Experimental Yersinia enterocolitica infection in mice: Kinetics of growth

Infection of several strains of laboratory mice with a virulent strain of Yersinia enterocolitica was followed by performing viable bacterial counts on homogenates of selected tissues at intervals after intragastric, aerogenic, or intravenous infection. It is observed that CD-1 mice are more susceptible to Y. enterocolitica infection than either the C(57)B1/6 or B6D2 strains. Development of an enteric infection is dose dependent; less than 5 x 10(7) organisms by mouth yields sporadic, low levels of systemic infection, with many of the animals showing no apparent infection. Increasing the challenge inoculum by a factor of 10 eliminates the variability among the animals, giving rise to an enteric infection in all of the mice that moves quickly to the mesenteric lymph node. The bacterial population in the lymph node multiplies rapidly, and the infection is disseminated to the spleen, liver, and lungs, ultimately killing most of the animals. Exposure to an aerogenic challenge of less than 1,000 organisms resulted in a fulminating pneumonitis with an invariably fatal outcome. Intravenous challenge with 500 organisms caused a rapidly fatal, systemic infection. The growth of the bacteria in the intravenously infected mouse depends upon the temperature at which the challenge inoculum had been grown in vitro. At temperatures below 26 C, the bacteria are cleared from the blood at a slower rate and are more resistant to intracellular killing, as compared to organisms grown at 37 C. This effect results in the inoculum increasing to greater numbers in the tissues in a shorter period of time.     

Yersinia enterocolitica infection in resistant and susceptible strains of mice.

We investigated natural resistance in mice to Yersinia enterocolitica, an enteric bacterial pathogen of humans, with a view to determine host genetic factors that are important in resistance. Most mouse strains studied (C3H/HeN, BALB/c, BALB.B, DBA/2, A, Swiss, and SWR) were highly susceptible to infection (50% lethal dose [LD50], 2 X 10(2) to 6 X 10(2) Y. enterocolitica administered intravenously [i.v.]). In contrast, C57BL/6 mice were highly resistant (LD50, 2 X 10(5) Y. enterocolitica administered i.v.). Resistance to i.v. Yersinia infection did not appear to be related to the Ity locus (which codes for resistance to Salmonella typhimurium and other pathogens) because Ityr mice (C3H/HeN, DBA/2, A, and SWR) were more susceptible to Y. enterocolitica than were Itys (C57BL/6) mice. In addition, because BALB.B mice (congenic to C57BL/6 mice at the H-2 locus) were susceptible, resistance was probably not H-2 linked. BALB/c X C57BL/6 F1 mice were intermediate in their resistance to Y. enterocolitica infection (LD50, 3 X 10(4) organisms administered i.v.), suggesting that resistance to Y. enterocolitica depends on a gene dosage affect or a resistance gene(s) interaction between susceptible and resistant parents. Further studies with C57BL/6 and BALB/c mice as prototype resistant and susceptible strains were undertaken. A time course study of Y. enterocolitica growth in various organs following i.v. infection revealed no strain difference in bacterial growth during the first 48 h of infection. Thereafter, however, C57BL/6 mice were capable of restricting Y. enterocolitica growth in all tissues (liver, lung, spleen, kidneys), whereas extensive bacterial proliferation occurred in BALB/c mice tissues. BALB/c mice were also more susceptible to oral Y. enterocolitica infection than were C57BL/6 mice, demonstrating increased mortality and greater numbers of bacteria in the Peyer's patches. Finally, whereas thymus-bearing C57BL/6 X BALB/c F1 mice were resistant to infection, athymic (nude) C57BL/6 X BALB/c F1 mice were susceptible. These studies provide a model to investigate natural immunity to enteric pathogens at mucosal surfaces, as well as provide the basis for clarifying the role of host genotype in Y. enterocolitica resistance.     

Susceptibility of HLA-B27 transgenic mice to Yersinia enterocolitica infection

The majority of patients with reactive arthritis have the major histocompatibility complex class I gene HLA-B27. The development of arthritis in these patients often occurs following infection with one of several enteric bacteria, including Yersinia enterocolitica. In this study, transgenic mice expressing HLA-B27 and their negative full sibs were infected intravenously with Yersinia enterocolitica 0:8 WA in an attempt to develop an experimental model of reactive arthritis. To date, no reactive arthritis has been observed; however, a significantly higher incidence of paralysis was observed in the HLA-B27⁺ transgenic mice. Injection of 10⁵ organisms induced hind limb paralysis in 8 out of 30 of the HLA-B27 transgenic mice (27%) and in only 1 of the 24 negative siblings (4%). Paralysis occurred in 14 out of 30 HLA-B27⁺ mice (47%) at a dose of 10⁴ organisms. Only 2 of the 25 negative siblings (8%) were affected at this dose. Paraspinal abscesses were found in all of the paralyzed animals. At the 10⁴ dose most of the HLA-B27⁺ mice (70%) succumbled to the disease within 4 weeks, while the mortality in their B27⁻ full sibs was less than 10%. Thus, HLA-B27 transgenic mice have higher mortality and morbidity from infection with Y. enterocolitica 0:8 WA than corresponding HLA-B27⁻ littermates.     

Yersinia enterocolitica infection of mice reveals clonal invasion and abscess formation

Yersinia enterocolitica is a common cause of food-borne gastrointestinal disease leading to self-limiting diarrhea and mesenteric lymphadenitis. Occasionally, focal abscess formation in the livers and spleens of certain predisposed patients (those with iron overload states such as hemochromatosis) is observed. In the mouse oral infection model, yersiniae produce a similar disease involving the replication of yersiniae in the small intestine, the invasion of Peyer's patches, and dissemination to the liver and spleen. In these tissues and organs, yersiniae are known to replicate predominantly extracellularly and to form microcolonies. By infecting mice orally with a mixture of equal amounts of green- and red-fluorescing yersiniae (yersiniae expressing green or red fluorescent protein), we were able to show for the first time that yersiniae produce exclusively monoclonal microcolonies in Peyer's patches, the liver, and the spleen, indicating that a single bacterium is sufficient to induce microcolony and microabscess formation in vivo. Furthermore, we present evidence for the clonal invasion of Peyer's patches from the small intestine. The finding that only very few yersiniae are required to establish microcolonies in Peyer's patches is due to both Yersinia-specific and host-specific factors. We demonstrate that yersiniae growing in the small intestinal lumen show strongly reduced levels of invasin, the most important factor for the early invasion of Peyer's patches. Furthermore, we show that the host severely restricts sequential microcolony formation in previously infected Peyer's patches.     

Yersinia enterocolitica infection in diarrheal patients

In this study, we hoped to provide valuable clinical information on yersiniosis for clinicians. Two thousand six hundred stool samples were collected from in- and outpatients with diarrhea, which were tested with both culture method and real-time polymerase chain reaction (RT-PCR). In total, 188 positive samples were detected by RT-PCR (178) and culture method (160), while the incidence was about 7.23%. The detection rate of RT-PCR was significantly higher than culture method and a higher incidence in autumn-winter was also noticeably identified than in spring-summer. Infection sources mostly focused on unboiled foods (101) and pets (45), while clinical manifestation mainly presented as gastroenteritis (156), pseudoappendicitis (32), and extraintestinal complications (46). The morbidity of extraintestinal complications in adults was significantly higher than in children and it was the same for high-risk patients between adults over the age of 60 years (4.7%) and children under the age of 3 years (1.4%), whereas the constituent ratio of children versus adults with yersiniosis in different systems was not significant. Of 160 isolates tested for antimicrobial susceptibility, the majority were susceptible to third-generation cephalosporins, aminoglycosides, fluoroquinolones, and trimethoprim-sulfamethoxazole, whereas only a small portion was susceptible to the first-generation cephalosporins and penicillins. During autumn-winter months, clinicians should pay more attention to clinical manifestation, early diagnosis, and treatment with susceptible antibiotics of yersiniosis and its complications, targeting high-risk patients.     

Long-term fecal excretion and resistance induced in mice infected with Yersinia enterocolitica.

European isolates of Yersinia enterocolitica serotypes O3 and O9 were shown to infect but not kill mice, which became fecal excreters for up to 135 days. The mice challenged with 500 50% lethal doses of the virulent WA strain of Y. enterocolitica serotype O8 survived, and some excreted the virulent strain. This rodent model may be of value in assessing the ecological significance of rodents in the maintenance of Y. enterocolitica.     

Characterization of oral Yersinia enterocolitica infection in three different strains of inbred mice

Several studies have highlighted differences in the resistances of various mouse strains to intravenous (i.v.) infection with Yersinia enterocolitica. In particular, differences in resistance and immunological response between BALB/c and C57BL/6 mouse strains have been determined. Following i.v infection, C57BL/6 mice are more resistant to Y. enterocolitica than are BALB/c mice. However, because Y. enterocolitica is typically a food-borne pathogen, the oral route of infection more accurately reflects the natural route of infection. Therefore, it was of interest to ascertain if the differences in resistance between mouse strains observed for an i.v. infection can be recapitulated following an oral infection. C57BL/6j, BALB/cj, and 129X1/Svj mouse strains presented no differences in 50% lethal dose (LD(50)) following oral infection with Y. enterocolitica. Subsequent analysis of cytokine levels, bacterial colonization and immune cell populations following oral infection confirmed characteristics previously described following i.v. Y. enterocolitica infection. All tissues analyzed from each mouse strain demonstrated a polarized Th1 cytokine profile and inflammatory cell influx throughout a 7-day course of infection. This immune response was present in all tissues and increased as bacterial colonization progressed. The lack of a differing LD(50) phenotype and common trends in immunological response among the three mouse strains tested suggests that oral infection is a useful model for studying the host response to Y. enterocolitica infection.     

Role of plasmid-encoded antigens of Yersinia enterocolitica in humoral immunity against secondary Y. enterocolitica infection in mice

In the present study the role of Yersinia enterocolitica antigens in humoral immunity against secondary Y. enterocolitica infection has been investigated. For this purpose, BALB/c mice, 4 weeks after immunization by primary infection with a sublethal dose of various Y. enterocolitica strains and mutant strains, were challenged with a lethal dose of highly virulent Y. enterocolitica strains of serotype O:8. As evident from the survival rate and protection against bacterial growth in the spleens of mice, only immunization with wild-type or attenuated strains harbouring an intact virulence plasmid mediated protection against a lethal challenge. Protection by immunization with plasmid-harbouring strains correlated with persistence of the bacteria in spleens for at least 7 days after immunization and high serum titres of Yersinia-specific antibodies directed against both chromosomal and plasmid-encoded determinants. Furthermore, the adoptive transfer of the purified IgG fraction of a rabbit serum specific for the adhesin YadA encoded by virulence plasmid pO8 from Y. enterocolitica O:8 mediated protection against a lethal challenge with the serotype O:8 strain harbouring the virulence plasmid pO8 but did not mediate protection when this strain harboured the virulence plasmid pO9 from serotype O:9. In summary, the results demonstrate that in our experimental mouse infection model: (i) the presence of the intact virulence plasmid in an immunizing Y. enterocolitica strain is essential for induction of protection against a lethal challenge with highly virulent Y. enterocolitica and (ii) that antibodies against plasmid-encoded determinants of Y. enterocolitica, especially YadA, are of major importance for achievement of protective immunity in mice.     

Pathogenecity of Yersinia enterocolitica for mice.

A laboratory infection of Yersinia enterocolitica in mice which closely resembles the naturally acquired human infection is described Intravenous inoculation of mice with small numbers of Y. enterocolitica gives rise to a systemic, pyogenic infection involving primarily the spleen, liver, and lungs. Massive neutrophil infiltration of these organs occurs early in the infection, eventually leading to large abscesses and pulmonary consolidation. Mice infected intragastrically show neutophil infiltration in the Peyer's patches of the distal ileum less than 24h postinfection. The Peyer's patches are unable to contain the infection which spreads to the mesenteric lymph node, causing large abscesses in the medullary regions. Soon after, the infection becomes systemic with abscesses forming in the liver, spleen, and lungs, and the total peripheral leukocyte count rises dramatically to over 30,000/mm2. A serological response, in the form of agglutinating antibody, begins to appear 2 weeks after infection. Possible causes of death and the usefulness of this infectious disease model are discussed.     

Persistent Yersinia enterocolitica infection in three rat strains

Yersinia enterocoliticamay persist for prolonged periods of time in humans sometimes resulting in the development of reactive arthritis. To elucidate factors predisposing for persistence we developed animal models. In Lewis and Fischer rats, viable bacteria could be demonstrated for prolonged time and abscesses could be found in the liver, spleen and lungs. Splenic abscesses were observed for more than 20 weeks.Yersinia enterocoliticapersisted in Lewis and Fischer rats, but only Lewis rats developed reactive arthritis. In Brown Norway rats abscesses developed early during infection but in contrast to the other strains disappeared after 3 weeks. Culture of homogenized abscess-containing tissue of all three rat strains yieldedYersiniae. Immunofluorescence studies of the abscesses showed diffuse staining inside the abscesses only, indicating the presence ofYersinia enterocoliticaantigen. Brown Norway rats, in contrast to Lewis and Fischer rats, developed a different serological reaction pattern againstYersinia enterocoliticaantigens and this correlated with the disappearance of the abscesses.     

The pathogenesis of Yersinia enterocolitica infection in gnotobiotic piglets

Yersinia enterocolitica is an important cause of enteritis and mesenteric adenitis in many countries. However the pathogenesis of the disease caused by this organism has not been fully elucidated. Most isolates from clinical material possess two independent properties associated with virulence whose relative contribution to the development of disease is not known. These are the ability to penetrate the intestinal wall, which is thought to be controlled by a plasmid gene, and the production of heat-stable enterotoxin, which is controlled by a chromosomal gene. In this study, we infected neonatal gnotobiotic piglets with strains of Y. enterocolitica expressing these two properties in various combinations. The suitability of the piglet model was shown in experiments in which piglets fed virulent Y. enterocolitica serogroup O3 developed a clinical illness related to the size of the inoculum, which was accompanied by intestinal lesions similar to those reported in naturally and experimentally infected people and animals. The results confirmed the key role of a 47 X 10(6)-mol. wt plasmid in the pathogenicity of Y. enterocolitica, but suggested that penetration of the intestinal wall may be governed by chromosomal rather than plasmid-borne genes. No role for enterotoxin in the pathogenesis of yersiniosis was shown, although there was evidence that enterotoxin may promote intra-intestinal proliferation of Y. enterocolitica, thus favouring increased shedding of bacteria and encouraging their spread between hosts.     

Kinetics of B cell response during Yersinia enterocolitica infection in resistant and susceptible strains of mice

In this study we analyze the B-cell response in murine yersiniosis. To this end, we determined whether polyclonal activation of B-lymphocytes occurs during infection of susceptible (BALB/c) and resistant (C57BL/6) mice with Y. enterocolitica O:8 and compared the immunoglobulin (Ig) isotypes produced in response to the infection by the two strains. The number of splenic cells secreting nonspecific and specific immunoglobulins was determined by ELISPOT. The presence of anti-Yersinia antibodies in serum was detected by ELISA. In both strains, the number of specific Ig-secreting cells was relatively low. Polyclonal B-cell activation was observed in both strains of mice, and the greatest activation was observed in the BALB/c mice, mainly for IgG₁- and IgG₃- secreting cells. The C57BL/6 mice showed a predominance of IgG_2a-secreting cells. The peak production of anti-Yersinia IgG antibodies in the sera of BALB/c mice was seen on the 28th day after infection. The greatest increase in IgM occurred on the 14th day. A progressive increase of specific IgG antibodies was observed in C57BL/6 mice up to the 28th day after infection while IgM increased on the 21st day after infection. The production of specific IgA antibodies was not detected in either BALB/c or C57BL/6 mice. We conclude that polyclonal activation of B lymphocytes occurs in both the Yersinia-resistant and Yersinia-susceptible mice and that the more intense activation of B lymphocytes observed in the susceptible BALB/c mice does not enhance their resistance to Y. enterocolitica infection.     

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF- or anti-IFN- antibodies (serotherapy) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF- treatment. The later treatment with anti-TNF- or anti-IFN- antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF- and IFN- is required for the restriction of a primary Y. enterocolitica infection in mice.     

Development of an experimental animal model for reactive arthritis induced by Yersinia enterocolitica infection.

We attempted to induce experimental arthritis in rats by systematically testing the effect of Yersinia infections in five strains of rats, using the intragastric, intraperitoneal, and intravenous routes of inoculation. We observed that Lewis rats which were given 10(4) to 10(5) Yersinia enterocolitica WA organisms via the intravenous route consistently developed arthritis. The arthritis was most severe at 3 weeks and subsided at 6 weeks. No arthritis was observed when this bacterial strain was administered to Buffalo, Fisher, DA, and LDA rats. No replicable bacteria were detected in the joints. This aseptic characteristic parallels that seen in the human condition and establishes this as an animal model of Yersinia-induced arthritis. The probable reason for arthritis development in only the Lewis rats became apparent when we analyzed the numbers of live bacteria in the spleens and livers of these infected animals. The arthritis-susceptible Lewis rats harbored 10-fold more bacteria than the arthritis-resistant rat strains, and this systemic infection also persisted for a significantly longer period. Speculations as to why human subjects who develop Yersinia-induced arthritis are genetically predisposed have been centered principally around the role of the HLA-B27 histocompatibility antigens. The present study reveals a heretofore unrecognized critical factor: the susceptibility of the hosts to the virulence of the infectious organisms. In addition, the present animal model will provide the necessary tool for the investigation of this and other important host and bacterial factors.     

Histopathological and ultrastructural changes associated with herpesvirus infection in waterfowl

Duck virus enteritis is an acute contagious viral disease affecting birds of the order Anseriformes (ducks, geese and swans). The disease agent is a member of the Herpesviridae family (Anatidae herpes virus 1). A group of Anseriformes waterfowl from a Nature Reserve and Centre for the Recovery of Endangered Species in Spain suffered an outbreak of the disease, affecting adults, young and newborns. Other non-Anseriformes waterfowl such as coots, from the family Rallidae, order Gruiformes, were also affected. Histopathological and ultrastructural findings confirmed the viral infection. The present study provides evidence that birds different from the order Anseriformes can be affected, suggesting that the virus has the ability to infest other non-Anseriformes waterfows.