Detection and Quantification of Ehrlichia ruminantium DNA in Amblyomma spp. and Blood of Domestic Ruminants in the Sudan by Plexor® qPCR System Assay Based on pCS20 Gene

Abstract

Heartwater is a tick-borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma. A pCS20 quantitative real-time PCR (qPCR) was applied for detection and quantification of E. ruminantium in Amblyomma spp. (170) and blood (120) of cattle, sheep and goats from some localities in the Sudan. The infection rate of E. ruminantium was 29.2% (35/120) (r: 0.9802 p-value: 0.0001) and 42% (21/50) (r = -0.9184 p-value = 0.2590) in A. lepidum and A. variegatum, respectively, while in the blood it was 8% (4/50), 8.6% (3/35) and 14.3% (5/35) from cattle, sheep and goats, respectively. There is no statistically significant difference in infection rates among the three ruminant species in the analyzed localities (ꭓ2 = 0.19, df = 2, P = 0.9074). The detection limit was 5 pCS20 copies/µl which was at least 20-times more sensitive than that of conventional PCR. Furthermore, the qPCR was compared to the conventional pCS20 PCR and nested MAP1 PCR. Out of 50 samples positive with qPCR, 49 (98%) were positive by nested MAP1 PCR and 42 (84%) by conventional pCS20 PCR. The Chi-square test revealed a statistically significant association between diagnostic methods and the detection of positive samples (ꭓ2 = 13.48, df = 2, P = 0.0012) indicating that the performance of the three PCR-based techniques varied considerably. The specificity of the qPCR was, also, determined using E. ruminantium Welgevonden as a reference strain from South Africa, four local E. ruminantium stocks (Dinder, Abonama, Nyala and Gdarif) and two other Rickettsiales namely E. canis and Anaplasma marginale. The qPCR specifically detected all E. ruminantium stocks and was positive with E. canis but negative with A. marginale. The qPCR applied for E. ruminantium is species specific and sensitive to as few as 5 gene copies/µl. The assay was found more sensitive than the standard pCS20 PCR assay and nested MAP1 PCR assay.