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Journal of Clinical Microbiology, March 1998, p. 777-782, Vol. 36, No. 3
Instituto Nacional de Tecnología
Agropecuaria, EEA Rafaela, Rafaela, Santa Fe,
Argentina,1 and
Animal Disease
Research Unit,
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.
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
*
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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