Development and evaluation of phenol red LAMP assay for the diagnosis of bovine tropical theileriosis

Bovine tropical theileriosis (BTT), a globally distributed disease, is caused by Theileria annulata and transmitted by Hyalomma species of ticks. While polymerase chain reaction (PCR)-based assays are sensitive and specific for detection of this disease, their practical application in the field is limited by the requirement for sophisticated instruments. The loop-mediated isothermal amplification (LAMP) assay emerges as a highly efficient, sensitive, and rapid nucleic acid amplification method that also requires minimal complex instrumentation for operation. Therefore, the present study aimed to develop a LAMP assay targeting a partial, cytochrome b gene of T. annulata, facilitating the visualization of LAMP products with the naked eye using pH-sensitive dye (phenol red). Positive LAMP products were confirmed by a ladder-like pattern in agarose gel electrophoresis. No amplification was detected with the DNA of other haemoprotozoans. The results of the LAMP assay were compared with those of blood microscopy and PCR assays. Among the 200 samples tested, LAMP identified 23 cases of infection, while blood microscopy and PCR detected infections in 16 and 20 samples, respectively. The findings of this study suggest that the LAMP assay exhibited higher sensitivity and specificity compared to PCR and blood microscopy, which may enable field-level diagnosis of BTT.