T.congolense IAEA/ELISA kits

The average OD values and %P of the upper control limit (UCL) and lower control limit (LCL) of the control sera showed in Table I. If the %P decision value was double the mean %P of the pool negative control serum, the %P of the positive sample was equal or over 60 (30 x 2). The percent of positive samples were 78.9% (576/730) in group 1 and 98.3% (118/120) in group 2. Nevertheless 81 samples (67.5%) of group 2 had the OD values more than 2.0.

3.3. T.evansi NIAH/ELISA

The average %P of PBS, the negative control serum and the positive control serum were 20.6 (13–28), 38.0 (28–58) and 100 respectively. If the %P decision value was double the mean %P of the disease free group, the %P of the positive sample was equal or over 76 (38.0 x 2). Percent of positive sample was 99.2% (119/120) in group 1 and 79.3% (579/730) in group 2. Six positive samples by blood smear test were also positive by the IAEA-ELISA and the NIAH/ELISA. The range %P in the IAEA/ELISA and NIAH/ELISA were 90–200% and 97-164% respectively.

4. DISCUSSION

Since Luckins [9]; developed an indirect ELISA for the diagnosis of Trypanosomosis in cattle, various sensitive and specific ELISA techniques had been reported. Recently a simple ELISA test kit is required but T.congolense IAEA/ELISA kit was not. In the present study the sensitivity of T.congolense IAEA/ELISA kit and T.evansi NIAH/ELISA for the detection of T.evansi antibodies was 100% (6/6) because six positive samples by blood smear test were positive by both ELISA techniques (Table II). The percentage of the positive sample in group 2 was high (79.3%) using T.evansi NIAH/ELISA, though T.evansi was not detected on blood smear and the cattle showed no signs. It was suspected that the cattle on group 2 were infected with T.evansi. Because the samples were collected from January to May after high population of Tabanus sp. and other blood suckling flies [10].

The specificity of the NIAH/ELISA test required more cattle sera samples from a T.evansi free zone. Antibody cross-reaction between cattle infected with T.evansi and other blood parasites using NIAH/ELISA have to be studied also. About 97% of the positive and negative samples obtained from T.congolense IAEA/ELISA test matched to the result obtained from NIAH/ELISA test (Table III). It is understood that specificity studies using this kit have been made and that no cross reactions occur with antibodies against problem organisms (Babesia sp., Anaplasma sp. and Theileria).

In addition to this study, ten microplates of IAEA/ELISA kits, coated with denatured T.vivax were tested. The maximum and minimum OD values of ULC and LCL of the C++ were 0.652-0.150 and 0.627-0.138 where the recommended UCL-LCL was 1.568-0.688. Thus test sera could not be evaluated.

In conclusion the T.congolense IAEA/ELISA and T.evansi NIAH/ELISA are suitable for the diagnosis of surra in cattle in Thailand. The use of denatured antigens in the IAEA kit has been shown to bed suitable for diagnosis of Trypanosomes in general and this confirms the use for T.evansi in Thailand.

TABLE I. COMPARISON THE%P OF UCL AND LCL BETWEEN THE CONTROL SERA OBTAINED FROM T.CONGOLENSE IAEA/ELISA KIT AND IAEA REFERENCE VALUES

Present study IAEA reference Control sera

UCL LCL UCL LCL

C++ (OD values) 1.312-0.827^a 1.255-0.792^b 1.845 0.508

C++ (%P) 104 96 103 97

C+ (%P) 63 58 54 35

C- (%P) 23 19 17 5

Cc (%P) 5 4 4 0

a = max. and min. OD values of the UCL on 18 plates

b = max.and min. OD values of the LCL on 18 plates

TABLE II. COMPARISON OF THE SENSITIVITY OF T.CONGOLENSE IAEA/ELISA AND T.EVANSI NIAH/ELISA FOR THE DETECTION OF T.EVANSI ANTIBODIES IN DAIRY CATTLE

OD value (%P)

Sample No. ^c IAEA NIAH

1 >2 0.745 (97%)

2 1.375 (90%) 0.822 (108%)

3 >2 1.075 (148%)

4 >2 0.999 (138%)

5 >2 1.054 (159%)

6 >2 1.089 (164%)

c = Serum sample was surra positive by blood smear test.

TABLE III. PERCENT POSITIVE SAMPLE BY IAEA/ELISA AND NIAH/ELISA

Diagnostic test %positive samples

IAEA/ELISA (T. congolense) Group 1 98.3 (576/730) ^d Group 2 78.9 (118/120) NIAH/ELISA (T.evansi) Group 1 99.2 (579/730) Group 2 79.3 (119/120)

d = number of reacted sample/total examined sample

REFERENCES

[1] TUNTASUVAN, D., LUCKINS, A.G. Status of surra in livestock in Thailand. J.

Protozool. Res., 8: 162–170 (1998).

[2] TEERAPRASERT, E., CHAICHANAPUNPOOL, I., TRONGWONGSA, L., SAKDASIRISATHAPORN, A., WONGKASEMCHIT, S. A case report of Trypanosoma evansi infection in pigs. Proc. 11th Annual Conf. Thai Vet. Med.

Assoc., Thailand. p. 53–64 (1984).

[3] LOHR, K.F., PHOLPARK, S., UPATUM, N., LEIDL, K., STAAK, C.

Trypanosoma evansi infection, a frequent cause of abortion in buffaloes. Trop.

Anim. Hlth. Prod., 18: 103–108 (1986).

[4] TUNTASUVAN, D., SARATAPHAN, N. AND NISHIKAWA, H. Cerebral Trypanosomiasis. Vet. Parasitol., 73: 357–363 (1997).

[5] TUNTASUVAN, D., MIMAPHAN, S., SARATAPHAN, N., TRONGWONGSA, L., INTRARAKSA, R., LUCKINS, A.G. Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine-biotin immunohistochemistry. Vet. Parasitol., 87: 223–230 (2000).

[6] TUNTASUVAN, D., CHOMPOOCHAN, T., WONGPAKORN, M.,

MOHKAEW, K. Development on serodiagnosis of Trypanosomosis in pigs by enzyme linked immunosorbent assay. J. Thai Vet. Med. Assoc., 47(2): 45–53 (1996).

[7] TUNTASUVAN, D., JARABRUM, W., VISESHAKUL, N, MOHKAEW, K., BORISUTSUWAN, S., THEERAPHAN, A., KONGKANJANA, N.

Chemotherapy of surra in horses and mules with diminazene aceturate. Vet.

Parasitol., 110 (3-4): 227–233 (2003).

[8] JOINT FAO/IAEA PROGRAMME TRYPANOSOMOSIS: Indirect Enzyme Immunoassay for Detection of Bovine Serum Antibody to Trypanosoma congolense. Animal Production Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria, 48 pages (2000).

[9] LUCKINS, A.G. Detection of antibodies in Trypanosome infected cattle by means of a microplate enzyme-linked immunosorbent assay. Trop. Anim. Hlth.

Prod., 9: 53–62 (1977).

[10] ITO, Y., BONNCHIT, S., SARATAPHAN, N. AND TUNTASUVAN, D.

Species composition and seasonal abundance of horseflies (Diptera: Tabanidae) on a cattle farm in Pathum Thani. J. Thai Vet. Med. Assoc., 50: (1-2): 17–28 (1999).

MOLECULAR DIAGNOSIS OF TRYPANOSOME SPECIES G. VILJOEN, J.M. ROMITO

Biotechnology Division, Onderstepoort Veterinary Institute, Onderstepoort, South Africa

Abstract

Various parameters for handling samples, extracting nucleic acid and protocols for use of PCR to diagnose trypanosomes have been examined. The use of a commercial kit (Nucleon BACC 2, Amersham) was superior to other extraction methods. After comparisons, a touch-down thermocycling protocol was the procedure used. PCR testing protocols were successfully implemented using Kin primers that bind to an internal transcribed spacer region (ITS1) situated between the 18S and the 5.8S ribosomal subunit genes on nuclear DNA. Their sensitivity was lower than that of satellite DNA primers, particularly for T.vivax. A Kin primer-based PCR could detect and distinguish between a number of Trypanosome species in blood samples. The use of an ITS1 binding primer was 3–5 x cheaper than using classical species-specific primers since the number of PCR reactions per sample is reduced to one.

1. INTRODUCTION