A quantitative real-time PCR

assay for Ehrlichia ruminantium

using pCS20

A quantitative realA quantitative real--time PCR time PCR

assay for Ehrlichia ruminantiumassay for Ehrlichia ruminantium

using pCS20using pCS20

Helena Steyn

Onderstepoort Veterinary Institute,

South Africa

Introduction• Ehrlichia ruminantium is the causative

agent of heartwater

• Obligate intracellular parasite that infects endothelial cells of the host

E. ruminantium colonies in endothelial cells

E. ruminantium organisms

Cell nucleus

Heartwater infects

livestock and game

Amblyomma hebraeum tick

Male Female

Distribution of heartwater

Endemic areas of heartwater

in South Africa

Clinical symptoms of heartwater

Leg stiffness and hanging head

Post mortem signs

pCS20 probe assay

• Fragment contains two overlapping genes:

– rnc and ctaG

Welgevonden genome sequence (Collins 2005) et al.,

rnc ctaG

HH1F HH2Rprobe (AB128-129)

279 bp

Aims of study

• pCS20 real-time PCR TaqMan probe method to detect the presence of E. ruminantium in blood and ticks

• Quantify parasites in cell cultures and experimentally infected sheep and ticks

• Identify and characterise new genotypes of Ehrlichia ruminantium in South Africa

• Development of Heartwater vaccine

Real-time PCR TaqMan Probe

ProbeForward Reverse

29 bp22 bp 18 bp

Plasmid standard curve

Comparison of the sensitivity of three pCS20

assays using Welgevonden infected cell

culture material

Dilution Copy Number

(copies/µl) PCR

PCR/32

P-

probe TaqMan

10-1

7 x 107 + + +

10-2

7 x 106 + + +

10-3

7 x 105 + + +

10-4

7 x 104 + + +

10-5

7 x 103 + + +

10-6

7 x 102 + + +

10-7

7 x 101 - + +

10-8

7 x 100 - - +

Specificity of pCS20 assays

using genomic DNA

Samples PCR 32P-probe TaqMan

Probe

Copy Number

(copies/µl)

Welgevonden + + + 7 x 108

Mara87/7 + + + 1 x 108

Vosloo + + + 3 x 106

Blaauwkrans + + + 1 x 109

Kwanyanga + + + 4 x 108

Ball3 + + + 4 x 106

E. canis + + + 1 x 104

E. chaffeensis + + + 2 x 103

T. parva - - - 3 x 106

A. centrale - - - 4 x 106

Detection of pCS20 in

experimentally infected sheep

Day PCR PCR/32P-

probe

Real-time

PCR

0-10 - - -

11 - 5+/5 -

12 - 5+/5 5+/5

13 5+/5 5+/5 5+/5

14 5+/5 5+/5 5+/5

15 n/d n/d n/d

Parasites determined in

experimentally infected sheep

Sheep number Average parasite copies per ml blood

861 1 x 109

862 2 x 109

869 4 x 108

870 7 x 108

871 4 x108

Conclusions

�The pCS20 real-time PCR was more sensitive than the standard PCR and 32P-probe when applied to cell

culture and tick samples.

�A faster diagnosis can be made within 2 hours instead

of 5 days.

� It is also potentially less expensive to use on a routine basis since it is less time consuming.

�DNA in the samples could be quantified, making it

suitable for diagnostic and epidemiological studies.

Acknowledgements

• ARC-OVI:

– Dr A Pretorius

– Dr M van Kleef

– Dr E Zweygarth

– Mrs A Josemans

– Ms E Faber

• Funding:

– European Union

– Red meat Research and Development Trust, SA

• Other:

– Prof C McCrindle,

University of Pretoria

– Prof C Steinmann,

Tshwane University of Technology,

Pretoria

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