Characterization of Brucella abortus Mutant A19mut2, a Potential DIVA Vaccine Candidate with a Modification on Lipopolysaccharide.

BACKGROUND: Brucella abortus is the main causative agent for bovine brucellosis. B. abortus A19 is a widely used vaccine strain to protect cows from Brucella infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent Brucella strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel Brucella DIVA vaccine candidate. STUDY DESIGN AND METHODS: The B. abortus mutant A19mut2 with the formyltransferase gene wbkC is replaced by an acetyltransferase gene wbdR from E. coli O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type Brucella LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. RESULTS: The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against B. abortus S2308 NalR infection was similar to that of A19 immunization. CONCLUSION: The A19mut2 has potential as a novel DIVA vaccine candidate in the future.

Isolation, Molecular Characterization, and Antibiotic Resistance of Avian Pathogenic Escherichia coli in Eastern China.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avians, resulting in considerable losses in the poultry industry. APEC showed zoonotic potential initially related to the fact that APEC serves as the reservoir of virulence genes and antibiotic resistance genes for other E. coli. Thus, we determine the serotypes, phylogenetic groups, virulence genes distribution, and antibiotic resistance profiles of APEC isolates in eastern China. A total of 230 APEC were isolated from diseased chicken and duck with typical colibacillosis symptoms. Serotyping identified that O78 (44.78%) was the predominant serotype. The majority of APEC isolates were classified into B2 (29.57%), A (26.96%), D (20.00%), and B1 (18.26%), respectively. Among the 15 virulence genes, a high prevalence of ibeB (99.57%), fimC (91.74%), mat (91.30%), ompA (83.04%), and iss (80.43%) genes was observed. Except for low resistance rates for imipenem (1.7%) and polymyxin B (0.4%), most of the APEC isolates were resistant to erythromycin (98.7%), enrofloxacin (96.1%), tetracycline (95.2%), doxycycline (93.9%), lincomycin (90.0%), and streptomycin (90.0%). Moreover, all APEC exhibit multi-drug resistance. This study indicated that APEC isolates harbor a variety of virulence genes and showed multi-antibiotic resistance profiles, providing proof for understanding the epidemiological background and zoonotic potential of APEC in poultry farms.

The novel LysR-family transcriptional regulator BvtR is involved in the resistance of Brucella abortus to nitrosative stress, detergents and virulence through the genetic regulation of diverse pathways.

Brucella is a facultative intracellular bacterium lacking classical virulence factors; its virulence instead depends on its ability to invade and proliferate within host cells. After entering cells, Brucella rapidly modulates the expression of a series of genes involved in metabolism and immune evasion. Here, a novel LysR-family transcriptional regulator, designated Brucellavirulence-related transcriptional regulator (BvtR), was found to be associated with Brucella abortus virulence. We first successfully constructed a BvtR mutant, ΔbvtR, and a complemented strain, ΔbvtR-Com. Subsequently, we performed cell infection experiments, which indicated that the ΔbvtR strain exhibited similar adhesion, invasion and survival within HeLa cells or RAW264.7 macrophages to those of the wild-type strain. In stress resistance tests, the ΔbvtR strain showed enhanced sensitivity to sodium nitroprusside and sodium dodecyl sulfate, but not to hydrogen peroxide, cumene hydroperoxide, polymyxin B and natural serum. Mouse infection experiments indicated that the virulence of the ΔbvtR strain significantly decreased at 4 weeks post-infection. Finally, we analyzed differentially expressed genes regulated by BvtR with RNA-seq, COG classification and KEGG pathway analysis. Nitrogen metabolism, siderophore biosynthesis and oligopeptide transport were found to be the predominantly altered functions, and key metabolic and regulatory networks were delineated in the ΔbvtR mutant. Thus, we identified a novel Brucella virulence-related regulator, BvtR, and demonstrated that BvtR regulation affects Brucella resistance to killing by sodium nitroprusside and sodium dodecyl sulfate. The differentially expressed genes responding to BvtR are involved in diverse functions or pathways in Brucella, thus, suggesting the breadth of BvtR's regulatory functions. This study provides novel clues regarding Brucella pathogenesis.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.