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.

Investigations into the natural infection rate of Haemaphysalis qinghaiensis with Piroplasma using a nested PCR

“…Despite this, staining the salivary glands of the ticks or vector transmission experiments can deWnitely show the infection of the ticks; but the methods are lacking in sensitivity and speciWcity and/or are time-consuming. The detection of the parasite DNA in ticks using speciWc PCR has been applied to T. annulata (d' Oliveira et al 1997;Kirvar et al 2000), T. parva (Watt et al 1997;Ogden et al 2003), T. lestoquardi (Kirvar et al 1998) and T. ovis (Aktas et al 2006). Based on the knowledge and availability of 18S rRNA gene sequences of Piroplasma, we have established a nested PCR for detection of the Piroplasma pathogens from Weld ticks.…”

Prevalence of Theileriosis in Red Hartebeest (Alcelaphus buselaphus caama) in Namibia

“…For the detection and identification of Theileria species several methods have already been used such as microscopical (Walker et al 1979(Walker et al , 1997, serological (Gray et al 1980;Leemans et al 1997) and molecular biology methods-polymerase chain reaction (PCR), reverse line blot hybridization (RLB) (Watt et al 1997;Sparagano et al 1999;Gubbels et al 1999, Schnittger et al 2004). These methods were developed separately for each species or simultaneously for the detection of several parasites that could be present in the blood and tick samples.…”

Cloning, expression and partial characterization of a Haemaphysalis longicornis and a Rhipicephalus appendiculatus glutathione S‐transferase

“…Haemaphysalis longicornis is distributed in Australia, New Zealand and eastern Asia and is the major vector of Theileria sp., which is the most economically important protozoan infectious disease affecting cattle in Asia (Onuma et al ., 1998). Rhipicephalus appendiculatus , present in eastern and southern African countries, is the principal vector of Theileria parva (Watt et al ., 1997). Therefore, control strategies are aimed to reduce tick infestation.…”