Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis

摘要

Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed aserology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of as antigens. DNA corresponding to each antigen was clonedinto a pET32a vector and expressed in . Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95%homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated (1:1,000) on a Western blot. Subsequently, wedemonstrated that both recombinants could capture the antibodies present in goat with naturally occurring melioidosis (optimized titer 1:40) while not cross-reacting with the serum samplesof goats naturally infected by or . Finally, an enzyme-linked immunosorbent assay (ELISA) using 20goat serum samples without melioidosis and 10 goat serum samples with melioidosis demonstrated that the infected group has significantly higher antibody titer levels than the normal group( <0.001) when using either OmpA or FliC as an antigen. However, the sensitivity (100%) of the assay using OmpA was superior to that (90%) from using FliC. Serologicaltests that are commonly used often rely on antigens from crude cell extracts, which pose risks for laboratory-acquired infections and inconsistency in their preparation; however, use ofrecombinant OmpA is safe; it can potentially be used as a reagent in testing for goat melioidosis.