Epidemiology of drug resistance and evaluation of possible mechanical transmission of Trypanosoma congolense by haematophagous insects in Zambezia Province Mozambique

Abstract

African animal trypanosomosis is a debilitating tsetse-transmitted parasitic disease of sub-Saharan Africa. The cyclical vectors of the disease are tsetse flies (Glossina spp). However, trypanosomosis can be mechanically transmitted by haematophagous flies from the family Tabanidae (Diptera). Therapeutic and prophylactic drugs used in the control of the disease were introduced more than 50 years ago, and drug resistance is increasingly reported and very few studies regarding the distribution of the disease vectors have been conducted in Mozambique after the country’s independence that took place in 1975. The aim of this study was to assess trypanosomosis and trypanocidal drug resistance prevalence in Nicoadala district, Zambezia province, Mozambique, and to investigate the presence of trypanosomosis vectors in three different habitats in the same district, as well as the trypanosome infection rates in these vectors. To study drug resistance, a cross-sectional study was conducted in five villages (Botao, Mungama, Zalala-Electrosul, Zalala-Madal, and Namitangurine), where 467 cattle were microscopically screened for trypanosomes, followed by a block treatment study, to evaluate treatment efficacy. Trypanosome-positive animals were randomly assigned to two groups; one treated with 0.5 mg/kg b.w. isometamidium (Inomidium®) and the other with 3.5 mg/kg b.w. diminazene (Inomazene®). Cattle were microscopically monitored at days 0, 14 and 28 post-treatment. At day 28, trypanocides were swapped to investigate single or multiple resistance. Microscopically-negative samples from the monitoring days were tested using 18S-PCR-RFLP. At the end of the block treatment experiment, day 42, six animals (9%) in Botao and two animals (9.5%) in Mungama remained positive after drug swap. No relapses occurred in Namitangurine. The presence of single and multi-drug resistance in Nicoadala district, Zambezia province was thus confirmed. To investigate the composition of Tabanidae and Glossinidae, vectors of trypanosomosis, an entomological survey was conducted. For 365 days, 55 traps (20 NGU, 20 H and 15 Epsilon) were deployed in three grazing areas of Nicoadala district, namely, Namitangurine (25), Zalala (15) and Botao (15). Flies belonging to the above-mentioned families were collected weekly, preserved in 70% ethanol and later identified using identification keys and manuals. Molecular identification of tabanids and tsetse flies, and a phylogeny study of tabanids, were conducted using cytochrome oxidase 1 gene. Additionally, trap efficiency was assessed to compare the performance of the three types of trap in the capture of individuals from both families. Furthermore, seasonal abundance of tabanids was also investigated. As result, individuals belonging to ten tabanid species and three tsetse species were caught. The use of molecular tools confirmed taxonomic identification as well as revealing monophyly in Tabanidae. NGU traps were identified as the most suitable for the capture of both tabanids and glossina. It was also observed that the rainy season was the most appropriate for the capture and study of tabanids. The vector composition lead to the conclusion that the role of tabanids in the transmission of trypanosomes in Nicoadala district may be greater than of tsetse flies. To assess trypanosome infection rates, DNA was extracted from 480 tabanids and 24 glossina. From the 480 tabanids, 160 were captured in Zalala, 160 in Botao and 160 in Namitangurine. Moreover, the tabanids used belonged to the four most captured species in the three areas namely Tabanus par, T. taeniola, Atylotus agrestis and Ancala africana. The 24 glossina used belonged to the species Glossina brevipalpis, G. morsitans and G. pallidipes. DNA extracted was submitted to 18S PCR-RFLP and was found to be positive for the presence of Trypanosoma congolense and T. theileri. The overall infection rates were 70.8% (n=24) for glossina and 13% (n=480) for tabanids. Similarly to what was observed in the block treatment, the only pathogenic trypanosome detected was T. congolense, strengthening the idea of this being the only pathogenic Trypanosoma species circulating in cattle in the area. The present study revealed the presence of drug resistance in Nicoadala district. It also demonstrated that there is a small Glossina population with an extremely low apparent density surviving in the area as well as a large homogenous population of tabanids cohabiting there, both participating in the epidemiology of trypanosomosis.