Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks

Abstract

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches.