Characterization of humoral and cell-mediated immunity in rabbits orally infected with Encephalitozoon cuniculi.

Encephalitozoonosis is a common infectious disease widely spread among rabbits. Encephalitozoon cuniculi, is considered as a zoonotic and emerging pathogen capable of infecting both immunocompetent and immunocompromised hosts. The aim of the study was to describe in detail the spread of the E. cuniculi in a rabbit organism after experimental infection and the host humoral and cellular immune response including cytokine production. For that purpose, healthy immunocompetent rabbits were infected orally in order to simulate the natural route of infection and euthanised at 2, 4, 6 and 8-weeks post-infection. Dissemination of E. cuniculi in the body of the rabbit was more rapid than previously reported. As early as 2 weeks post-infection, E. cuniculi was detected using immunohistochemistry not only in the intestine, mesenteric lymph nodes, spleen, liver, kidneys, lungs and heart, but also in nervous tissues, especially in medulla oblongata, cerebellum, and leptomeninges. Based on flow cytometry, no conspicuous changes in lymphocyte subpopulations were detected in the examined lymphoid organs of infected rabbits. Cell-mediated immunity was characterized by ability of both CD4+ and CD8+ T cells to proliferate after stimulation with specific antigens. Th1 polarization of immune response with a predominance of IFN-γ expression was detected in spleen, mesenteric lymph nodes and Peyer's patches. The increased expression of IL-4 and IL-10 mRNA in mixed samples from the small intestine is indicative of balanced control of IFN-γ, which prevents tissue damage. On the other hand, it can enable E. cuniculi to survive and persist in the host organism in a balanced host-parasite relationship. The Th17 immunity lineage seems to play only a minor role in E. cuniculi infection in rabbits.

Immunohistochemical Detection of Encephalitozoon cuniculi in Ocular Structures of Immunocompetent Rabbits.

Encephalitozoonosis is a common infectious disease widely spread among rabbits. Its causative agent, Encephalitozoon cuniculi, is considered as a zoonotic and emerging pathogen capable of infecting both immunocompetent and immunocompromised hosts, including humans. In rabbits, clinical signs include neurological, kidney and ocular disease. The aim of this study was to detect E. cuniculi in ocular structures in immunocompetent rabbits after experimental oral infection using immunohistochemistry. In infected animals, E. cuniculi spores were present in periocular connective tissue, sclera, cornea, choroidea, iris, retina and lens, as a round to ovoid organism reacting with a specific anti-E. cuniculi monoclonal antibody as early as 2 weeks after infection. There were no signs of inflammatory lesions in any of the ocular tissues examined at 2, 4, 6 and 8 weeks after infection. In the present study, E. cuniculi was also detected in the lenses of adult rabbits, which indicates that ways of lens infection other than intrauterine and haematogenic are possible.

Aspects of intradermal immunization with different adjuvants: The role of dendritic cells and Th1/Th2 response.

Intradermal (i.d.) application of vaccine is promising way how to induce specific immune response against particular pathogens. Adjuvants, substances added into vaccination dose with the aim to increase immunogenicity, play important role in activation of dendritic cells with subsequent activation of lymphocytes. They can, however, induce unwanted local reactions. The aim of the study was to determine the effect of i.d. administration of model antigen keyhole limped hemocyanine alone or with different adjuvants-aluminium hydroxide and oil-based adjuvants-on local histopathological reaction as well as dendritic cell activation at the site of administration and local cytokine and chemokine response. This was assessed at 4 and 24 hours after application. Selection of the adjuvants was based on the fact, that they differently enhance antibody or cell-mediated immunity. The results showed activation of dendritic cells and both Th1 and Th2 response stimulated by oil-based adjuvants. It was associated with higher expression of set of genes, incl. chemokine receptor CCR7 or Th1-associated chemokine CXCL10 and cytokine IFNγ. Application of the antigen with aluminium hydroxide induced higher expression of Th2-associated IL4 or IL13. On the other hand, both complete and incomplete Freund´s adjuvants provoked strong local reaction associated with influx of neutrophils. This was accompanied with high expression of proinflammatory IL1 or neutrophil chemoattractant CXCL8. Surprisingly, similarly strong local reaction was detected also after application of aluminium hydroxide-based adjuvant. The best balanced local reaction with sufficient activation of immune cells was detected after application of oil-based adjuvants Montanide and Emulsigen.

Ovarian disorders in immature rats after postnatal exposure to environmental polycyclic aromatic hydrocarbons.

The study investigated the effects of postnatal exposure to polycyclic aromatic hydrocarbons (PAHs) on the development of the rat ovary. Neonates were injected on each postnatal day 1-14 with benzo(a)pyrene (BaP), benz(a)anthracene (BaA) and benzo(k)fluoranthene (BkF) (0.1, 1.0, 5.0 or 10.0 mg kg(-1)), ethynylestradiol (EE; 1.0 µg kg(-1)) or a vehicle (control group). The rats were killed on day 23. Postnatal exposure to BaP increased the total number of antral follicles in ovaries (P < 0.05) and the number of nonatretic follicles (P < 0.01) as a result of a lower degree of apoptosis of granulosa cells, and the thickness of theca cell layers (P < 0.01). Similar histological findings were observed after BaA administration. Conversely, BkF exposure caused a decrease in the number of antral follicles, but did not alter the other investigated parameters. Degeneration of primordial oocytes after exposure to PAHs was observed only after exposure to BaP. Treatment with BaP at doses of 1.0 and 10.0 mg kg(-1) impaired 28.1 and 60.3% of the primordial follicles, respectively. Substantial alterations in ovarian ERß expression were detected in the rats; their intensity differed with the type of PAH. Response of the ovaries to EE (three injections of 1.0 µg kg(-1) on postnatal days 20-22) in rats exposed to PAHs was suppressed in contrast to the controls. The study showed that postnatal exposure to BaP, BaA and BkF altered ovarian ERß expression, disturbed morphological development of the ovaries and caused ovarian dysfunction in immature rats.

Experimental infection of gnotobiotic piglets with Escherichia coli strains positive for EAST1 and AIDA.

The virulence factors EAST1 and AIDA are often detected in ETEC/VTEC strains isolated from pigs and their role in diarrhoeal infections is discussed. In order to elucidate the pathogenesis of AIDA, the colonisation patterns of F4 positive and AIDA positive strains were investigated. Two wild-type Escherichia coli strains AIDA/EAST1 and F4/EAST1 isolated from diarrhoeal piglets were used for animal experiment to evaluate the ability of the EAST1 toxin to be involved in induction of diarrhoea. Gnotobiotic piglets were supplemented with normal porcine serum and orally inoculated with the strains. Faecal bacterial shedding of the challenge strains was observed during the experiment. Light microscopy and scanning electron microscopy were used to detect the colonisation pattern of both challenge strains. Although bacterial isolation demonstrated shedding of the challenge strains until the end of the experiment, diarrhoea did not develop in any piglet. Based on histological examination, piglets were more heavily colonised in the case of infection with E. coli O149/F4/EAST1 strain. Scanning electron microscopy showed bacterial cells of F4/EAST1 E. coli adhering to enterocytes, in contrast to AIDA/EAST1 which were poorly present on the intestinal surface. The EAST1 toxin alone was not able to induce diarrhoea in animals. Therefore our results demonstrate that the function/role of EAST1 and AIDA in colibacillosis of pigs remains to be elucidated.

Host response in rabbits to infection with Pasteurella multocida serogroup F strains originating from fowl cholera.

Although Pasteurella multocida serogroup F has been described as an avian-adapted serogroup, it was recently found in rabbit nests in the Czech Republic. Therefore, the ability of 2 avian P. multocida serogroup F strains to induce disease in rabbits was investigated. Two groups of 18 Pasteurella-free rabbits were intranasally challenged with strains isolated from chickens and turkeys. Half of the animals in each challenge group were immunosuppressed using dexamethasone. All of the challenged rabbits exhibited clinical signs of peracute septicemic disease, ending with shock, and died or were euthanized in the terminal stages of the disease 1 to 2 d post-infection. Gross pathological changes included systemic vascular collapse and vascular leak syndrome. Hyperemia, hemorrhage, edema, inflammatory cell infiltrates, focal necrosis, and degenerative changes were observed histologically in parenchymatous organs. This is the first study directly demonstrating that avian P. multocida serogroup F strains are highly virulent in rabbits and that avian hosts cannot be excluded as a possible source of rabbit infection with serogroup F.

Influence of 5 major Salmonella pathogenicity islands on NK cell depletion in mice infected with Salmonella enterica serovar Enteritidis.

BACKGROUND: In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants. RESULTS: The virulence of S. Enteritidis for Balb/C mice was exclusively dependent on intact SPI-2. Infections with any of the mutants harbouring SPI-2 (including the mutant in which we left only SPI-2 but removed SPI-1, SPI-3, SPI-4 and SPI-5) resulted in fatalities, liver injures and NK cell depletion from the spleen. The infection was of minimal influence on counts of splenic CD4 CD8 T lymphocytes and gammadelta T-lymphocytes although a reduced ability of splenic lymphocytes to respond to non-specific mitogens indicated general immunosuppression in mice infected with SPI-2 positive S. Enteritidis mutants. Further investigations showed that NK cells were depleted also in blood but not in the caecal lamina propria. However, NK cell depletion was not directly associated with the presence of SPI-2 and was rather an indicator of virulence or avirulence of a particular mutant because the depletion was not observed in mice infected with other attenuated mutants such as lon and rfaL. CONCLUSIONS: The virulence of S. Enteritidis for Balb/C mice is exclusively dependent on the presence of SPI-2 in its genome, and a major hallmark of the infection in terms of early changes in lymphocyte populations is the depletion of NK cells in spleen and blood. The decrease of NK cells in circulation can be used as a marker of attenuation of S. Enteritidis mutants for Balb/C mice.

Virulence potential of five major pathogenicity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens.

BACKGROUND: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host. RESULTS: The mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of S. Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs. CONCLUSIONS: SPI-1 and SPI-2 are the two most important pathogenicity islands of Salmonella Enteritidis required for the colonisation of systemic sites in chickens.

Morphological and functional disorders of the immature rat uterus after postnatal exposure to benz[a]anthracene and benzo[k]fluoranthene.

We investigated the effects of postnatal exposure to benz[a]anthracene (B[a]A) and benzo[k]fluoranthene (B[k]F) on the development of the rat uterus. Neonates were injected on each postnatal days 1-14 with B[a]A (0.1, 1.0 or 10.0mg/kg), B[k]F (0.1, 1.0 or 5.0mg/kg), ethynylestradiol (EE; 1.0µg/kg) or a vehicle. The rats were killed on day 23. All doses of B[a]A and B[k]F induced a reduction of the uterine weight, a reduction of the estrogen receptor α expression in the luminal epithelium, glandular epithelium and stromal cells. Moreover, the uterotrophic response to EE (3-day administration of 1.0µg/kg on postnatal days 20-22) in rats exposed to B[a]A, B[k]F and EE was lower than in controls. The study showed that postnatal exposure to B[a]A and B[k]F resultes in morphological and functional disorders of the immature rat uterus.

Estrogenic activity of environmental polycyclic aromatic hydrocarbons in uterus of immature Wistar rats.

Polycyclic aromatic hydrocarbons (PAHs) are an important group of environmental pollutants, known for their mutagenic and carcinogenic activities. Many PAHs are aryl hydrocarbon receptor (AhR) ligands and several recent studies have suggested that PAHs or their metabolites may activate estrogen receptors (ER). The present study investigated possible estrogenic/antiestrogenic effects of abundant environmental contaminants benzo[a]pyrene (BaP), benz[a]anthracene (BaA), fluoranthene (Fla) and benzo[k]fluoranthene (BkF) in vivo, using the immature rat uterotrophic assay. The present results suggest that BaA, BaP and Fla behaved as estrogen-like compounds in immature Wistar rats, when applied for 3 consecutive days at 10mg/kg/day, as documented by a significant increase of uterine weight and hypertrophy of luminal epithelium. These effects were likely to be mediated by ERalpha, a major subtype of ER present in uterus, as they were inhibited by treatment with ER antagonist ICI 182,780. BaA, the most potent of studied PAHs, induced a significant estrogenic effect within a concentration range 0.1-50mg/kg/day; however, it did not reach the maximum level induced by reference estrogens. The proposed antiestrogenicity of the potent AhR agonist BkF was not confirmed in the present in vivo study; the exposure to BkF did not significantly affect the uterine weight, although a weak suppression of ERalpha immunostaining was observed in luminal and glandular epithelium, possibly related to its AhR-mediated activity. The PAHs under study did not induce marked genotoxic damage in uterine tissues, as documented by the lack of Ser-15-phoshorylated p53 protein staining. With the exception of Fla, all three remaining compounds increased CYP1-dependent monooxygenation activities in liver at the doses used, suggesting that the potential tissue-specific antiestrogenic effects of PAHs mediated by metabolization of 17beta-estradiol also cannot be excluded. Taken together, these environmentally relevant PAHs induced estrogenic effects in vivo, which might affect their toxic impact and carcinogenicity.

Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits.

The role of Pasteurella multocida serogroup F in inducing disease in rabbits was investigated in this study. Three groups of 12 Pasteurella-free rabbits each were intranasally (i.n.), subcutaneously (s.c.), and perorally (p.o.) challenged, respectively. Six rabbits of each group were immunosuppressed using dexamethasone. Eight rabbits (four of them immunosuppressed) inoculated i.n. showed symptoms of respiratory distress resulting in respiratory failure and died or were euthanized in the terminal stage of the disease 3-6 days post-infection (p.i.). The main pathological findings were fibrinopurulent pleuropneumonia (immunocompetent rabbits) or diffuse haemorrhagic pneumonia (immunosuppressed rabbits). Septicemic syndrome ending with shock occurred in 11 rabbits (6 of them immunosuppressed) inoculated s.c., which died or were euthanized in the terminal stage of the disease 2-3 days p.i. The most significant pathological findings were extensive cutaneous and subcutaneous lesions. All of the p.o. inoculated rabbits survived the challenge showing no clinical signs of the disease and no macroscopic lesions. The observations in this study indicate that in addition to serogroups A and D of P. multocida, serogroup F also can be highly pathogenic for rabbits and therefore might be a cause of considerable economic loss in commercial rabbit production.

The effect of intramuscular administration of colistin on the development and course of experimentally induced oedema disease in weaned piglets.

Shiga-toxigenic E. coli (STEC) strains that produce Shiga toxin Stx2e cause oedema disease in weaned piglets. The purpose of the present study was to investigate the impact of Stx2e released in mesenteric lymph nodes on disease pathogenesis. Colistin and ampicillin were intramuscularly administered to piglets of the experimental group simultaneously challenged with STEC strain, type O139:F18ab, Stx2e+. Piglets of the control group were challenged with STEC only. The strain was naturally resistant to ampicillin and susceptible to colistin. After the challenge, colonisation of the intestines was observed in both antibiotic-treated piglets and control piglets without antibiotic treatment. Histochemistry and scanning electron microscopy revealed sporadic colonisation of the small intestine in the piglets. STEC was detected in the mesenteric lymph nodes of untreated piglets. The clinical manifestations of oedema disease were observed in both groups. In the antibiotic-treated group (11 piglets), oedema disease developed in 10 piglets, eight of which died or were euthanized ante finem. In the untreated group (11 piglets), oedema disease developed in five piglets, four of which died or were euthanized ante finem. We therefore propose that the STEC lysed by colistin suddenly released the toxin from bacterial cells immediately after their passage through the intestinal wall. That could explain a more severe course of oedema disease in the treated piglets. Even though high amounts of STEC were present in the lymph nodes of untreated piglets, the toxin was not released abruptly because the bacterial cells were not damaged.

Effect of postnatal exposure to benzo[a]pyrene on the uterus of immature rats.

The objective of this study was to investigate the morphological effects of postnatal exposure to benzo[a]pyrene (B[a]P) on the development of the uterus, uterine estrogen receptor (ERalpha) expression, and the uterine response to estrogen stimulation using the uterotrophic bioassay in rats. Neonates were injected on each postnatal day (PND) 1-14 with B[a]P (0.1, 1.0 and 10.0mg/kg), ethynylestradiol (EE; 1.0 microg/kg) or vehicle (control group). All animals were killed on PND 23. Postnatal administration of B[a]P with doses of 1.0 and 10.0 mg/kg induced significant (P<0.01) reduction of uterine weight and significantly lowered (P<0.05) ERalpha expression in the luminal epithelium. The increase in uterine weight and luminal epithelium heights after EE stimulation (1.0 microg/kg) on PND 20-22 was significantly higher (P<0.01) in all groups in comparison with corresponding non-stimulated groups. However, the uterotrophic response in rats postnatally exposed to EE and B[a]P was significantly lower (P<0.01) than in controls. In the control and EE groups, EE stimulation on PND 20-22 induced a significant (P<0.01) decrease in ERalpha immunoreactivity of the luminal epithelium. In contrast, rats postnatally treated with B[a]P showed no change in the density of ERalpha immunostaining when detected after estrogenic stimulation. The present study showed that postnatal exposure to B[a]P caused pathological changes in constitution and maturation of uterine ERalpha resulting in disturbed morphological development and uterine dysfunction in immature rats.