A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

Abstract

Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/microl corresponding to blood parasitaemias of 9.2 x 10(-6)%, 2.8 x 10(-5)% and 8.3 x 10(-5)%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle.

Detection of tropical bovine theileriosis by polymerase chain reaction in cattle

“…The present study is in agreement with Rady et al (2010) and Odongo et al (2010). Altay et al (2005), Durrani et al (2008), Azizi et al (2008), Bami et al (2009) also reported that PCR was more sensitive than the traditional method of blood smear examination who also used the same primers for detection of Theileria organisms in blood.…”

A review of Theileria diagnostics and epidemiology

“…This included conventional PCR followed by agarose gel electrophoretic analysis (Bishop et al., 1992; Pienaar et al., 2011a), PCR-RFLP methods (Bishop et al., 1992; Geysen et al., 1999; Heidarpour Bami et al., 2009; Zaeemi et al., 2011), nested-PCR (Odongo et al., 2010), PCR followed by dot blotting, capillary blotting or slot-blotting and hybridisation using radio-isotope labelled probes (Bishop et al., 1992; Allsopp et al., 1993; Collins et al., 2002; Skilton et al., 2002). The latter was improved by the non-radio-active reverse line blot method that used chemiluminescence (Gubbels et al., 1999; Schnittger et al., 2004), with recent variation on this technique using a DNA bead-based suspension array (Ros-García et al., 2012a, 2013).…”

“…In contrast, the Marikebuni strain maintained the carrier-state indefinitely well above the LOD (Bishop et al., 1992; Skilton et al., 2002). Prevalence of T. parva in cattle from East Coast fever endemic regions range between 37 and 42% using nested PCR (Odongo et al., 2010), compared to ~80% in buffalo (Pienaar et al., 2011a), suggesting that the former is not compatible with an endemic stable situation. In East Coast fever, the carrier-state may therefore be strain dependent and intermittent.…”

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission

“…DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to instructions. The p104 gene PCR parameters and the primers used in the primary PCR were as described previously [25, 26]. All reactions were performed in 15 μl volumes using 5 μl of DNA extracted from blood for the primary reaction or 5 μl of the primary PCR reaction for the nested PCR reaction with 0.25 units of Taq DNA polymerase, (Promega, Madison, USA), 1× PCR buffer (Promega), 200 mM of each dNTP (Promega) and 25 ng of primers and 1.5 mM of MgCl 2 .…”