Molecular Epidemiology of Heartwater (Ehrlichia ruminantium Infection) in Domestic Ruminants in the Sudan

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Sayed Mohammed Suliman, Mohammed
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This study was to investigate molecular epidemiology of heartwater in the Sudan by assessing prevalence of infection in domestic ruminants and vector of the genus Amblyomma using the standard PCR and quantitative PCR based on the pCS20 gene as well as to isolate Ehrlichia ruminantium stocks in the Sudan. A total of 460 adult Amblyomma spp., A. lepidum (290), A. variegatum (100) and blood samples (170) from cattle (60), sheep (60) and goats (50) were tested with pCS20 PCR. Twenty nine out of 190 (15.3%) and 14 out of 100 (14%) were positive for E. ruminantium in A. lepidum and A. variegatum, respectively. Four out of 60 (6.7%), 2 out of 50 (4%) and 5 out of 60 (8.3%) blood samples from cattle, sheep and goats, respectively were positive for E. ruminantium. A qPCR was applied for detection and quantification of E. ruminantium in Amblyomma spp. and blood samples. The sensitivity of the qPCR was determined through standard curve generated from the Welgevonden strain. The detection limit was 5 pCS20 copies/µl which was at least 20-times more sensitive than that of standard PCR. Furthermore, the qPCR was compared to the standard pCS20 PCR and nested MAP1 PCR. Out of 50 samples positive with qPCR, 49 (98%) were positive with nested MAP1 PCR and 42 (84%) with standard pCS20 PCR. 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 mean copy number of pCS20 was determined. The copy numbers ranged from 7x10 6 to 7x10 15 in A. lepidum with the highest (7x10 15 ) copy numbers in Abonama and the lowest (7x10 6 ) in Elhoush. In A. variegatum, the copy numbers ranged Please purchase PDFcamp Printer on to remove this watermark. XII from 1x10 7 to 4x10 11 with the highest (4x10 11 ) copy number in Kass, followed by (4x10 9 ) in Aidelfersan and the lowest (1x10 7 ) was in Nyala. Blood samples from cattle, sheep and goats were tested by qPCR for detection of E. ruminantium in Singa, Tamboul and Nyala. Four out of 50 (8%), 3 out of 35 (8.6%) and 5 out of 35 (14.3%) blood samples from cattle, sheep and goats, respectively were positive. In vivo isolation of three E. ruminantium isolates (Dinder, Abonama and Nyala) was carried out using Nubian goats reared in Amblyomma free zone north of Khartoum. Dinder and Abonama stocks were isolated by inoculating A. lepidum homogenates in goats and Nyala stock isolated from A. variegatum were inoculated in a goat. Goats developed clinical signs of heartwater by 6 to 12 days. Estimation of copy numbers of E. ruminantium infection in Amblyomma tick homogenates and blood stabilates of experimentally infected goats during febrile periods were established using qPCR. Nyala stock showed hyperacute form of heartwater. The load of pCS20 copies number in the infective dose was (5 x 10 4 ) and the animal died on day 9 post infection while the load in Dinder stock was (1 x 10 4 ) and underwent an acute form and the animal died on day 14. Abonama stock showed a mild form of the disease and the animal survived although the load was the highest (2 x 10 5 ). The copies number of pCS20 in the blood stabilates of goats inoculated with tick homogenates was, also, estimated during the febrile periods in each stock. The copies number of pCS20 ranged from 2x10 8 to 1x10 11 copies/µl and the highest (1x10 11 ) copies number was in Nyala stock coincided with the highest body temperature (42.2°C) followed by (1x10 10 ) in Abonama stock and the lowest (2x10 8 ) copies number was reported in Dinder stock. It conclude that the study defined the spatial distribution and prevalence of E. ruminantium in the vector population and domestic ruminant hosts and provided an accurate situation of the potential risk of heartwater that pose livestock in the endemic regions in the Sudan. The qPCR applied for E. ruminantium is species specific and sensitive to as few as 5 gene copies/µl. The assay was found to be Please purchase PDFcamp Printer on to remove this watermark. XIII more sensitive than the standard pCS20 PCR assay and nested MAP1 PCR assay. The successful in vivo isolation of E. ruminantium new stocks (Dinder, Abonama and Nyala) confirmed the high infection rate of ticks and wide distribution of the disease in different localities in the Sudan. This study has opened the way for a complete investigation of the epidemiology of heartwater in the Sudan. The new technique contributes the knowledge of the diversity of E. ruminantium stocks and will facilitate vaccine studies to develop specific vaccines in problem areas.
Molecular Epidemiology,Heartwater,Domestic Ruminants,Sudan