Abstract
Brucellosis is one of the major zoonoses in South Korea and has been recognized as a cause of reproductive failure in dairy cattle, thereby causing significant economic losses through calf loss and in costs for regulatory and eradication programs. The present study was designed to delineate the clinicopathological profile, vertical transmission, serological characteristics of a bovine pathogenic strain of Brucella abortus biotype 1 isolated from bovine supra mammary lymph node in South Korea and to assess the protective immunity of B. abortus strain RB51 (SRB51) in Sprague-Dawley (SD) rats.
SD rats were inoculated subcutaneously with B. abortus biotype 1 at the dose rate of 1.0×109 colony forming units (cfu)/500μl during early gestation (7 days), mid gestation (14 days) and late gestation (18 days) groups of primiparous and multiparous rats, respectively. The infectivity was determined by clinical, bacteriological and pathological examination and by AMOS PCR (Brucella abortus, Brucella melitensis, Brucella ovis, Brucella suis Polymerase Chain
Reaction). The maximum rectal temperature was recorded as 38°C until 3 days in infected group whereas in the control group temperature remained normal (36°C). However, there were no stillbirths, abortions or premature birth and relapsing fever found in the infected SD rats. The pathological findings of infected SD rats were splenomegaly, metritis, swelling of lymph node and placentitis associated with lymphocytic and macrophage infiltration. This study confirmed the suitability of the SD rat as an experimental model for brucellosis.
To verify the vertical transmission of B. abortus biotype 1 in SD rat, PCR, serodiagnostic and bacteriological methods were applied. Brucella free SD rats were injected subcutaneously containing 1.0×109 cfu
with 500
ul
suspension of B. abortus biotype 1 in physiological saline solution and allowed to mate with noninfected opposite sex partner. B. abortus biotype 1 was detected bacteriologically and by AMOS PCR assay from testicular tissues of infected male rats and from spleens of infected female rats. Both in the Rose Bengal test (RBT) and the plate agglutination test (PAT), the reciprocal antibody titer was 1:800 at 7 days post-infection. After parturition of pregnant SD rats the titer increased to 1:1600. The reciprocal antibody titer of one month old litter mates were measured 1:400 by RBT and 1:200 by PAT, and 1:800 in mother rat both measured by RBT and PAT. The infected female and male SD rats were capable of transmitting of Brucella organism to their opposite sex partner and to their offspring. Fetuses born from infected dams were found infected and the AMOS PCR was shown to be a valuable tool for verification of the vertical transmission of Brucella species in animals.
In order to investigate the serological characteristics, B. abortus biotype 1 was inoculated subcutaneously to female SD rats at the dose rate of 1.0×109 cfu suspension in physiological saline. The SD rats were monitored at regular intervals by serological and bacteriological methods. There was no reciprocal antibody titer in sera of 24 weeks post-infection both through PAT and TAT (tube agglutination test) despite the presence of bacteremia but the reciprocal antibody titers were 1:25 through RBT and 1:100 through mercaptoethanol test (MET), respectively in infected rats. Whereas the infected rats revealed positive for brucellosis until 24 weeks post-infection through enzyme linked immunosorbent assay (ELISA). PAT, TAT, RBT and MET using B. abortus strain 1119-3 (S1119-3) whole cell antigen and ELISA using S1119-3 whole cell and cytoplasmic protein antigens were evaluated for the first time using brucellosis infected sera in rat. ELISA using S1119-3 cytoplasmic protein antigen was a potential candidate as an improved diagnostic method for early diagnosis of brucellosis in rats.
To investigate the protective immunity of SRB51 in SD rat, female SD rats were orally administered with 1.0 x 107 cfu suspension of SRB51 and half of these SD rats were challenged on 4 weeks post with 1.0 x 109 cfu suspension of B. abortus biotype 1 isolated in South Korea. Sera of SD rats were monitored at regular intervals by TAT, PAT and RBT using S1119-3 whole cell antigen and dot-blot assay using whole cell antigens of SRB51 and S1119-3 and bacteriological examination of blood. The reciprocal antibody titers were 1:100 in inoculated challenged rat through TAT, PAT, and 1:200 through RBT on 8 weeks post whereas the reciprocal antibody titer was 1:1600 through RBT, PAT, TAT, respectively in non inoculated challenged rat on that time and there were no antibody titer in inoculated and non inoculated control rats. There were no clinical sign in inoculated, inoculated challenged and control rats and it was possible to isolate SRB51 from blood of inoculated rat until 1 week post inoculation. The highest immune response was observed in sera of 4 weeks post inoculated SD rats by dot-blot assay using SRB51 whole cell antigen and higher antigen-antibody reaction was observed in inoculated challenged rat than inoculated rat through western blot. This research will be useful for the eradication of animal