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Journal of Clinical Microbiology, March 1998, p. 777-782, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Susana Torioni de Echaide,1 Donald P. Knowles,2,3 Travis C. McGuire,3 Guy H. Palmer,3 Carlos E. Suarez,3 and Terry F. McElwain3,4,*

Instituto Nacional de Tecnología Agropecuaria, EEA Rafaela, Rafaela, Santa Fe, Argentina,1 and Animal Disease Research Unit, U.S. Department of Agriculture, Agricultural Research Service,2 Department of Veterinary Microbiology and Pathology, Washington State University,3 and Washington Animal Disease Diagnostic Laboratory,4 Pullman, Washington

Received 21 August 1997/Returned for modification 15 October 1997/Accepted 17 December 1997

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. 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 of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.

* Corresponding author. Mailing address: Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA 99164-7034. Phone: (509) 335-3045. Fax: (509) 335-7424. E-mail: tfm{at}vetmed.wsu.edu.

Journal of Clinical Microbiology, March 1998, p. 777-782, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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