Submitted to: Veterinary Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/19/2007
Publication Date: 7/27/2008
Citation: Scoles, G.A., Goff, W.L., Lysyk, T.J., Lewis, G.S., Knowles Jr, D.P. 2008. Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates. Veterinary Microbiology. 130:184-190. Interpretive Summary: Anaplasmosis is a serious disease of cattle caused by infection of the red blood cells with Anaplasma marginale. Domestic sheep are frequently infected with a closely related pathogen, Anaplasma ovis, which is generally not considered to cause severe disease. However, due to the lack of a good diagnostic assay, studies to determine the actual economic impact of A. ovis infection of sheep have not been done. Anaplasma infections have also been found in several wild ruminant species (bison, elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer, etc), but the importance of these infections has not been determined. In this study we have validated a commercially available A. marginale cELISA kit for diagnosing Anaplasma infections in sheep and wild ruminants. We evaluated the commercial test using the positive and negative controls provided with the kit for cattle as well as with our own controls from domestic sheep. The test results for domestic sheep were confirmed with a polymerase chain reaction test (PCR); an indirect immunofluorescent assay (IFA) was used to confirm both the sheep and the wild ruminant samples. The predicted threshold inhibition for the cELISA using the kit-supplied controls on domestic sheep samples was 19.2; the sensitivity was 98.2%, specificity was 96.3%. When we used our own sheep positive and negative controls the predicted threshold inhibition was decreased to 14.3; the sensitivity was 96.5% and specificity 98.1%. For sheep a >90% concordance was observed between the cELISA and IFA or PCR, when the kit-supplied bovine controls were used with 19% as the inhibition cutoff. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64 to 100%. We conclude that this cELISA test kit can be used very effectively to test domestic sheep for A. ovis using the kit-supplied controls, and that it may also be useful for detecting Anaplasma infections in wild ruminants.
Technical Abstract: A commercially available cELISA kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A. ovis infection in sheep using bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from hand-raised pathogen free sheep (OcELISA). ROC analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2 and the area under the ROC curve was 0.993 (95% CI = 0.965 - 0.999). The sensitivity was 98.2%, specificity was 96.3% and positive and negative predictive values were 98.76% and 94.69% respectively. The predicted threshold inhibition decreased to 14.3 for the OcELISA and the area under the ROC curve was 0.995 (95% CI = 0.968 - 0.999). The sensitivity was 96.5% and specificity 98.1% with corresponding positive and negative predictive values of 99.35% and 90.33% respectively. A high degree of concordance (>90%) was observed between an indirect immunofluorescent assay (IFA) and PCR, and the BcELISA at 19% inhibition cutoff and IFA and PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64 - 100%.