Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model.

Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.

Experimental Burkholderia pseudomallei infection of pigs.

Experimental infection with Burkholderia pseudomallei was successfully produced after a single intravenous challenge of 2-month-old pigs with a dose of 5.0 x 10(9) bacterial cells. Clinical, paraclinical and morphological findings of the infectious process and post-infectious immunity were examined up to day 30 post infection (p.i.). A transient and short hyperthermia accompanied by enhanced and longer demonstrated pulse frequency. An increased erythrocyte sedimentation rate and tachypnea were observed too after clinical examination. The infection starts with significant leucopenia, and a reduced number of alveolar and peritoneal macrophages which have been overcome in the latest intervals of infection. In contrast, the phagocytic activity of leucocytes was statistically increased during the course of infection and up to day 15 p.i. in the case of alveolar macrophages. Burkholderia pseudomallei was able to colonize the lungs during the whole experiment and was only present 3 days in the spleen and mesenterial lymph nodes (MLN). Significant antibody response was developed as early as day 7 p.i. Hyperaemia, haemorrhages and necrotic foci were found in the brain, liver spleen and MLN. Lung tissue was also hyperaemic, with formation of small abscesses and signs of catarrhal pneumonia. Data obtained in this study revealed that B. pseudomallei causes a chronic generalized infection in pigs, even after intravenous challenge.

Experimental infections with wild and mutant Yersinia pseudotuberculosis strains in rabbits.

Experimental oral infections of rabbits with a wild-type Yersinia pseudotuberculosis strain (pIB102), and two null-mutants (yopK and ypkA) were carried out with the aim to explore the possibility to use mutant strains of Y. pseudotuberculosis as live carrier vaccine strains. The infectious process of the three strains proceed with passing hyperthermia, leucocytosis with granulocytosis, moderate monocytosis and a transient lymphopenia, better demonstrated at mutant strain infections. Short-term bacterial dissemination into the brain and viscera was observed at yopK infection. An augmented resistance to bactericidal activity of leucocytes at the initial phase of infection was followed by an increased sensitivity discovered earlier in case of yopK strain accompanied by at least 70- and 20-fold, respectively, for ypkA lower virulence for mice. The level of attenuation of yopK was accompanied by significant Yersinia specific IgG and IgM antibody response. Inflammatory foci were found by morphological examination in brain, lung and small intestines after infection with the wild-type strain, while such foci were only observed in brain and mesenterial lymph nodes after infection with the yopK mutant. After infection with the ypkA mutant foci were found in brain and spleen of the infected animals. Morphological changes in the lymphatic tissue of rabbits infected with mutant strains were consistent with induction of immunogenesis. The data suggest that genetically constructed yopK null-mutant exhibits characteristics that makes the strain suitable to be used as a live carrier vaccine to deliver heterologous antigens.

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits.

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.

Protective effect of Oxadin on experimental Yersinia enterocolitica infection in rats.

The effect of Oxadin (a new Bulgarian antimicrobial chemotherapeutic agent) on some parameters of non-specific immune response was investigated in a rat model of infection. After mimicking natural Yersinia enterocolitica systemic infection the number and functional activity of blood leucocytes and peritoneal macrophages were compared between groups of animals treated with Oxadin before and after infection. A significant immunostimulating effect of Oxadin was found in both experimental groups but was better expressed when administered before Yersinia infection. Bactericidal response of peritoneal macrophages (killing ability) and phagocytic activity of polymorphonuclear leucocytes from animals treated with Oxadin and thereafter infected with Yersinia enterocolitica were significantly activated during the first week of study. These findings correlated with the enhanced number of both types of phagocytic cells and the higher glycolytic activity of peritoneal macrophages.

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.

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.

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.

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.

Treatment of adjuvant arthritis in mice with yeast superoxide dismutase.

Yeast Cu/Zn superoxide dismutase (SODy) was used for treatment of adjuvant-induced arthritis in mice. SODy was applied intraperitoneally (i.p.) in doses of 10 mg/kg (30,000 U/kg) and 30 mg/kg (90,000 U/kg) one or three times daily on consecutive days. It was very effective in reducing the paw swelling whether administered before or immediately after induction or when the treatment began at the onset of inflammation or at the peak of the arthritic process. The effect of yeast SOD was compared to that of commercial SOD from bovine erythrocytes (SODb), as well as with indomethacin treatment. Histological data confirmed the antiinflammatory effect of yeast SOD. The schedules and doses tested did not elicit anti-SOD antibodies in serum.

Studies of Yersinia enterocolitica 0:3 experimental infection in pigs.

Y. enterocolitica (serotype 0:3, pYV+, biotype 4) infection of 20-day-old pigs challenged per os with a total dose of 5 x 10(10) CFU was studied. Clinical, paraclinical and morphological findings were examined in dynamics from 1st to 25th days post infection (p.i.). Augmentation of body temperature and erythrocyte sedimentation rate during the first days p.i. were established. The number of leucocytes, peritoneal (pMa) and alveolar (aMa) macrophages was increased significantly from 4th to 15th days p.i. Phagocytic activity of pMa and aMa examined in vitro was maximal on days 15 and 25 p.i. The enhanced phagocytic activity of macrophages was in correlation with the observed histological changes--purulent meningoencephalitis, necrotic tonsillitis, peribronchial lymphoid-leucocytic cell infiltration and catarrhal enteritis. Extensive colonization of internal organs was detected at necropsy till the end of trial. Analysis of the results shows that this orally caused infection runs slowly with dissemination and persistency of Y. enterocolitica 0:3 in the macroorganism, like a generalized infection.

Experimental mixed infection with Yersinia enterocolitica and Listeria monocytogenes in guinea pigs.

The pathogenesis and the cell immune response (CIR) of guinea pigs after mixed infection with Y. enterocolitica and L. monocytogenes was investigated. The guinea pigs were infected per os with 1.1 x 10(9) CFU Y. enterocolitica 0:3, (pYV+) and four days later with 1.1 x 10(9) CFU L. monocytogenes 4B. Clinical, paraclinical and morphological findings attending the infectious process were followed in dynamics up to the 28th day post infection (p.i.) with L. monocytogenes. The phagocyting activity of alveolar macrophages (aMa) was suppressed against Y. enterocolitica, in contrast to peritoneal macrophages (pMa) engulfing yersiniae more actively at the end of the study. Moreover, the tendency of augmented entering in both phagocytes of L. monocytogenes cells was well demonstrated, starting at the earlier intervals of examination. Histopathological studies showed a purulent meningoencephalitis and a catarrhal pneumonie, non-reactive micronecroses in the spleen and lymphadenitis catarrhalis in the mesenteric lymph nodes. Analysis of the T-cell immune response (T-CIR) showed maximal values in the spleen lymphocytes after Y. enterocolitica and L. monocytogenes mixed infection. The B-CIR occurred early (at the 7th day p.i.) and was maximal at the 28th day p.i. in blood lymphocytes. The results obtained demonstrated that the mixed infection of guinea pigs with Y. enterocolitica and L. monocytogenes runs has a non lethal, generalized illness with a dominant role of L. monocytogenes cells.

Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats.

Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

Immunological and electronmicroscopic studies in pigs infected with Yersinia enterocolitica 0:3.

The cellular immune response after an experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, harbouring the virulence plasmid-p YV) was studied in pigs. A maximal stimulation of the T cell population in the thymus, spleen and peripheral blood was stated on the 15th day post infection (p.i.) by the rosette forming cell (RFC) test. The hemolysins (produced by B cells and detected by the plaque forming cell test-PFC) were significantly increased on the 15th day p.i. among the thymus and spleen lymphocytes and on the 25th day p.i. among the blood lymphocytes. Blood and thymus lymphocytes were activated faster by the infectious agent in comparison to the spleen cells. The electronmicroscopic studies revealed an intracellular presence of the bacteria in alveolar macrophages (aMa) and peritoneal macrophages (pMa) as well as in Peyer's patches and tonsils as early as on the 4th day p.i. Extracellularly located bacteria were observed, too. The results have shown that inspite of the activation of T and B cell immune response, this infectious agent persisted in the porcine organism.

Experimental melioidosis in hens.

Experimental intramuscular infection of hens with Pseudomonas pseudomallei, strain 2796 (1 x 10(9) CFU from a 24-h culture) was reproduced. Clinical, paraclinical and pathomorphological findings were followed from 1 to 30 days after challenge. Haemagglutinin titre, bacterial dissemination in the viscera, number of leucocytes, alveolar (aMa) and peritoneal (pMa) macrophages and their phagocytic activity in vitro were studied. During the course of infection a leucocytosis as well as an increased haemagglutinin titre (1:256) were established. The number of bacteria per gram tissue in the spleen and liver was highest at 1 day post-infection (p.i.). Melioidose bacteria from egg yolk were isolated at 15 and 30 days p.i. Leucocyte and pMa phagocytic activity was maximal at 3 days p.i. unlike the activity of aMa which increased gradually until the end of the study. Inflammatory-necrotic changes were found in the viscera and brain at 3 and 15 days p.i. The investigation of experimental melioidosis infection in hens showed that they are susceptible to P. pseudomallei and this disease takes a generalized subacute course.

Experimental melioidosis in inbred mouse strains.

Experimental infection was induced in three inbred mouse strains (BALB/c, BDF1 hybrid and C57BL) by i. p. inoculation with Pseudomonas pseudomallei. The bacterial load in the viscera and the host response induced in different compartments (blood, peritoneal cavity and organs) were determined. Blood cell parameters and peritoneal exudative cell populations were evaluated during the infection with the aid of an automated haematology analyser Technicon H-1. It was found that all mouse strains produced a similar intraperitoneal inflammatory response with predominance of granulocytes at the early stage of infection and subsequent increase of macrophages especially in BDF1 hybrid and BALB/c mice. The highest bacterial count found in the liver and spleen of C57BL was associated with corresponding tissue damage (purulent pneumonia, abscesses in liver, karyorrhexis of hepatocytes and meningoencephalitis). The degree of bacterial load and histological changes found in BALB/c and BDF1 hybrid mice were lower than in C57BL mice. The results show that the variations in the infection magnitude among inbred mouse strains are host-dependent.

Responsiveness of susceptible inbred mice to Yersinia pseudotuberculosis serovar III infection.

The susceptibility of BALB/c, C57BL and BDF1-hybrid mouse strains to Yersinia pseudotuberculosis serovar III infection was studied. The bacterial load in the viscera and brain and the host responses at different levels, i.e. blood, peritoneal cavity and organs were determined. Blood cell parameters and peritoneal exudate cell population were evaluated during the infection using the automated hematologic analyzer Technicon H-1. It was found that BDF1-hybrid mice produced an early peritoneal inflammatory response, while in BALB/c and C57BL mice it was not observed. The high susceptibility of C57BL was associated with a great number of microorganisms in the organs and with the corresponding histological changes. It was shown that the magnitude of the inflammation induced by Y. pseudotuberculosis varied among the host strains used. The variations of the susceptibility to Y. pseudotuberculosis among inbred mouse strains suggest the possible role of genetic factors regulating the host defence.

Role of iron in pathogenesis of guinea pigs infected with Yersinia enterocolitica 0:3.

Examined have been the pathogenesis of guinea pigs preliminarily overload with iron, and infected with Yersinia enterocolitica 0:3 in a logarithmic and stationary phase of development, cultivated at 25 degrees C and 37 degrees C. Dextrofer-100 (Fedex-100) medicine have been used as an iron source. The results show that in spite of the phase of development and cultivation temperature, the iron excess does not increase the bacterial virulence of the strain used so far. The morphological changes in the mesenterial lymphatic nodes, small intestines, spleen and liver of guinea pigs treated with iron, and then, infected with Y. enterocolitica, are more slightly expressed as compared with animals infected with Y. enterocolitica only. The investigations suggested so far, have attested that Yersinia enterocolitica 0:3 does not contain a gene responsible for the synthesis of the protein participating in iron uptake.