the enrichment were applied before subculturing on Cefsulodin Irgasan Novobiocin (CIN) agar. Presumptive Y. enterocolitica colonies were subjected to urease, API 20E and agglutination test. Yersinia enterocolitica isolates were biotyped, serotyped, and tested for pathogenicity using a TaqMan PCR directed towards the ail-gene that is associated with human pathogenic strains of Y. enterocolitica.

Results

The samples collected from 99 sheep yielded 567 presumptive Y. enterocolitica colonies. Eighty urease positive isolates, from 35 sheep, were identified as Y. enterocolitica by API 20E. Thirty-four of 35 further subtyped Y. enterocolitica isolates, all from faecal samples, belonged to biotype 1A serotype O:5, O:6. O:13,7 and O:10. One strain was Yersinia mollaretii serotype O:62. No human pathogenic strains of Y. enterocolitica were found in the investigated sheep. Other species identified were Y. kristensenii (n = 4), Y. frederiksenii/intermedia (n = 3), Providencia rettgeri (n = 2), Serratia marcescens (n = 1) and Raoultella ornithinolytica (n = 1).

Conclusions

This study does not support the hypothesis that sheep play an important role in transmission of the known human pathogenic Y. enterocolitica in the studied geographical region. However, because there are studies indicating that some strains of Y. enterocolitica biotype 1A may cause disease in humans, the relative importance of sheep as carriers of human pathogenic strains of Y. enterocolitica remains unclear. Tonsils do not appear to be favourable sites for Y. enterocolitica biotype 1A in sheep.     

Experimental studies on the serological relationships between Yersinia enterocolitica and Brucella abortus.

Yersinia enterocolitica O9 was shown to provoke O, OH and H antibody response in calves. Brucella abortus failed to generate Yersinia H antibody response and generated Brucella O antibodies that cross-reacted with Yersinia O and OH antigens. The presence of Yersinia H agglutinins along with a higher titre of Yersinia OH antibody than Brucella O antibody is suggestive of subclinical infection with yersiniosis rather than brucellosis. Cross-absorption of sera from calves with established Yersinia infections indicated that absorption of sera with Brucella O antigens, although completely removing Brucella O antibodies, failed to remove completely Yersinia O, OH and H antibodies, and thus provides an additional method of distinguishing between the two infections.     

Yersinia enterocolitica infection in goat. A serological and bacteriological investigation

The distribution of agglutinating antibodies to Yersinia enterocolitica type 2 in sera from goats in an infected herd, and in 190 other animals from different parts of the country was studied. The faecal excretion of Yersinia enterocolitica from the same animals was also examined. Experimental inoculations of two goats were carried out. The serological results indicate that subclinical cases had occurred in the infected herd during the enzooty and during the following months. Faecal excretion of the organism was observed during the first month after the acute phase of the disease. It was found to be difficult to induce the disease experimentally, but clinical signs were observed in 1 goat after injection of live Yersinia enterocolitica intraperitoneally. The Widal-titre rose to 1/1250 during the first 2 weeks after the inoculation and then fell to 1/80 during the next 2 months. The serological results indicate infections with Yersinia enterocolitica, if a Widal-titre of at least 1/80–1/160 in the first month after the acute phase of a disease and a fall to a low level during the following months are found. Among animals from different partst of the country 16.3 % had a Widal-titre which indicated infection with Yersinia enterocolitica during the previous 3–6 months.