The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay

Abstract

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

Quantitative Analysis of Cytokine mRNA Expression and Protozoan DNA Load in Theileria parva-Infected Cattle

“…It is reported that PCR primers based on p104 gene specifically amplify T. parva DNA, but DNA of T. annulata, T. buffeli, T. lestoqoardi, T. mutans and T. taurotragi was not amplified using the p104 primers. Furthermore, p104 is encoded by a single-copy gene conserved among different T. parva stocks [25]. Cycling condition included an initial heat denaturation at 95°C for 10 sec, followed by 40 cycles of 5 sec at 95°C, 15 sec at 65°C and 15 sec at 72°C.…”

Acquisition and transmission of Theileria parva by vector tick, Rhipicephalus appendiculatus

“…5'-TAAGATGCCGACTATTAATGACACC-3' which produce a 496-bp product as described previously (Kaba et al, 2005;Skilton et al, 2002). T. parva p104 gene is highly conserved within the T. parva strains (Skilton et al, 2002).…”

“…T. parva p104 gene is highly conserved within the T. parva strains (Skilton et al, 2002). Cycling conditions involved an initial 5 min denaturation at 95°C, followed by 30 cycles, each consisting of a 30 s denaturation at 94°C, a 30 s annealing at 65°C, and 1 min extension at 72°C.…”

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

“…Primers designed from the p104 and p67 genes have been successfully used for PCR amplification and detection of T. parva infections (Skilton et al, 2002;Musoke et al, 2005). Prior to this study several LAMP primer sets were designed from the p67 and p104 genes of T. parva.…”

“…However, serological assays are unable to differentiate between current and past infections due to the persistence of antibodies. PCR diagnostic assays have been developed for the sensitive and specific detection of T. parva, including the detection of very low levels of the parasite in carrier animals (Bishop et al, 1992;Gubbels et al, 1999;Skilton et al, 2002). These assays are not in widespread use in the resource poor countries where ECF occurs due to the relatively complex nature of the assays, and the need for expensive equipment and well-trained personnel (Kuboki et al 2003;Poon et al 2006).…”