Identification And Antigenicity Analysis Of Brucella Rough Mutants

Brucellosis is an important zoonosis caused by Bucella spp.that re-emerged globally in recent years with severe public health challenges and major economic burdens.Lipopolysaccharide(LPS)is an important virulence factor of Brucella as well as the most important surface antigen that plays a major role in the diagnosis,prevention and control of brucellosis,therefore it becomes one of the research hotspots in the study of Brucella molecular pathogenic mechanism.At present,vaccines combined with serological detection and culling of infected animals were primarily used to control animal brucellosis.However,vaccines currently have a series of problems including strong virulence,poor safety,unable to differentiate from animals with vaccine immunized to animals infected.Therefore,it is the key to research and develop vaccines with high protection,low pathogenicity and no interference with existing serological diagnosis of new vaccines for effective control of brucellosis.In this research,12 monoclonal antibodies(MAbs)against smooth Brucella LPS were prepared and characterized.The MAbs were specific respectively for four epitopes on O-chain,including M,C(M>A),C/Y(M = A)and C/Y(A>M).Based a MAb 3F9 against the C(M>A)epitope,competitive ELISA for detection of bovine brucellosis and ovine brucellosis were established respectively with higher specificity and good sensitivity.To analyze the effect of gene mutation on LPS,95 rough type mutants screened out by serum agglutination from a genome-wide transposon-derived mutant array were classified into four types,i.e.RS,R1,R2 and R3,according to the reactivity with three MAbs for different epitopes on O-chain,that revealed the subtle discrepancy in antigenicity among rough mutants.Meanwhile,40 novel genes related with rough phenotype were found.RS type rough mutants reacted with smooth and rough anti-sera and MAbs,and R3 type mutants with no reactivity with smooth anti-sera but reacted MAbs may be available in vaccine research.The virulence and immune protection of the two tpyes of rough mutants were evaluated on mouse model.The virulence of RS and R3 type mutants decreased significantly compared with the parent strain and the immune protection against challenge with virulent Brucella meletensis were inferior to the vaccine strain M5-90,that indicated the significance of complete O-chain for the immune protection.One mutant was proved to be good immune protection against challenge with Brucella canis,implying that the wbkD gene interrupted in this strain may be a candidate gene for vaccine research for canis brucellosis.In conlusion,our study made an analysis and classification for Brucella rough mutants with epitopic specific MAbs for LPS,providing references for the slection of candidate genes for the research of novel vaccines.