Polymerase Chain Reaction (PCR) using T.b.rhodesiense specific primers PCR assay using primers(SRA-R 5' ATA GTG ACA AGA TGC GTA CTC

AAC GC ^3' and SRA-F ^5' AAT GTG TTC GAG TAC TTC GGT CAC GCT ^3') specific for T.b.rhodesiense serum resistance associated (SRA) gene was conducted to identify T.b.rhodesiense as described by [16]. PCR amplification was performed using 50 ng of DNA extracted from purified T.b.rhodesiense Trypanosomes as control DNA, or 10 μL of DNA extracted from domestic animal blood. The DNA amplifications was carried out in 25 μL reaction mixture containing final concentrations of 20 mM Tris-HCl, pH 8.7, 100 mM KCl, 50 mM (NH4)2SO4, 1.5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, 0.5 μM each primers and 2.5 units of HotStar Taq DNA polymerase.

SRA-PCR was performed using a GeneAmp PCR System 9700 from Applied

Biosystems with the initial incubation for 15 minutes at 95^oC, followed by 45 cycles involving denaturation for 1 min at 94^oC, annealing at 68^oC for 1 min, extension at 72^oC for 1 min; and a final extension for 10 min at 72^oC. The absence of contaminants was routinely checked by inclusion of negative control samples in which the DNA sample was replaced with sterile water. All samples were subjected to an initial PCR, followed by a second PCR using 1ul of the first PCR product in order to increase sensitivity.

Twenty microlitres of each sample were electrophoresed in a 2% agarose containing 1 μg/mL ethidium bromide. The amplified products were observed by UV transillumination. Similarly, PCR assay using primers designed by [17]; for T.b.rhodesiense serum resistance associated (SRA) gene was performed using the same T.b.rhodesiense isolates in order to compare the results with those obtained in SRA-PCR designed in [16].

3. RESULTS

The human serum resistance associated (SRA) gene was identified in 28 (80%) of the 35 T.b.rhodesiense Trypanosomes from parasitologically confirmed sleeping sickness cases, using the primers designed in [16] and in 27 (77.2%) of the same 35 T.b.rhodesiense Trypanosomes using the primers designed by Gibson (Table I; Fig. 1).

In the Serere area, 4 of the 11 T.b.rhodesiense isolates were SRA-PCR negative using primers [16]; and three of the same isolates were again SRA-PCR negative using [17];

primers (Table I). Similarly in Tororo area, 2 of the 22 T.b.rhodesiense isolates were SRA-PCR negative using [16]; primers and four including the two of the same isolates were again SRA-PCR negative using Gibson [17]; primers (Table I). However, about 20% of the 35 T.b.rhodesiense Trypanosomes could not be detected by SRA-PCR even when an aliquot of the first PCR (Table III) was used in the second PCR, indicating that the SRA gene may be absent in those Trypanosomes or the Trypanosomes could be having another variant of SRA not detectable by these primers since three variants of SRA genes have so far been identified or the amount of Trypanosomal DNA extracted from infected blood was too low to be detected. Since SRA genes resemble variable surface glycoprotein (VSG) genes, it may be that these isolates which are SRA-gene negative are indicative of some T.b.rhodesiense Trypanosomes with modified or lack SRA genes. The number of T.b.rhodesiense isolates which are SRA gene negative are too many to be accounted for as miss identification, but could be explained by simple loss of the SRA gene from the expression site in which it resides during antigenic variation.

Analysis of Trypanosomes derived from domestic animals showed that, 79 (90.8%) of the 87 Trypanosomes isolated from cattle (Table II) were positive by TBR-PCR, indicating that they are Trypanozoon while 8 (9.2%) were negative, suggesting that they could be T.vivax, T.congolense or T.theileri. When subjected to SRA-PCR, 10 (11.5%) of the 87 Trypanosomes isolates derived from cattle were positive, indicating that they could be T.b.rhodesiense circulating in cattle which is similar to the percentage of T.b.rhodesiense previously obtained in cattle in Serere, Soroti district [18]. Some of the SRA-PCR negatives could as well be T.b.rhodesiense since this technique appears to miss some of the parasitologically confirmed cases of T.b.rhodesiense sleeping sickness which could be due to modified SRA genes or loss of the SRA genes from the expression sites in which they reside during the gene rearrangements associated with antigenic variation.

TABLE I. RESULTS OF PCR AMPLIFICATION OF TRYPANOSOMAL DNA ISOLATED FROM INFECTED HUMANS FROM THREE DIFFERENT AREAS

Primers used in PCR

Origin Host No. of

isolates examined

TBR 1 and 2 + -

SRA(Radwanska) + -

SRA (Gibson) + - Serere Humans 11 11 0 7 4 8 3 Tororo Humans 22 22 0 20 2 18 4 Kaberamaido Humans 2 2 0 1 1 1 1 Total (%) 35 35 (100) 0 (0) 28 (80.0) 7 (20.0) 27(77.2) 8

(22.8)

TABLE II. RESULTS OF PCR AMPLIFICATION OF TRYPANOSOMAL DNA ISOLATED FROM INFECTED DOMESTIC ANIMALS FROM FOUR DIFFERENT AREAS

Primers used in PCR

FIG. 1. Agarose gel electrophoresis showing results obtained SRA-PCR (Gibson et al.2002, primers).

1 = Marker; 2 = negative control; 3 = positive control SRA T.b.rho; 4 = positive SRA T.brucei from domestic animal; 5-12 = negative T.brucei from domestic animals

460 bp

TABLE III. MOLECULAR DIFFERENTIATION OF T.B.RHODESIENSE TRYPANOSOMES FROM INFECTED CATTLE BLOOD SAMPLES FROM KABERAMAIDO, NEW FOCUS OF SLEEPING SICKNESS

Radwanska [16]; analysed 25 different T.b.rhodesiense strains from Uganda, Kenya and Rwanda and found out that 24 strains were SRA-PCR positive, but one strain STIB 884 was negative and was thought as a miss identification of the strain. In the present study 8 (20.0%) of the 35 T.b.rhodesiense from parasitologically confirmed sleeping sickness cases were SRA-PCR negative, indicating that there may be more than one factor involved in resistance to lysis by normal human serum (NHS).

Furthermore, [16]; he also showed that strain TREU 927/4 used as the reference T.brucei strain for the Trypanosome genome sequencing project (TIGR database) was both SRA gene and SRA transcript negative, but resistant to lysis by NHS, again

indicating that resistance to NHS may be conferred by other factors not yet identified besides the SRA gene. Similar analysis using the same 35 T.b.rhodesiense strains and primers developed by Gibson [17]; showed that 7 (22.8%) were SRA-PCR negative (Table I). Since SRA gene resembles Variable Surface Glycoprotein (VSG) gene, it may be these T.b.rhodesiense isolates, which are SRA-gene negative, are true T.b.rhodesiense with modified SRA gene or lack SRA gene. The number of T.b.rhodesiense isolates which are SRA gene negative are too many to be accounted for as miss identification of the isolates, but the SRA-PCR negativity could be explained by simple loss of the SRA gene from the expression site in which it resides in the Ugandan T.b.rhodesiense [13]; during the gene rearrangements involved in antigenic variation. It is intended that DNA hybridization will be done on some of the T.b.rhodesiense isolates that are SRA gene negatives to confirm their status.

Analysis of Trypanosomes derived from domestic animals showed that 79 (90.8%) of the 87 Trypanosomes isolated from cattle (Table II) were positive by TBR-PCR, indicating that they were Trypanozoon while 8 (9.2%) were negative, suggesting that they could be T.vivax, T.congolense or T.theileri. When subjected to SRA-PCR, 10 (11.5%) of the 87 Trypanosomes isolates derived from cattle were positive, indicating that they could be T.b.rhodesiense circulating in cattle which is similar to the percentage of T.b.rhodesiense previously obtained in cattle in Serere, Soroti district [18].

On the other hand, some of the SRA-PCR negatives could as well be T.b.rhodesiense since this technique appears to miss some of the parasitologically confirmed cases of T.b.rhodesiense sleeping sickness which could be due to modified SRA gene or loss of the SRA genes from the expression sites in which they reside during gene rearrangements associated with antigenic variation. Alternatively, SRA gene negative Trypanosomes isolates could represent a group truly T.b.brucei which are non pathogenic to humans; and is similar to what was found in [17]; where the SRA gene was absent from West African T.b.brucei.

ACKNOWLEDGEMENTS

Thanks to the Director of LIRI, Uganda, for logistic support and for allowing me to attend this meeting. I thank all district and field staff and local council members for assistance in the research activities.

This investigation was supported by the IAEA.

REFERENCES

[1] GIBSON, W.C., MARSHALL, T.F. DE C., GODFREY, D.G. Numerical analysis of enzyme polymorphism: a new approach to the epidemiology and taxonomy of Trypanosomes of the subgenus Trypanozoon. Advances in Parasitology, 18: 175–

246 (1980).

[2] MEHLITZ, D., ZILLMANN, U., SCOTT, C.M., GODFREY, D.G. Epidemiological Studies on the animal reservoir of Gambian sleeping sickness part III.

Characterization of Trypanozoon stocks by isoenzymes and sensitivity to human serum.Tropenmedizin und Parasitologie, 33: 113–118 (1982).

[3] MIHOK, S., OTIENO, L.H., DARJI N. Population genetics of Trypanosoma brucei and the epidemiology of human sleeping sickness in the Lambwe Valley, Kenya, Parasitology, 100: 219–233 (1990).

[4] ENYARU, J.C.K., ODIIT, M., GASHUMBA, J.K., CARASCO, J.F., RWENDEIRE, A.J.J. Characterization by isoenzyme electrophoresis of Trypanozoon stocks from sleeping sickness endemic areas of South East Uganda.

Bulletin of the World Health Organisation, 70 (5): 631– (1992).

[5] ENYARU, J.C.K., ALLIGHAM, R., BROMIDGE, T., KANMOGNE, G.D., CARASCO, J.F. The isolation and genetic heterogeneity in Trypanosoma brucei gambiense from North West Uganda. Acta Tropica 54: 31–39 (1993).

[6] HEISCH, R.B., MC MAHON, J.P., MANSON-BAHR, P.E.C. The isolation of Trypansosoma rhodesiense from a bushbuck. British Medical Journal, 2: 1203–1204 (1958).

[7] ONYANGO, R.J., VAN HOEVE, K., DE RAADT, P. The epidemiology of Trypanosoma rhodesiense sleeping sickness in Alego location, Central Nyanza, Kenya. I: Evidence that cattle may act as reservoir hosts of Trypanosomiasis infective to man. Transactions of the Royal Society of Tropical Medicine and Hygiene, 60: 175–182 (1966).

[8] HIDE, G., BUCHANAN, N., WELBURN, S.C., MAUDLIN, I., BARRY, J. D., TAIT, A. Trypanosoma brucei rhodesiense: characterization of stocks from Zambia, Kenya and Uganda using repetitive DNA probes. Experimental Parasitology, 72: 430–439 (1991).

[9] BITEAU, N., BRINGAUD, F., GIBSON, W., TRUC, P., BALTZ, T.

Characterization of Trypanosoma brucei isolates using genetic microsatellite and minisatellite markers. Molecular Biochemical Parasitology, 105: 185–201 (2000).

[10] MACLEOD, A., WELBURN S., MAUDLIN, I., TURNER, C.M.R., TAIT, A.

Evidence for multiple origins of human infectivity in Trypanosoma brucei revealed by minisatellite variant repeat mapping. Journal of Molecular Evolution, 52: 290–301 (2001).

[11] DE GREEF, C., IMBERECHTS, H., MATTHYSSONS, G., VAN MERIVENNE, N., HAMERS, R. A gene expressed only in serum resistant variants of Trypanosome brucei rhodesiense . Molecular Biochemical Parasitology, 36: 169–

176 (1998).

[12] DE GREEF, C., HAMERS, R. The serum resistance associated (SRA) gene of Trypanosome brucei rhodesiense encodes a VSG like protein. Molecular Biochemical Parasitology, 68: 277–284 (1994).

[13] XONG, H., VANHAMME, L., CHAMEKH, M., CHIMFWEMBE, C.E., VAN DEN ABBEELE, J., PAYS, A., VAN MEIRVENNE, N., HAMERS, R., DE BAETSELIER, P., PAYS, E. A VSG expression site-associated gene confers resistance to human serum in Trypanosome rhodesiense, Cell 95: 839–846 (1998).

[14] VANHAMME, L., PATURIAUX-HANOCQ, F., POELVOORDE, P.,NOLAN, D. P., LINS, L., VAN DEN ABBEELE, J., PAYS, A., TEBABI, P., VAN XONG, H., JACQUET, A., MOGUILEVSKY, N., DIEU, M., KANE, J.P., BAETSELIER, P.D., BRASSEUR, R., PAYS, E. Apolipoprotein L-1 is the Trypanosome lytic factor of human serum. Nature, 422: 83–67 (2003).

[15] PENCHENIER, L., DUMAS, V., GREBAULT, P., REIFENBERG, J.M., CUNY, G. Improvement of blood and fly gut processing for PCR diagnosis of Trypanosomiasis. Parasite 4: 387–389 (1996).

[16] RADWANSKA, M., CHAMEKH, M., VANHAMME, L., CLAES, F., MAGEZ, S., MAGNUS, E., BAETSELIER, P., BUSCHER, P., PAYS, E. The serum resistance associatated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense. American Journal of Tropical Medicine and Hygiene, 67:

684–690 (2002a).

[17] GIBSON, W.C., BACKHOUSE, T., GRIFFITHS, A. The human serum resistance associated gene is ubiquitous and conserved in Trypanosoma brucei rhodesiense throughout East Africa. Infection, Genetics and Evolution, 25: 1–8 (2002).

[18] WELBURN, S.C., PICOZZI, K., FEVRE, E.M., COLEMAN, P.G., ODIIT, M., CARRINGTON, M., MAUDLIN, I. Identification of human infective Trypanosomes in animal reservoirs of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene. Lancet 358: 2017–2019 (2001).

THE USE OF ITS1 RDNA PCR IN DETECTING PATHOGENIC