Trypanosomes from domestic animals from North West Uganda

A total of 58 (89.2%) of the 65 (53 from cows and 12 from pigs) Trypanosome isolates from domestic animals were positive by TBR-PCR, indicating that they were Trypanozoon Trypanosomes; and 7 (10.8%) Trypanosome isolates were TBR-PCR negative, indicating that they were possibly not T.brucei ssp and could be T.congolense, T.vivax or T.theileri. From the 58 Trypanozoon Trypanosomes, 31 Trypanosome isolates from 25 cows and from 6 pigs were TgsGP-PCR negative, suggesting they were not from T.b.gambiense Trypanosomes (Table V and Fig. 2). Additionally, all the 31 Trypanosomal DNA samples were also SRA-PCR negative, indicating that they were not from T.b.rhodesiense Trypanosomes.

TABLE IV. RESULTS OF PCR STANDARDIZATION USING 3 DIFFERENT PRIMERS FOR AMPLIFICATION OF TRYPANOSMAL DNA

Serial No

Code No

Inoculum Origin Host Tryp Species

T.b.g. = Trypanosoma brucei gambiense; T.b.rh = Trypanosoma brucei rhodesiense TBR = Primers for (Trypanozoon) Trypanosoma brucei

TgsGP = Primers for Trypanosoma gambiense surface glycoprotein gene SRA = Primers for Serum resistance Associated gene

+++ = Very strongly positive;

++ = Strongly positive;

+ = weakly positive;

- = negative

TABLE V. SUMMARY OF THE RESULTS OF POLYMERASE CHAIN REACTION USING TBR, TGSGP AND SRA PRIMERS FOR THE AMPLIFICATION OF TRYPANOSOMAL DNA ISOLATED INFECTED DOMESTIC ANIMALS IN NORTH WEST UGANDA

Serial

TBR = Primers for (Trypanozoon) Trypanosoma brucei subspecies TgsGP = Primers for Trypanosoma gambiense surface glycoprotein gene SRA = Primers for Serum resistance Associated gene

+++ = Very strongly positive ++ = Strongly positive + = Weakly positive - = Negative

TABLE VI. RESULTS OF PCR AMPLIFICATION OF TRYPANOSOMAL DNA ISOLATED FROM INFECTED HUMANS FROM THREE DIFFERENT AREAS IN SOUTH EAST UGANDA

TABLE VII. RESULTS OF PCR AMPLIFICATION OF TRYPANOSOMAL DNA ISOLATED FROM INFECTED DOMESTIC ANIMALS FROM 4 DIFFERENT AREAS IN SOUTH EAST UGANDA

4. DISCUSSION AND CONCLUSION

ITS1-PCR could not differentiate all the 106 Trypanosome isolates into different Trypanosome species because 29 Trypanosome isolates were negative by ITS1-PCR, but positive by TBR-PCR. The 29 Trypanosome isolates were TBR-PCR positive, indicating that they were Trypanozoon Trypanosomes, but could not be amplified by ITS-PCR possibly due to low concentration of DNA in the extracts. The sensitivity of ITS-PCR could be improved by carrying out a second PCR using an aliquot of the first PCR or a bigger volume of infected blood samples is to be used to increase DNA concentration. However, TBR-PCR was able to resolve all the 106 Trypanosome isolates in 88 Trypanozoon and 18 others, which were identified and confirmed as 6 T.congolense and 12 T.vivax Trypanosomes.

Animal Trypanosomosis results from North West Uganda

The combined infection rate of animal Trypanosomosis in Arua and Moyo districts varied from 0 in goats, 5% in dogs, 58% in cattle, 6.4% in sheep and 30.9% in pigs, indicating that pigs were more susceptible to Trypanosome infection. A total of 58 (89.2%) of the 65 (53 from cows and 12 from pigs) Trypanosome isolates from domestic animals were positive by TBR-PCR, indicating that they were Trypanozoon Trypanosomes. However, 7 (10.8%) of the 65 Trypanosome isolates were TBR-PCR negative, indicating that they were possibly not T.brucei ssp and could be T.congolense, T.vivax or T.theileri. Indeed, the 7 Trypanosome isolates were confirmed to be T.congolense (2) and T.vivax (5) using species specific primers. Furthermore, the 31 from the 58 TBR-PCR positive Trypanosomal DNA samples were TgsGP-PCR negative, indicating that they were not T.b.gambiense and hence no domestic animals identified as reservoir of T.b.gambiense. Additionally, all the 31 Trypanosomal DNA samples so far tested were SRA-PCR negative, indicating that there is probably no mixed infection of the two diseases, T.b.gambiense and T.b.rhodesiense in this area.

Human Trypanosomosis -T.b.rhodesiense identification using SRA gene

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 by [11] and in 27 (77.2%) of the same 35 T.b.rhodesiense Trypanosomes using the primers designed by Gibson et.al (Table VI).

In Serere area 4 of the 11 T.b.rhodesiense isolates were SRA-PCR negative using primers as in [11] and three of the same isolates were again SRA-PCR negative using [5] primers (VI).

Similarly in Tororo area, 2 of the 22 T.b.rhodesiense isolates were SRA-PCR negative using [11] primers and four including the two of the same isolates were again SRA-PCR negative using [5]; 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 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 VII) 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. Some of the SRA-PCR negatives could 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.

5. CONCLUSIONS

ITS1-PCR could only resolve 72.6% of the 106-Trypanosome isolates into different Trypanosome species. These samples may require the use of an aliquot of the first PCR in the second PCR or increase the volume of infected blood sample used in the DNA extraction in order to increase the ITS1-PCR sensitivity. Trypanosome infection rate in domestic animals was highest in pigs (30.9%), followed by sheep (6.4%), cattle (5.8%) and dogs (5.0%).From the few isolates so far analysed, TgsGP primers appear to be specific for T.b.gambiense from North West Uganda. All the 31 Trypanosomal DNA samples from domestic animals were TgsGP-PCR negative, indicating that they are not T.b.gambiense; hence no domestic animals have been identified as harbouring pathogenic parasites to humans. So far no SRA genes found in T.b.rhodesiense Trypanosomes were detected in Trypanosomes from domestic animals from northwest Uganda, possibly no mixed infections of the two parasites. Screen the remaining DNA samples for presence of T.b.gambiense and isolate more Trypanosomes from domestic animals especially pigs, which appear to have more infections.

M N P 1 2 3 4 5 6 7 8 9

FIG. 1. Agarose gel electrophoresis showing results of standardization of TgsGP-PCR protocol for the detection of T.b.gambiense.

M = Marker; N = Negative control; P = Positive control; lanes 1-9, T.b.gambiense (lanes 3, 5, 6 and 9 are weakly positives while lanes 1, 2, 4, 7 and 8 are stronger positives)

308bp

M 1 2 3 4 5 6 7 8 9 10 N

FIG. 2. Agarose gel electrophoresis showing results obtained using Tgs

FIG. 2. GP-PCR for T.b.gambiense.

M = Marker; N = Negative control; Lane1-3 = T.b.gambiense, positive controls- Lanes 4-10 T.brucei from cattle (Arua)

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 3. Agarose gel electrophoresis showing results obtained using ITS-PCR for the detection of animal Trypanosomes from cattle in Sembabule district, Uganda.

Lane 1 = T.brucei from cow ; 2 = T.brucei from cow ; 3 = T.vivax from cow ; 4 = T.brucei from cow; Controls 5 = T.simae; 6 = T.evansi; 7 = T.congo kilifi; 8 = T.congo forest; 9 = T.congo savannah; 10 = T.brucei; 11 = Negative control; 12 = DNA Marker

308bp

360bp 460bp 550bp 230bp

ACKNOWLEDGEMENTS

Thanks to the Director of Livestock Health Research Institute (LIRI) Uganda, for logistic support and for allowing me to attend this meeting. I am gratefully indebted to IAEA for sponsorship the research activities. I thank all district and field staff and local council members for assistance in the research activities

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DETECTION OF T.B.RHODESIENSE TRYPANOSOMES IN HUMANS