A rat model for dose-response relationships of Salmonella Enteritidis infection.

AIMS: To develop an animal model to study dose-response relationships of enteropathogenic bacteria. METHODS AND RESULTS: Adult, male Wistar Unilever rats were exposed orally to different doses of Salmonella enterica serovar Enteritidis after overnight starvation and neutralization of gastric acid by sodium bicarbonate. The spleen was the most sensitive and reproducible organ for detection of dose-dependent systemic infection. Illness was only observed in animals exposed to doses of 10(8) cfu or more. At lower doses, histopathological changes in the gastro-intestinal tract were observed, but these were not accompanied by illness. Marked changes in numbers and types of white blood cells, as well as delayed-type hyperresponsiveness, indicated a strong, dose-dependent cellular immune response to Salm. Enteritidis. CONCLUSION: The rat model is a sensitive and reproducible tool for studying the effects of oral exposure to Salm. Enteritidis over a wide dose range. SIGNIFICANCE AND IMPACT OF THE STUDY: The rat model allows controlled quantification of different factors related to the host, pathogen and food matrix on initial stages of infection by food-borne bacterial pathogens.

Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice.

We evaluated the side effects induced by injection of Freund's adjuvant (FA) and alternative adjuvants combined with different antigens. Rabbits and mice were injected subcutaneously, intramuscularly (rabbits) and intraperitoneally (mice) with different adjuvants (FA, Specol, RIBI, TiterMax, Montanide ISA50) in combination with several types of antigens (synthetic peptides, autoantigen, glycolipid, protein, mycoplasma or viruses). The effects of treatment on the animals' well-being were assessed by clinical and behavioural changes (POT and LABORAS assays) and gross and histopathological changes. In rabbits, treatment did not appear to induce acute or prolonged pain and distress. Mice showed behavioural changes immediately after (predominantly secondary) immunization. Injection of several adjuvant/antigen mixtures resulted in severe pathological changes, depending on adjuvant, type of antigen, animal species used and route of injection. Both rabbits and mice showed pathological changes ranging from marked to severe after injection of FA, and ranging from minimal to marked after Specol and Montanide injections. Pathological changes after RIBI injections were severe in rabbits, though slight in mice. After TiterMax injections, pathological changes were moderate in rabbits, though severe in mice. In conclusion, injection of FA according to present guidelines resulted mostly in severe pathological changes, whereas only very few clinical and behavioural signs indicated prolonged severe pain. Our findings indicate that Montanide ISA50 and Specol induce acceptable antibody titres, and cause fewer pathological changes than FA. Thus they are effective alternatives to FA.

Immune responses and side effects of five different oil-based adjuvants in mice.

In this study, five different oil based adjuvants were compared to assess efficacy and side effects. Mice were injected subcutaneously (s.c.) or intraperitoneally (i.p.) with a weak immunogen (synthetic peptide) emulsified in Freund's adjuvant (FA), Specol, RIBI, TiterMax or Montanide ISA50. Efficacy of adjuvants was evaluated based on their properties to induce peptide specific IgG1, IgG2a and total IgG antibodies, native protein cross-reactive antibodies and cytokine production. Side effects were evaluated based on clinical and behavioural abnormalities, and (histo)pathological changes. Although marked differences in isotype profile and height of titre are observed among the different adjuvants used, we found that FA, Montanide ISA50 and Specol worked equally well in the s.c. and i.p. route, TiterMax functioned only when given i.p. and RIBI also did not perform up to par. The number of cytokine (interferon-gamma and interleukin-4) producing spleen cells was significantly higher after injection of RIBI compared with other adjuvants. Injection of FA or TiterMax resulted in severe pathological changes while after RIBI injection minimal changes were observed. In conclusion, high peptide specific antibody levels with limited side effects can be obtained by s.c. injection of peptide combined with Montanide ISA50 or Specol as alternatives to FA.

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa.

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".

Comparison of adjuvants for immune potentiating properties and side effects in mice.

Four types of adjuvants were evaluated as alternatives to the use of Freund's complete adjuvant in mice. The adjuvants evaluated included a water-in-oil emulsion (Specol), a microorganism (Lactobacillus), performed immune-stimulating complexes (ISCOM) containing rabies virus glycoprotein and a saponin, Quil A. The adjuvants and saline were combined with three weak immunogens (a synthetic peptide, a self antigen and a particulate antigen) and given by three different routes (intraperitoneal, subcutaneous and dorsal in the foot). The evaluation was based on clinical observations, behavioural studies, pathological lesions and capacity to support immunological responses to weak immunogens. Lesions were most severe after injection of antigen combined with Freund's adjuvant or Quil A, mild to moderate with Specol and minimal with Lactobacillus, iscom conjugates or saline. Despite pathological changes, no signs of prolonged pain or distress could be demonstrated based on clinical observations and behavioural studies. Minimal immunological responses were found after injection of antigen in combination with saline or Lactobacillus. T-cell activation and high antibody responses were found after injection of antigen-iscom conjugates or antigen in Freund's adjuvant emulsions. After Specol/antigen immunisations T-cell activation was demonstrated and high antibody titres were found except for Specol/self antigen immunisations. Presented data suggest that Specol is a possible alternative to Freund's complete adjuvant for the induction of an immune response against weak immunogens except possibly self antigens, for which performed iscoms seem very suitable.

Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits.

In three experiments we evaluated several types of adjuvants as an alternative to Freund's adjuvant (FA). In the first experiment three adjuvant preparations (a water-in-oil emulsion (Specol), a combination preparation of monophosphoryl lipid A + trehalose dimycolate + cell wall skeleton and a non-ionic block polymer surfactant (TiterMax)) were evaluated. The adjuvants were combined with three different types of weak immunogenic antigens (synthetic peptide, glycolipid and particulate antigen) and administered following the intramuscular and subcutaneous route. The evaluation was based on clinical, pathological and immunological parameters. The animals did not appear to be severely or chronically impaired by the experiment. After injection of the RIBI adjuvant, side effects of the same severity as with FA were induced, while low antibody titers were produced. TiterMax caused few side effects, while antibody responses were very low. In comparing Specol and FA, Specol had far fewer adverse effects than FA. However, Specol had immunostimulating properties of the same level as FA. In the second experiment, the effect of injected volume of FA on side effects and antibody titer was studied. Immunization of rabbits with a total of 0.5 ml FA at different sites does not seem to increase the immune response when compared with the immune response seen after injection of 0.5 ml FA at one site. However side effects were seen in all the animals. In the third experiment, the side effects following intradermal (i.d.) injection of the adjuvants were studied. After i.d. injection of FA or RIBI, undesirable effects were found. No side effects occurred after i.d. injection of Specol or TiterMax. From the studies it is concluded that Specol is an alternative to FA for hyperactivation of the immune response in rabbits.

[Eosinophilic granuloma of the oral cavity in a Siberian husky]. / Eosinofiel granuloom in de mondholte bij een Siberische Husky.

An ulcerative sublingual inflammation was found to be present in a Siberian Husky dog. Histological examination revealed an eosinophilic granuloma. Treatment with corticosteroids was successful.