DETECTION OF CATTLE NATURALLY INFECTED WITH ANAPLASMA-MARGINALE IN A REGION OF ENDEMICITY BY NESTED PCR AND A COMPETITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY USING RECOMBINANT MAJOR SURFACE PROTEIN-5

A competitive enzyme-linked immunosorbent assay using recombinant majo r surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validate d in a naturally infected cattle herd in an area of eastern Oregon whe re A. marginale is endemic. The true positive and negative A. marginal e infection status of 235 randomly selected cattle was determined by u sing a nested PCR (nPCR) coupled with msp5 sequence analysis and hybri dization. Judgment of the reliability of the nPCR and hybridization fo r detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per mi. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A . marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point o f 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively a nd specifically detect cattle with naturally acquired persistent A. ma rginale infections and suggest that it is an excellent assay for epide miological studies, eradication programs, and regulation of internatio nal cattle movement.