Diagnosis and mapping of diminazene aceturate resistance in Trypanosoma congolense, Broden 1904, strains circulating in cattle in Matutuíne district, Mozambique

Abstract

Trypanocidal Drug Resistance is presently recognized as an important constraint for trypanosomosis control efficiency in the endemic areas. No recent and up to date report is available for the Matutuíne District, which is preventing the establishment of an appropriate control strategy in this endemic area. The present study was carried out in order to assess and map the diminazene resistance in T. congolense populations circulating in bovines in the Matutuíne District. Between 2009 and 2011, a longitudinal survey was performed, allowing an updated vision of the disease prevalence and of the drug resistance situation. A total of 2427 bovine blood samples were collected on filter paper in all five administrative areas in Matutuine (Belavista, Zitundo, Catuane, Katembe and Machangulo). The parasitological (buffy coat) prevalence was 17.3% while using the molecular test 18S PCR, a prevalence of 46% was recorded. Four trypanosome species were detected: the pathogenic T. congolense (savannah and Kilifi type), T. vivax, T. b. brucei and the non-pathogenic T. theileri. The T. congolense savannah type was the predominant species with a parasitological prevalence of 12.7% (309) (although including the Kilifi type which was not possible to distinguish by buffy coat) and using the molecular test was 22.3% (540) as single infection; 1.6% (149) as mixed infection with T. theileri. These results confirm the Matutuíne District as a trypanosomosis-endemic area and even indicate stable disease status, since the prevalence is almost identical to that reported 13 years before. Additionally, it indicates that the T. congolense savannah type remains the main etiologic agent of bovine trypanosomosis in the region. Concerning the diminazene resistance in Matutuíne District, the 689 T. congolense savannah 18S PCR positive samples were submitted to the Ade2 PCR followed by the DpnII-PCR-RFLP. Only 21% (143) of the samples were amplified on the Ade2 PCR, confirming the low sensitivity of this PCR tool compared to the 18S PCR.. Ade2 positive PCR s were from four administrative areas in Matutuíne s i.e. Belavista (55.9%), Zitundo (25.2%), Catuane (15.4%) and Katembe (2.1%). Machangulo was not evaluated. The DpnII-PCR-RFLP revealed the occurrence of all three possible profiles of the TcoAT1/TcoNT10 gene: sensitive homozygous (17.5%), sensitive heterozygous or mixed (44.8%), and resistant (37.8%). Such TcoAT1/TcoNT10 gene allele distributions have been reported in areas without or with moderate trypanocidal drug usage. Therefore we can assume that this is the status of the Matutuíne District and also that, diminazene resistance is not a major problem in the region, allowing the use of the sanative pair strategy for trypanosomosis control in the area. For the evaluation of the correlation between the molecular DpnII-PCR-RFLP and the in vivo standardized mouse test for diminazene resistance diagnosis, 24 T. congolense savannah type isolates were evaluated by both tests. All of them (24/24) were sensitive to the drug at the dose of 10 mg/kg and 20 mg/kg bw in the in vivo drug sensitivity mouse test with the relapses being checked only by parasitological methods. The molecular test revealed 5/24 homozygous sensitive, 8/24 heterozygous sensitive and 11/24 homozygous resistant isolates.. Based on these data, the K statistic revealed a slight agreement between both tests (K= 0.15), supporting the idea of possibly misleading information in the drug sensitivity mouse test when the relapses are detected by parasitological methods. The molecular tool for detection of resistance to diminazene should not be used at the individual level but rather considering the distribution of the resistance alleles as indicator of drug use intensity. This correlation should be further explored. However, the result of the mouse test also indicates that diminazene resistance in this region has not yet been established.