Molecular Detection of Anaplasma marginale Infection in Carrier Cattle by MSP5 Gene-Based Semi-Nested PCR

Abstract

Bovine anaplasmosis, caused by Anaplasma marginale, is a major tick-borne haemoparasitic disease that causes significant economic losses to the cattle industry by reducing productivity and increasing mortality. Early detection of persistently infected carrier animals is essential for effective disease surveillance and control; however, conventional diagnostic methods often fail to identify low-level infections. The present study was undertaken to develop and evaluate a semi-nested polymerase chain reaction (PCR) assay targeting the major surface protein 5 (msp5) gene for the sensitive detection of A. marginale infection in carrier cattle. A total of 31 blood samples were collected from cattle in Jabalpur district, Madhya Pradesh. Microscopic examination of Giemsa-stained blood smears detected A. marginale in only 16.1% (5/31) of the samples. In contrast, primary PCR amplified a 458-bp fragment of the msp5 gene in 41.93% (13/31) of the samples, whereas semi-nested PCR amplified a 345-bp fragment in 70.96% (22/31) of the samples, demonstrating substantially higher diagnostic sensitivity. The assay successfully detected infections in apparently healthy carrier animals with low rickettsaemia that were missed by microscopy and primary PCR. These findings indicate that the developed semi-nested PCR assay is a rapid, sensitive, and reliable molecular diagnostic tool for detecting A. marginale in carrier cattle and has considerable potential for routine diagnosis, epidemiological surveillance, and effective control of bovine anaplasmosis.