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.     

Evaluation of Brucella abortus S19 vaccine strains by bacteriological tests, molecular analysis of ery loci and virulence in BALB/c mice.

Two Brucella abortus S19 commercial vaccine strains used for vaccination against brucellosis in India and three S19 strains available as international reference were examined by microbiological assays and molecular analysis of the ery loci involved in erythritol metabolism, and tested for residual virulence in BALB/c mice. According to the sensitivity to penicillin and i-erythritol, the five strains tested had the phenotypic characteristics of strain S19. However, on culture medium containing i-erythritol, all strains developed spontaneous i-erythritol resistant colonies at mutation rates ranging from 1.42x10(-2) to 1.33x10(-6). The S19 characteristic 702 bp deletion in the erythrulose 1-phosphate dehydrogenase gene of the ery locus was present only in the three reference strains but not in the two commercial vaccines. Both commercial strains and one of the reference strains showed reduced virulence in BALB/c mice. The presence or absence in S19 strains of the 702 bp deletion in the ery locus had no correlation with either the rates of spontaneous mutation to erythritol resistance or the residual virulence in mice.     

布氏菌104M变异株毒力及保护力研究

目的研究皮上划痕人用布氏菌活疫苗104M变异株的残余毒力及保护力。方法对布氏菌104M变异株进行菌种检定。通过测定布氏菌104M变异株与未变异株的半数致死量(50%lethal dose,LD50),比较两者的残余毒力;用布氏菌104M变异株与未变异株分别免疫小鼠,4周后用羊型弱毒或强毒布氏菌皮下攻击,4周后测定脾脏细菌数,比较布氏菌104M变异株与未变异株的保护效果。结果菌种检定结果显示,布氏菌104M变异株与未变异株分别符合变异及未变异布氏菌株特征。104M未变异株LD50为1.1×109,变异株LD50为2.2×1010。用羊型弱毒及强毒布氏菌攻击后,布氏菌104M变异株与未变异株均具有较好的保护效果,但104M变异株保护力低于未变异株,差异具有统计学意义(P0.05)。结论布氏菌104M变异株残余毒力比未变异株明显降低,保护力略低于未变异株。     

Genome sequence of the Chlamydophila abortus variant strain LLG

Chlamydophila abortus is a common cause of ruminant abortion. Here we report the genome sequence of strain LLG, which differs genotypically and phenotypically from the wild-type strain S26/3. Genome sequencing revealed differences between LLG and S26/3 to occur in pseudogene content, in transmembrane head/inc family proteins, and in biotin biosynthesis genes.     

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.     

Production and control of freeze-dried Brucella abortus strain 19 vaccine by using cells produced by fermentation units

Summary A method of producing freeze-dried Brucella abortus Strain 19 vaccine by using batch cultures on fermentation units is described. Details of the equipment and technique employed, results on microbiological and biological controls are given.     

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.     

Characterization of Brucella abortus S19 as a challenge strain for use in a mouse model of brucellosis

The objective of this project was to conduct a feasibility study to determine whether the Brucella abortus S19 vaccine infects and persists in mice and determine whether S19 can be used as a challenge strain for vaccine trial studies. Groups of BALB/c mice were inoculated (intraperitoneally, subcutaneously, intranasally) and euthanized to determine colonization titers in the spleens and lungs. This study showed that S19 does infect and persist in the tissues of mice for 8 weeks and demonstrates that S19 can be used, safely and economically under BSL2 containment, as the challenge strain for future trials to evaluate vaccine efficacy.     

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 and efficacy of Brucella abortus strain RB51 vaccine in captive pregnant elk

Brucella abortus strain RB51 is a laboratory-derived rough mutant of virulent B. abortus strain 2308 used as a vaccine because it induces antibodies that do not react on standard brucellosis serologic tests. Strain RB51 vaccine was evaluated in pregnant captive elk (Cervus elaphus) to determine (1) if it induced abortion and (2) if it protected against abortion following subsequent challenge. The time period of this study (February-June, 1998) was similar to field conditions where elk are vaccinated and possibly exposed to B. abortus. Fourteen elk were randomly and equally divided into vaccinated and control groups. The vaccinated group was vaccinated intramuscularly with 1.03 x 10(10) colony-forming units (CFU) of strain RB51 and seroconverted postvaccination. Antibodies to strain RB51 were detected by a modification of an existing dot-blot assay. Both groups were challenged 40 days postvaccination with 9.8 x 10(6) CFU of B. abortus strain 2308 administered intraconjunctivally. The first abortion occurred 38 days postchallenge. Abortion occurred in all control elk and in five of seven vaccinated elk 5 to 12 wk postchallenge (P = 0.23). Mixed strain RB51 and 2308 infections were present in fetuses and vaginas from the vaccinated group whereas only strain 2308 was cultured from control group fetuses and vaginal swabs. Further evaluation of strain RB51 will be necessary to determine if it will be safe and efficacious in free-ranging pregnant elk.