Experimental infection of Brucella canis in mice

Abstract

Canine brucellosis is a reproductive infectious disease which constitutes a serious problem not only in kennels but also in various canine populations worldwide [1]. B. canis can also be eventually transmitted to humans. The use of a vaccine may be the most convenient and efficacious method for the control of this disease. Part of the limitation in testing a vaccine is the lack of a suitable laboratory animal permissive to the infection by B. canis. Studies on infection, transmission and pathogenia of this bacterium have been carried out in dogs but information about vaccines is scarce. The mouse model has been developed for determining the efficacy of vaccines against B. ovis and smooth brucellae. The aim of this study was to evaluate mouse model for B. canis infection with different I) doses, II) routes and III) times of sacrifice. Female 5-6 weeks old BALB/c mice were challenged with virulent B. canis RM6/66. This strain was grown for 24 h on Brucella agar (BA) slants. Bacteria was harvested in sterile saline, photometrically adjusted to the desired concentration and inoculated into mice. The exact dose expressed as colony forming unit (c.f.u) was retrospectively determined by dilution and spreading on plates of BA. After slaughter, spleens were aseptically removed, homogenized, serially diluted, and plated. Colonies were counted after incubation at 37°C for 72 h and the results were represented as the mean log c.f.u. ± standard deviation (SD) per group. Mice were randomly distributed in different groups and were inoculated using following protocols: I) intraperitoneal (i.p.) inoculation with tenfold dilution starting at 7x102 up to 7x107 c.f.u., respectively and slaughter 30 days after challenge, II) intravenous (i.v.) or i.p. routes with 2.6x105 , 2.6x106 , or 2.6x107 c.f.u. and kill 30 days after inoculation and III) i.p. inoculation with 1.2x106 or 1.2x107 c.f.u. and sacrifice at 7, 14, 21 and 30 days post-inoculation. The results showed that bacterial burden per spleen increased significantly when increasing the dose up to 7x105 . After this dose, counts reached a plateau phase. No colonies were isolated when the minimum dose (7x102 ) was inoculated. Spleen infection of mice inoculated i.v. or i.p. did not show statistical differences for the doses used. Most mice would reach splenic infection after 2 weeks and the bacterial counts were maintained at least up to day 30. Our results are consistent with the kinetics of other Brucella strains infection in mice [2,3]. Mouse is a promising model to evaluate vaccines against B. canis.