Nonspecific Stimulation or Resistance of Mice to Infection by Lipopolysaccharide of Bordetella bronchiseptica Incorporated into Liposomes

Abstract

Toman M., J. TurAnek, P. HofavovA: Nonspecific Stimulation of Resistance of Mice to Infection by Lipopolysacharide of Bordetella bronchiseptica Incorporated into Liposomes. Acta vet. Bmo, 63,1994: 71-79. A phenolic extract containing lipopolysaccharide and residual nucleic acids (BBE-LPS) was prepared from selected strains of Bordetella bronchiseptica. Toxicity and immunostimulatory activity of the free extract and the extract incorporated into large unilamellar (LUV) and multilamellar (ML V) liposomes were tested in laboratory animals. The free extract significantly enhanced the resistance of mice to infections with P. multocida and pseudorabies virus at doses down to I \lg per animal. Incorporation into liposomes slightly reduced the toxicity of the extract. The incorporated extract enhanced the resistance to infection in mice, but its activity did not exceed that of the free extract. Immunostimulation, toxicity, lipopolysaccharide, liposome, mouse, resistance to infection Bordetella pertussis is one of the best known bacterial species showing a non-specific immunostimulatory activity described for the first time in the late forties (Green berg and Fleming 1947). This activity was later confirmed in several model systems in vitro and experiments aimed at the enhancement of resistance to infection or antibody responses to weak antigens (Athanasiades 1977; Kirchner et al. 1978; Bomford 1980; Winters etal. 1985; Bagget etal. 1986). In veterinary medicine, a similar immunostimulatory activity was demonstrated in the related species B. bronchiseptica. Adjuvant effects were described in mice immunized with tetanus toxoid (M a g y a r et al. 1983) and in cattle vaccinated against bovine virus diarrhoea (Chen et al. 1985). Recently, non-specific immunostimulatory activity of B. bronchiseptica has been described in mice (Wieler et al. 1990; Toman et al. 1990). Two strains of B. bronchiseptica with pronounced immunostimulatory effects were selected in our previous experiments (Toman et al. 1990). The strains markedly enhanced the resistance of mice to infections with virulent strains of Pasteurella multocida. The immunostimulatory effects were confirmed also in model experiments using sublethally irradiated mice. The treatment with B. bronchiseptica bacterin 24 h prior to irradiation resulted in an earlier and accelerated regeneration oflymphoid tissues, as demonstrated by higher spleen weights, a rapid increase of peripheral lymphocyte counts and a high chemiluminiscent activity on post-irradiation day 6. Effects of bacterin B. bronchiseptica at various doses and in combinations with other substances were also examined in 2-month-old clinically normal calves. The treatment induced an increase of peripheral leukocyte counts after 24 h and enhanced phagocytosis of synthetic particles and chemiluminiscent activity on post-treatment days 2-4. The aim of the experiments presented here was to determine to what degree lipopolysaccharides (LPS) were responsible for the immunostimulatory activity of the B. bronchiseptica strains under study. Regarding the known side effects ofLPS of Gram-negative bacteria, we compared the toxicity and die immunostimmulatory activity of a free extract and an extract incorporated into liposomes. Materials and Methods Extract of Bordetella bronchiseptica The strains of B. bronchiseptica used iii the experiments were obtained from the Czechoslovak Collection of Animal Pathogenic Microorganisms (codes CCM 6229 and CCM 6230). They showed growth in Phase I, agglutinated bovine erythrocytes and produced adenylate cyclase and cytotoxin. The strain 6229 was weak producer and the strain 6230 a strong producer of necrotoxin. The strains were propagated in the liquid medium BHI (Difco) and, for extraction, used as a mixture of two strains. A B. bronchiseptica extract (BBE-LPS) was prepared by phenolic extraction at 68°C (Westphal et al. 1952). The water phase was dialysed against flowing water, condensed by ultrafiltration (Amicon) through the membrane PM 10 and precipitated with ethanol in the presence of sodium acetate. The precipitate was washed witb methanol, acetone and ether.