Improvements in the fermenter growth of Brucella abortus strain 19

Summary The fermenter-growth conditions for a high yield of non-dissociated strain 19 Brucella abortus are described allowing a yield of close to 200×10⁹ viable bacteria ml^-1 of culture medium in less than 48 h. Details are also given of an improved growth-medium which may be sterilized by filtration or more advantageously by autoclaving in situ.     

Brucella abortus in Bison. II. Evaluation of strain 19 vaccination of pregnant cows

Protection against Brucella abortus induced abortion and infection provided by strain 19 (S19) vaccination was evaluated in American bison (Bison bison). Forty-eight pregnant bison were manually inoculated (MI) with S19 vaccine, 44 were ballistically inoculated (BI) with an absorbable hollow pellet containing lyophilized S19, and 46 were manually injected with buffered saline as non-vaccinated controls (NVC). All bison were Brucella spp. seronegative prior to the experiment, in the second trimester of pregnancy, and were randomly assigned to experimental groups. Approximately 60 days post-vaccination, abortions were observed in the vaccinated bison. Brucella abortus strain 19 was recovered from a bison that had recently aborted, her fetus, and from 11 of 12 other aborted fetuses. Fifty-eight percent (53 of 92) of vaccinated bison aborted, and no abortions were observed in the NVC bison. One cow aborted during her second post-vaccinal pregnancy and S19 was identified from the dam and fetus indicating that chronic S19 infections can occur in bison. Positive antibody titers were present 10 mo post-vaccination in 73% (66 of 91) of the bison. Thirteen mo post-vaccination, 30 MI vaccinates, 27 BI vaccinates, and 30 NVC bison were challenged during the second trimester of pregnancy with 1 x 10(7) CFU of B. abortus strain 2308 via bilateral conjunctival inoculation. Protection against abortion was 67% (P less than or equal to 0.0001) for vaccinated bison compared to 4% in NVC. Protection against B. abortus infection was determined to be 39% (P greater than or equal to 0.001) for vaccinates and 0% (zero of 30) for NCV. Persistent antibody titers, vaccine induced abortions, and chronic S19 infections indicate that the S19 vaccine doses used in this study are not suitable for pregnant bison.     

The Brucella abortus vaccine strain B19 carries a deletion in the erythritol catabolic genes

Abstract Brucella abortus B19, an avirulent strain obtained by spontaneous mutation, is used worldwide as a vaccine for the control of bovine brucellosis. B19 differs from other B. abortus strains in its sensitivity to erythritol. We took advantage of a previously obtained erythritol sensitive Tn 5 insertion mutant of B. abortus 2308 to clone the chromosomal region containing erythritol catabolic genes from this representative pathogenic strain and from the vaccine strain B19. Physical mapping with restriction endonucleases and nucleotide sequence determination revealed the existence of a 702 bp long deletion, occurring between two short direct repeats, in the chromosome of B19. This deletion rendered the B19 strain sensitive to erythritol. Two oligonucleotides whose sequences flank this deletion provided an easy method to differentiate B19 from all other B. abortus isolates.     

In vitro and in vivo characterization of smooth small colony variants of Brucella abortus S19

Brucella abortus is known to produce chronic infections in both humans and a variety of animal species. However, the mechanisms underlying the persistence of the bacteria in the presence of an ongoing immune response are still unknown. In this respect we made use of the observation that in vitro grown B. abortus S19 exhibits heterogenicity in colony size when plated onto TS agar, while experimental infection of mice uniformly results in the in vivo selection of the small colony variant. We demonstrate that the spontaneous smooth small colony variant is characterized not only by a slower growth rate in vitro but also by an increased tolerance to hyperosmotic medium and, most importantly, a less effective clearance from spleens and livers of experimentally infected mice. On a molecular level, a gene with homology to a formerly described galactoside transport ATP binding protein (mglA) was differentially expressed in small versus large colonies of B. abortus S19.     

Use of Mitomycin C to Distinguish Brucella abortus Field Strains from the Vaccinal Brucella abortus Strain 19

Field strains of Brucella abortus were resistant to mitomycin C, whereas strain 19 was sensitive; therefore, the antibiotic was used to distinguish B. abortus strain 19 from other strains.     

Safety of Brucella abortus strain RB51 in black bears

In two studies conducted from October 1999 to March 2000 and December 2000 to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4-3.1 x 10(10) colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n=12) or 2 ml of 0.15 M NaCl solution (saline, n=11). We did not detect a difference (P>0.05) in antibody titers to SRB51 in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study 1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51 did not appear to influence (P>0.05) reproductive performance.     

Safety of Brucella abortus strain RB51 in deer mice

Brucella abortus strain RB51 is an approved brucellosis vaccine for use in cattle that may have potential as an oral vaccine for use in elk (Cervus elaphus) and/or bison (Bison bison). This study was designed to determine effects of strain RB51 on deer mice (Peromyscus maniculatus), a nontarget species that could have access to treated baits in a field situation. In February 1994, 90 mice were orally dosed or intraperitoneally injected with 1 x 10(8) colony forming units strain RB51 and 77 controls were similarly dosed with sterile saline. At weekly intervals through early April 1994, 4 to 6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically. All orally inoculated mice cleared the infection by 6 wk post-inoculation (PI). While most of the injected mice cleared the infection by 7 wk PI, a few required 9 wk. There were minimal adverse effects attributable to strain RB51. Apparently, strain RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Also, deer mice appear to be able to clear the vaccine in 6 to 9 wk, thus the probability of these mice transmitting the vaccine to other animals is low.