Immunological Detection of Brucella Species

Introduction: Brucella causes brucellosis in animals and humans globally. In particular, pathogenic species like B. abortus and B. melitensis can be transmitted through milk. For isolation and detection, a BSL-3 laboratory setup and labor intensive culturing method is used. Based on the highly intraspecies homology of Brucella, lipopolysaccharides (LPS) of non-pathogenic B. neotomae was used as a model antigen to develop antibody and was further tested against pathogenic Brucella species.

Purpose: To develop antibody against B. neotomae LPS for detection of pathogenic Brucella species especially B. abortus and B. melitensis in milk.

Methods: B. neotomae LPS was extracted by butanol-water method, quantified by endotoxin assay kit, tested by silver staining and was used to immunize rabbits. The Protein-A purified antibody was tested in enzyme linked immunosorbent assay (ELISA) and Western blotting. Purified LPS of B. neotomae, B. abortus RB51, E. coli and 6 strains of pathogenic Brucella species and their whole cell lysates were used as antigen to evaluate the specificity of the antibody.   

Results: In ELISAs, there was significant difference in antibody reaction between B. neotomae and E. coli O157:H7 LPS, and between B. neotomae and whole cell lysates of other Gram negative species such as Salmonella. Immunofluorescence absorbance were 8168 ± 117(1:2000 diluted antibody) RFU for B. neotomae LPS vs. 264 ± 8 (1:2000) RFU for E. coli O157:H7 LPS. In Western blot, 6 strains of pathogenic Brucella species (B. abortus, B. melitensis) showed identical bands as B. neotomae, while B. abortus RB51, a LPS negative vaccine strain and E. coli O157:H7 showed negative result with no bands.

Significance: B. neotomae may be used as a model for antibody development and detection of pathogenic Brucella species.