Activities in 2009
Theileriosis
Dirk Geysen
Address of laboratory ITM, Nationalestraat 155
Address continued 2000 Antwerp, Belgium
Tel.: (+32 [0]3) 247.62.64, Fax: (+32 [0]3) 247.62.68
e-mail address: dgeysen@itg.be, website: www.itg.be
Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease at your laboratory
Test
For
Specificity
Total
ELISA
Antibody
Group
235
SELISA
Antibody
Group
0
IFAT
Antibody
Group
0
PCR
Antigen
18S
196
RT-PCR
Antigen
Cox III
388
Production and distribution of diagnostic reagents
Theileria parva antigen for IFAT and SELISA tests for internal use and to Congo.
Theileria parva recombinant PIM protein for ELISA tests for research purposes.
Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
Development of a programme to standardise methods for diagnosing Theilerias by accreditation of a newly
develop RT-PCR test and training staff at the molecular lab of the Dpt of Tropical Diseases, Pretoria University,
Onderstepoort, South Africa and at the Dept. of Microbiology and Parasitology of Sokoine University, Morogoro,
Tanzania.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
None.
5.
Research and development of new procedures for diagnosis and control
Development of a FRET based RT-PCR test enabling to differentiate the 6 commonly present Theileria species in
bovine animals with a sensitivity of 10-5 parasites. Further development of the FRET based RT-PCR test
incorporating differential diagnosis of Theileria sp. buffalo for buffalo samples. This will contribute to a better
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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Theileriosis
knowledge of the host range of the different Theilerias and enabling to collect epidemic data from cattle and
buffalo in a more directed way.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
Characterisation of recombinant profiles between cattle-derived and buffalo-derived Theileria parva parasites in
South Africa and Rwanda. These results question the hypothesis of re-introduction of classical Theileria parva
into South Africa through carrier buffalos.
7.
Provision of consultant expertise to OIE or to OIE Members
None.
8.
Provision of scientific and technical training to personnel from other OIE Members
Two students received training in RT-PCR, 1 MSc student from Tanzania and 1 PhD student from South Africa.
9.
Provision of diagnostic testing facilities to other OIE Members
One time to New Zealand.
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
11. Participation in international scientific collaborative studies
1. Validation of reverse line blot results against RT-PCR results of buffalo-derived Theileria spp in South Africa
(Sibeko et al. 2008). Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled
disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by
the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time
polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to
improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide
primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of
control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in
addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other
Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the
T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive
than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in
carrier animals with a piroplasm parasitaemia as low as 10 -5 %.
2. Molecular characterisation of T. parva strains isolated from buffalo and cattle in South Africa (Sibeko et al.
2009).
Previous studies characterizing the Theileria parva p67 gene in East Africa revealed two genotypes. Cattle-derived
isolates associated with East Coast fever (ECF) have a 129 bp deletion in the central region of the p67 gene (allele
1), compared to buffalo derived isolates with no deletion (allele 2). In South Africa, Corridor disease outbreaks
occur if there is contact between infected buffalo and susceptible cattle in the presence of vector ticks. Although
ECF was introduced into South Africa in the early 20th century, it has been eradicated and it is thought that there
has been no cattle to cattle transmission of T. parva since. The variable region of the p67 gene was amplified and
the gene sequences analyzed to characterize South African T. parva parasites that occur in buffalo, in cattle from
farms where Corridor disease outbreaks were diagnosed and in experimentally infected cattle. Four p67 genotypes
were identified, including alleles 1 and 2 previously detected in East African cattle and buffalo, respectively, as
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Theileriosis
well as two novel genotypes, one with a different 174 bp deletion (allele 3), the other with a similar sequence to
allele 3 but with no deletion (allele 4). Sequence variants of allele 1 were obtained from field samples originating
from both cattle and buffalo. Allele 1 was also obtained from a bovine that tested T. parva positive from a farm
near Ladysmith in the KwaZulu-Natal Province. East Coast fever was not diagnosed on this farm, but the p67
sequence was identical to that of T. parva Muguga, an isolate that causes ECF in Kenya. Variants of allele 2 were
obtained from all T. parva samples from both buffalo and cattle, except Lad 10 and Zam 5. Phylogenetic analysis
revealed that alleles 3 and 4 are monophyletic and diverged early from the other genotypes. These novel genotypes
were not identified from South African field samples collected from cattle; however allele 3, with a p67 sequence
identical to those obtained in South African field samples from buffalo, was obtained from a Zambian field isolate
of a naturally infected bovine diagnosed with ECF. The p67 genetic profiles appear to be more complex than
previously thought and cannot be used to distinguish between cattle- and buffalo-derived T. parva isolates in
South Africa. The significance of the different p67 alleles, particularly the novel variants, in the epidemiology of
theileriosis in South Africa still needs to be determined.
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)
 Presentations at international conferences and meetings
None so far
 Scientific publications in peer-reviewed journals
None so far
 Other communications
None so far
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
No
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
No
_______________
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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