Affects Of Deletion Of B0107and A1103Gene On The Virulence Of Brucella Melitensis Virulent Strain M28

The Brucella spp. are Gram-negative bacteria that cause economically important diseases in foodanimals worldwide. Brucella cause abortion and infertility in their natural hosts—goats and sheep, cattleand swine, respectively. Humans can also acquire a severe, debilitating febrile illness known asbrucellosis, or "undulant fever,"as the result of contact with infected animals or their products.Brucella melitensis vaccine strain M5-90originated after a virulent B.melitensis strain (M28)isolated from a sheep was serially passaged through chickens, treated with acriflavine, and passaged for90generations in chicken embryo fibroblasts. This attenuated vaccine is considered to be one of the keyfactors that led to a rapid decline in the incidence of brucellosis in animals and humans in China. Up tonow, mechanisms about the attenuation of M5-90are unknown.According to previous research, there are31indels and125SNPs located in35CDS regions of thewhole genome of M5-90including frameshift mutations in the B0107gene and A1103gene, coding forO-antigen polymerase and Quinine tRNA-ribosyltransferase, respectively. O-antigen has already beenproved to be one of the most important virulent factors for Gram negative bacteria while the role ofQuinine tRNA-ribosyltransferase involved in virulence of Brucella has yet to be defined.To investigate the affects of mutation of B0107and A1103gene on the virulence of Brucellamelitensis virulent strain M28, we constructed two M28artificial mutant strain (designatedM28-ΔB0107and M28-A1103) with the ORFs replaced by kanamycin resistance gene based on themethod of homologous recombination.The dynamics line in vitro revealed no distinction among M28-ΔB0107, M28-ΔA1103and M28.To further elucidate pathogenicity in vivo and the ability of replication in macrophage cells, we infecthost animals with mice model and cell line RAW264.7. The result showed that the M28-ΔB0107werestill able to cause the mouse spleen swollen, but twice smaller than that in the mice infected with theparental bacteria(P<0.05). In addition, the bacteria load in the spleen was ten times lower than that inthe mice infected with the parental bacteria at6week post infection(P<0.05). As for M28-ΔA1103, thepathogenicity it caused in mice showed insignificant change compared to the M28strain(P>0.05).However, the bacterial counts displayed that neither the survival rate of M28-ΔB0107nor M28-A1103had any difference to the parental strain in the murine macrophage cell line RAW264.7.These results indicate B0107is one of the virulent factors of B.melitensis and its mutation hadcontributed to the attenuation of M5-90to some degree.