A duplex PCR-based assay for simultaneous detection of Trypanosoma evansi and Theileria annulata infections in water buffaloes

Abstract

Trypanosomosis and bovine tropical theileriosis are important vector-borne protozoan diseases imposing some of the serious constraints on the health and productivity of domestic cattle in tropical and subtropical regions of the world. Following recovery from primary infection of both these conditions, animals become persistent carriers and act as reservoirs of infection thereby playing a critical role in disease epidemiology. The present study describes development and evaluation of duplex polymerase chain reaction (PCR) assays for simultaneous detection of Trypanosoma evansi and Theileria annulata in buffaloes. Following in silico screening for candidate target genes representing each of the pathogens, an optimized duplex PCR assay was established using TBR F/R and TAMS F/R as primer sets encoding for products of 164 and 721 bp for T. evansi and T. annulata, respectively. The results were compared and correlated with conventional Giemsa-stained thin blood smear examination and the single PCR assay. The duplex PCR detected each pathogen with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen. Moreover, single and duplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. evansi and T. annulata, and no evidence of nonspecific amplification from nontarget species was observed. The developed assay may be seen as a good tool for epidemiological studies aiming at assessing the burden of dual infections and improving control of the associated diseases in endemic regions.

Comparative Efficiency of Whole blood and FTA Card for Diagnosis of Theileriosis in Buffaloes of Nagpur Region, India

“…Several workers like Mahmood et al, (2010), Kolte et al, (2017), Paliwal et al, (2019), Kundave et al, (2015) have performed PCR using whole blood and also reported that PCR was most sensitive tool for diagnosis of theileriosis. Partial similar observations were recorded earlier by Ali and Radwan (2011), Ghanem et al, (2013), Chaisi et al, (2013, Sudan et al, (2015) and many other authors.…”

Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia

“…A total number of six animals were co-infected by both parasites 6 [3.5; 8.5] CI at 95% which was lower than cattle in India (11.25%) (Sudan et al 2015). The highest rate co-infection was observed in 2 to 5 years age group 8 [3; 13] CI at 95%.…”

First molecular evidence of the transplacental transmission of Theileria annulata

“…The disease is caused by an apicomplexan parasite Theileria annulata and involves Hyalomma anatolicum anatolicum as its vector in the Indian subcontinent (Gill et al 1978). BTT is responsible for high rates of mortality in dairy cattle and about 200 million animals are supposed to be exposed to its risk (Radostits et al 1994;Sudan et al 2014Sudan et al , 2015. The global annual losses due to BTT are estimated to be of US$800 million (Brown 1997) while in India alone, the disease accounts for an approximate annual loss of US$384.3 million (Minjauw and McLeod 2003).…”