Abstract
The Tsetse transmitted African animal trypanosomosis (Nagana) causes considerable
economic losses in sub-Saharan Africa. The use of trypanocidal drugs is the most widely
accepted means of controlling the disease. Since they have been on the market for more
than 40 years, resistance has developed against the two most important compounds
isometamidium chloride (ISMM) and diminazene aceturate (DIM). Under a BMZ-funded
project (1998 - 1999), the occurrence and distribution of drug resistant trypanosome
populations in cattle were investigated with parasitological methods in the province of
Kénédougou in southwest Burkina Faso.
The objective of the present study was to assess the polymerase chain reaction (PCR) as a
tool to monitor the efficacy of prophylactic treatment with ISMM and curative treatment with
DIM.
The PCR blood samples originated from cattle, which were examined during the block
treatment study of the BMZ-project. The study involved 10 villages with a high trypanoso-mosis
prevalence (>10%) where a total of 738 cattle were treated with ISMM (1mg/kg b.w.).
Cattle, parasitaemic during one of the fortnightly follow-up visits, were treated additionally
with DIM (3.5 mg/kg b.w.).
Before the ISMM treatment was administered, trypanosomes were diagnosed
parasitologically with the so-called "buffy coat technique" in 90 out of 738 cattle (12%). Blood
samples from the 90 BCT-positive cattle plus another 90 randomly selected blood samples
from 648 blood samples of BCT-negative cattle were analysed with the PCR using primers
for Trypanosoma brucei, T. vivax and T. congolense savannah. Out of the BCT positive
blood samples, 92.2% (83) were identified with the PCR. Out of the 90 BCT-negative
samples 37.8% (34) were PCR positive.
The efficacy of a prophylactic treatment was assessed a fortnight after the administration of
ISMM. Trypanosomes were again detected using either method. Parasitaemia, based on
BCT, occurred in 19.8% of the cattle. The detection rate of BCT-positives with the PCR
reached 97.1% (34/35) with the post-treatment samples. Furthermore, 28.8% (41/142) of the
BCT-negative samples were positive according to the PCR. A fortnight after an additional
curative DIM treatment of 34 cattle, which had been parasitologically positive after the
prophylactic treatment with ISMM, again trypanosome infections were diagnosed using either
method. All BCT-positive cases (5) were confirmed by the PCR. Another 55.2% (16) positive
cases were identified using the PCR.
Whereas about 30% more positive cases were identified with the PCR compared to the BCT
before the prophylactic treatment with ISMM, 114% more positive cases were detected after
the ISMM-prophylaxis and 320% after the DIM-therapy, respectively. The increasing
discrepancy in the number of positives with either method is almost certainly due to a larger
number of cattle with a lower parasitaemia beyond the detection limit of the BCT after
trypanocide treatment. The elimination of the sensible trypanosome populations led to
generally lower parasitaemias, although resistant populations survived.
In consequence, treatment failure remained parasitologically undetected in a number of
villages when compared with the PCR.
T. congolense was the most frequently diagnosed species with either method followed by T.
vivax and T. brucei. Before the prophylactic treatment, particularly mixed infections and
infections with T. brucei, which were mostly associated with the former, were identified more
frequently by PCR than by BCT. After the ISMM-prophylaxis, all previously identified
trypanosome species were detected again with the BCT, whereas only T. congolense
savannah and T. vivax were detected with the PCR. Both of the latest were identified again
with the PCR after the curative treatment with DIM, whereas the only parasitologically
identified species was T. congolense.
The PCR results were confirmed by specific DNA probes. Moreover, up to 10.7% signals
appeared with electrophoresis-negative PCR samples, leading to a further increase in the
PCR sensitivity.
In order to facilitate the procedure and reduce costs, a multiplex PCR was tested with the
three primer pairs. When samples with single infections were analysed, the multiplex PCR
results were exactly the same as the simplex PCR results. However, out of samples with
mixed infections only 53% were identified as such, whereas the other samples were
diagnosed as single infections. In some cases an unspecific product appeared.
For use in field studies, the multiplex PCR should be optimised in order to minimize the
competitive inhibition among the primer pairs and to avoid the amplification of unspecific
products.
The above mentioned results demonstrate that the simplex PCR seems to be ideally suited
to monitor the success of drug treatment in trypanosome-infected cattle in the field. In
combination with the results of the DNA probes, the existence of isometamidium- and
diminazene-resistant trypanosome populations in cattle in the province Kénédougou in
Burkina Faso could be confirmed. Since T. brucei might invade the central nervous system
where it is inaccessible to the drugs, the test may be curtailed for this species. |
