Detection of various Trypanosome species

All the PCR reactions described made use of the Kin primers which bind to the internal transcribed spacer region ITS1. We successfully implemented PCR testing protocols, using the Kin primers described in [14]. These primers bind to an internal transcribed spacer region (ITS1) situated between the 18S and the 5.8S ribosomal subunit genes on nuclear DNA [13].

The length of ITS1 is between 300 and 800 bp which varies between Kinetoplastida species, but is presumed to be constant within a species. Taxa, which can be distinguished by size include: T.vivax, T.theileri, T.simiae, T.congolense savannah, T.congolense forest, T.congolense Kilifi). In the case of the Trypanozoon species (T.equiperdum; T.brucei; T.evansi) species-specific differentiation is not possible [16].

The sensitivity of the Kin primers is regarded to be low for detecting T.vivax because of only a 75 to 90% sequence homology to the forward and reverse Kin primers [14]. The predicted amplicon sizes [14]for various Trypanosome species are tabulated as follows:

TABLE XIII. PREDICTED AMPLICON SIZES

Species Amplicon size

T.vivax 305 bp

T. simiae 435 bp

T.theileri 455 bp

Trypanozoon spp. 540 bp

T. brucei 540 bp

T.evansi 540 bp

T.equiperdum 540 bp

T.congolense Kenya 680 bp

T.congolense savannah 750 bp

T.congolense forest 780 bp

Samples of various species gave amplicon sizes in the expected ranges (Figs 4, 5 and 6) with the exception of several specimens that didn't always yield any results or gave unexpected sized products. The former poor test repeatability was probably due to operator inexperience as several students were being trained.

T.vivax has a predicted size of 305 bp, but amplicons seen were invariably larger in size (Fig. 4, lane 40; Fig. 5, lanes 7, 15; Fig. 6, lane 14) and comparatively weaker in strength.

Similarly, the T.theileri samples either failed to yield products of the expected size and/or were barely visible (Fig. 4: lane 3; Fig. 5: lane 4, 24; Fig. 6: lane 11). T.

lewesi-containing blood specimens, gave relatively weak products usually in the range expected for T.theileri (455 bp). T.theileri is regarded as a usually apathogenic organism of cattle. T. lewesi is a rodent Trypanosome. Samples containing T.congolense forest and savannah gave amplicons in the region expected for each viz. 780 bp and 750 bp respectively (Fig. 4: lanes 21, 39, 41; Fig. 5: lane 5; Fig. 6: lane 17).

More effective size determination with suitable control samples would be required to give greater certainty.

Three samples gave products in the size expected for T.congolense Kenya (680 bp) cf Fig. 7, lane Kenyan samples 1, 2, 10. Samples containing all T.brucei subspecies yielded amplicons of the expected size (540 bp) cf Fig. 1 lane 10, 12, 13, 15, 16 etc. Kin primers cannot distinguish Trypanozoon species (T. brucei, T.evansi, T.equiperdum) since all yield product sizes of 540 bp.

Species-specific primers are available for the latter two species and would be required for identification.

FIG. 4. Agarose gel (1) showing PCR amplicons from various Trypanosome-infected blood specimens.

Mr: 100 bp molecular size marker. T. lewisi: Lanes 1, 13, 27, 37; T.brucei gambiense:

Lanes 12; T.brucei rhodesiense: Lanes 2, 15, 24, 34; T.brucei brucei: Lanes 7, 14, 16, 17, 19, 20, 28, 38; T.theileri: Lane 3; T. simiae: Lanes: 5, 23, 33, 26, 36; T. congolense: 8, 9, 18, 21, 29, 31, 39, 41; T.vivax: Lane 30, 40, Water controls: Lanes: 10, 22, 32, 42.

FIG. 5. Agarose gel (2) showing PCR amplicons from various Trypanosome-infected blood specimens.

Mr: 100 bp molecular size marker. T. lewisi: Lanes 2, 14, 22; T.brucei gambiense: Lanes 1, 11; T.brucei rhodesiense: Lanes 8, 13, 23; T.brucei brucei: Lanes 3, 16, 21, 25, 26, 27;

T.theileri: Lane 4, 24; T. simiae: Lanes 6, 17; T. congolense: Lanes 5, 9, 12, 18, 19, 29;

T.vivax: Lane: 7, 15, 28. Water controls: Lane: 10, 20, 30.

Fig. 6. Agarose gel) showing PCR amplicons from various Trypanosome-infected blood specimens.

Mr: 100 bp molecular size marker. T. lewisi: Lanes 9; T.brucei rhodesiense: Lanes 13;

T.brucei brucei: Lanes 10, 15, 16; T.theileri: Lane 11; T. congolense: Lane 17; T.vivax:

Lane: 14. Water controls: Lane: 8, 18.

Blood samples derived from roan antelope were also tested using PCR. These had been shown to be positive serologically using CATT. The PCR products obtained (Fig. 7) suggested presence of Trypanozoon species (540 bp), although a previous gel had also given products suggestive of T.theileri (450 bp). The latter is a stercarian tabanid-transmitted parasite that occurs widely in cattle and is usually regarded as apathogenic.

FIG. 7. Agarose gel showing PCR amplicons obtained from roan antelope.

Blood samples (1 to 16) that were all CATT positive. Kenyan samples 1 to 10 were confirmed Trypanosome positive samples supplied by KETRI. Lane 1 and 2: T congolense savannah. Lane 3 supposedly T.vivax, but not according to gel. Samples 9 and 10 were controls viz. T.congolense savannah (750 bp) and T.congolense Kenya coast (680 bp).

3.5. ICATT

Sera from camels and horses from Mongolia were evaluated at OVI for presence of antibodies to Trypanosoma using ICATT. The basis for selecting these serum samples was not indicated. In total 96% of horse sera and 37% camel sera tested positive against T.evansi variable surface antigen using CATT. It is known that antibodies to other salivarian Trypanosomes can also react to such antigens.

TABLE XIV. RESULTS ANALYSING SERA FROM CAMELS AND HORSES USING ICATT

Five of these CAT positive sera samples were also tested using PCR. The resultant amplicons from two samples (Fig. 8), although of strong intensity, were smaller than any predicted sizes. Faint bands of a higher size are, however, visible in sample 2. It is known that a recent dourine outbreak occurred in China, Russia and Ethiopia [15]. Desquesnes states that no laboratory has a recent isolate definitely identified as T.equiperdum. Also many older isolates originally classified as T.equiperdum were confused with T.evansi. These aspects should be kept in consideration should these samples undergo further evaluation.

FIG. 8. Agarose gel showing PCR amplicons from camel and horse sera testing.

CATT positive Mr: molecular size marker; Lane 6 T.congolense control; Lane 7 water control.

4. FUTURE

We have made contact with researchers in several African countries e.g. Kenya, Burkina Fasso, Nigeria with whom further collaboration can now take place. This will include being a diagnostic referral centre for testing blood samples for presence of Trypanosomes. Other Trypanosome-binding primers will still be evaluated. An additional PCR method according to Dávila (pers comm), using other primers and thermocycling parameters will still be examined. In addition, species-specific primers will be used in selected cases viz. those described in [8] T.congolense savannah TCS1 and TCS2; T.congolense forest: TCF1 and TCF2; T.congolense Kenya Coast YCK1 and TCK2; T.vivax: TV [9].

Positive Negative Uncertain Total Horse 23

96%

1 4%

0 24 Camel 22

37%

32 53%

6 10%

60

REFERENCES

[1] Office International des Epizootique (OIE) World Organisation for Animal Health. Manual of Standards for Diagnostic Tests and Vaccines, 4th edition.

OIE, Paris, Pages 855–862 (2000).

[2] VAN DER BOSSCHE, P. The control of Glossina morsitans morsitans (Diptera:

Glossinidae) in a settled area in Petauke District (Eastern Province, Zambia) using odour-baited targets. Onderstepoort Journal of Veterinary Research, 64:

251–257 (1997).

[3] CONNOR, R.J. African Trypanosomiases. In: Infectious diseases of livestock with special reference to Southern Africa, edited by Coetzer J.A.W, Thomson, G. and Tustin R.C. Cape Town, Oxford University Press, 1: 167–205.

[4] KAPPMEIER, K., NEVILL, E.M., BAGNALL, R.J. Review of tsetse flies and Trypanosomes in South Africa. Onderstepoort Journal of Veterinary Research, 65: 195–203 (1998).

[5] MAJIWA, P.A.O., THATTHI, R., MOLOO, S.K., NYEKO, J.P.H., OTIENO, L.H., MALOO, S. Detection of Trypanosome infection in the saliva of tsetse flies and buffy coat samples from antigenaemic but aparasitaemic cattle.

Parasitology, 108: 313–322 (1994).

[6] KATAKURA, K., LUBINGA, C., CHITAMBO, H., TADA, Y (1997).

Detection of Trypanasoma congolense and T.brucei subspecies in cattle in Zambia by PCR from blood collected on filter paper. Parasitol. Res. 83: 241–

245.

[7] SOLANO, P., MICHEL, J.F., LEFRANCOIS, T., DE LA ROCQUE, S., SIDIBE, I., ZOUNGRANA, A., CUISANCE, D. Polymerase chain reaction as a tool for detecting Trypanosomes in naturally infected cattle in Burkina Faso.

Vet. Parasitology, 86: 95–103 (1999).

[8] MASIGA, D.K., SMYTH, A.J., HAYES, P., BROMIDGE, T.J., GIBSON, W.C.

Sensitive detection of Trypanosomes in tsetse flies by DNA amplification. Int. J.

Parasitol., 22: 909–918 (1992).

[9] CLAUSEN, P.H., WIEMANN, A., PATZELT, R., KAKAIRE, D., OESTZCH, C., PEREGRINE, A., MEHLITZ, D. Use of a PCR assay for the specific and sensitive detection of Trypanosoma Spp. in naturally infected dairy cattle in peri-urban Kampala, Uganda. Ann. N.Y. Acad. Sci., 849: 21–31 (1998).

[10] DESQUESNES, M., DÁVILA, A.M.R. Applications of PCR-based tools for detection and identification of animal Trypanosomes: a review and perspectives.

Veterinary Parasitology 2429: 1–19 (2002).

[11] CHOMCZYNSKI, P., SACCHI, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162: 156–159 (1987).

[12] ROMERO, C., PARDO, M., GRILLO, M.J., DIAZ, R., BLASCO, J.M., LOPEZ, I. Specific detection of Brucella DNA by PCR. J. Clinical Microbiology, 33 (12): 3198–3200 (1995).

[13] MCLAUGHLIN, G.L., SSENYONGA, S.S., NANTEZ, E., RUBAIRE-AKIKI., WAFUKLA, O., HANSEN, R.D., VODKIN, M.H., NOVAK, R.J., GORDON, V.R., MONTENEGRO-JAMES, S., JAMES ,M., AVILES, H., ARMIJOS, R., SANTRICH, C., WEIGLE, K., SARAVIA, N., WOZNIAK, E., GAYE, O., MDACHI, R., SHAPIRO, S.Z., CHANG, K.P., KAKOMA, I. PCR-based detection and typing of parasites. In: Azcel MA, Alkan MZ (Eds), Parasitology for the 21st Century. CAB International, Wallingford, Oxon UK, Chapter 25, 261–287 (1996).

[14] DESQUESNES, M., MCLAUGHLIN, G., ZOUNGRANA, A., DÁVILA, A.M.R. Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA. Int. J.

Parasitol., 31: 610–614 (2001).

[15] SOLANO, P., MICHEL, J.F., LEFRANCOIS, T., DELA ROCQUE, S., SIDIBE, I., ZOUNGRANA, A., CUISANCE, D. PCR as a diagnosis tool for detecting Trypanosomes on naturally infected cattle in Burkina Faso. Vet.

Parasitol. 86: 95–103 (1999).

[16] DESQUESNES, M AND DÁVILA A.M.R. Applications of PCR-based tools for detection and identification of animal Trypanosomes: a review and perspectives.

Veterinary Parasitology 2429: 1–19 (2002).

USING PCR FOR UNRAVELING THE CRYPTIC EPIZOOTIOLOGY