Development and evaluation of a lateral flow assay for detecting Anaplasma marginale infection in cattle

A rapid lateral flow assay using recombinant major surface protein 5 (MSP5) was developed and evaluated for detection of antibodies to Anaplasma marginale in cattle. Sera samples collected from A. marginale known positive and negative animals (as confirmed by microscopic examination of stained blood smears, MSP5 gene based nested PCR and rMSP5 antigen based indirect ELISA), when probed for anti-MSP5 antibodies in the LFA, gave distinct immunoreactivity with all the known positive sera in the test and control lines, while the known negative sera showed no immunoreactivity in the test line. The rMSP5 LFA showed no cross-reactivity with B. bigemina, T. annulata and T. evansi positive animals. The test was further validated using 314 sera samples collected from cattle of endemic regions of north India. The results indicated that the developed LFA had a sensitivity, specificity and accuracy of 86.72%, 86.36% and 83.43%, respectively with respect to nested PCR, and 89.81%, 85.71% and 88.53%, respectively, with respect to rMSP5 based indirect ELISA. A substantial agreement was observed between rMSP5-LFA and nested PCR (k = 0.6878) as well as between rMSP5-LFA and rMSP5-ELISA (k = 0.7387), illustrating LFA's applicability as rapid, simple and field-deployable diagnostic assay for the detection of A. marginale antibodies in naturally infected cattle.