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 hebraeum. The protocols currently used to detect heartwater
take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to
detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E.
ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A.,
Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E.,
Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A.,
2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy
number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20
PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood
from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the
most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis
and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and
pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile
reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus,
because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h
it is an effective assay for epidemiological surveillance and monitoring of infected animals.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:tut/oai:encore.tut.ac.za:d1000379 |
| Date | 10 April 2008 |
| Creators | Steyn, HC, Pretorius, A, McCrindle, CME |
| Publisher | Elsevier |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | |
| Rights | Elsevier |
| Relation | Veterinary Microbiology |
Page generated in 0.0019 seconds