Phylogeny of Brucella abortus strains isolated in the Russian Federation

Objective: To study Brucella(B.) abortus strains isolated in the Russian Federation, in order to identify their detailed position in the phylogenetic structure of the species global population as well as to determine genetic relationships for isolates from different geographical areas.Methods: Based on Bayesian method, the whole genome singlenucleotide polymorphism(SNP) analysis of 258 B. abortus strains from different geographical areas of the world including 20 B. abortus strains isolated in Russia was carried out. Results: The core genome SNP analysis of the B. abortus isolates allowed describing the main genetic lineages. The Russian strains entered two separate clades, including the basal branch and the C1 branch that is widely spread in Eurasia. The data on the isolation time was used for the dating of phylogenetic tree, and also the estimated time frame for the B. abortus genotype diversification was determined. There were sets of specific SNPs identified, which defined each of the genotypes and sub-genotypes.Conclusions: A significant genetic diversity of the brucellosis pathogen strains from Russia has been proven. The sets of unique specific SNPs described in our study may become one of the elements within a bio-informational analysis algorithm to be used for epidemiological study of brucellosis outbreaks, including those caused by new(atypical) genetic variants of B. abortus.

A case study investigating the efects of emergency vaccination with Brucella abortus A19 vaccine on a dairy farm undergoing an abortion outbreak in China

Brucellosis is an important zoonosis that results in substantial economic losses to the livestock industry through abortions and reduced milk yield.This study investigated an abortion outbreak in a dairy herd and then explored the efects of emergency vaccination with Brucella abortus A19 vaccine on the incidence of abortion and milk yield.A full dose of vaccine(6×10^(10)—12×10^(10)colony forming units,CFU)was administered subcutaneously to calves and non-pregnant heifers,and a reduced dose(6×10^(8)—12×10^(8)CFU)to adult cows and pregnant replacement heifers.Rose Bengal Test was used to screen Brucella infection status and then positive samples were tested with a C-ELISA.Animals that tested positive for both tests were considered positive to Brucella spp.The animal-level seroprevalence of brucellosis was 23.1%(95%CI:17.0,30.2),and the attributable fraction of abortions in seropositive animals was 89.1%(95%CI:64.3,96.7).The odds of seropositivity were signifcantly higher in cows that aborted compared to cows that calved normally(OR=21.4,95%CI:4.4,168.4).Cows in sheds A2 and C1 were 10.2(95%CI:1.4,128.0)and 17.0(95%CI:2.8,190.3)times more likely to be seropositive than cows in shed B1.Antibodies were not detectable in most heifers 12 months post-vaccination.The efectiveness of the vaccine in preventing abortions was estimated to be 56.8%(95%CI:15.8,77.8)for the entire herd,but increased to 86.7%(95%CI:4.4,98.1)when only primiparous heifers were considered.Furthermore,a signifcant increase in the average herd 305-day milk yield one-year after vaccination was also observed relative to that in the previous three years.It is concluded that emergency vaccination of a dairy herd undergoing an abortion outbreak with the A19 vaccine efectively reduced the incidence of abortion and indirectly increased milk yield one-year after vaccination.

Prokaryotic Expression and Antigenic Analysis of Wbkc Gene from Brucella abortus

[ Objective ] This study aimed to clone and express formyltransferase （Wbkc） gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase （Wbkc） was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors （TLRs） recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 （rBCSP31） to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases （MAPKs） by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-KB in macrophages. In addition, continuous exposure （〉24 h） of RAW264.7 cells to rBCSP31 significantly enhanced I FN-y-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4＋ T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2-/- and TLR4-/- mice was lower than that from C57BL/6 macrophages, and the activation of NF-KB and MAPKs was attenuated in macrophages from TLR2-/- and TLR4-/- mice. In addition, CD4＋ T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-y and IL-2 compared with CD4＋ T cells from TLR2-/- and TLR4-/- mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2-/- and TLR4-/- mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2-/- and TLR4-/- mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Thl immune response.

DNA vaccine encoding L7/L12-P39 of Brucella abortus induces protective immunity in BALB/c mice

Brucella abortus is a gram-negative, facultative, intracellular bacterium that infects both cattle and humans, causing abortion and infertility in the former and undulant fever, endocarditis, arthritis, and osteomyelitis. Resistance to Brucella depends on acquired cell-mediated immunity （CMI）. Live attenuated vaccines can stimulate strong CMI response, which are usually very effective against brucellosis and are used to control brucellosis in domestic animals. However, there is no safe and effective vaccine available for human because the vaccine strains used for animals are considered too virulent for humans. A vaccine that will be noninfectious to humans but effective in stimulating a broad protective immune response is needed.

Isolation and identification of Brucella abortus from patients in Guizhou Province for the first time

Objective To etiologically diagnose and analyze two suspected patients of brucellosis and provide a etiologic basis for confirmation of the patients and control of brucellosis in Guizhou Province.Methods Antibody levels of Brucella in the suspected patients were determined by agar plate agglutination test and tube agglutination test.