Trypanosoma Congolense Resistant to Trypanocidal Drugs Homidium and Diminazene and their Molecular Characterization in Lambwe, Kenya.

PURPOSE: African animal trypanosomiasis (AAT) is a disease affecting livestock in sub-Saharan Africa. The use of trypanocidal agents is common practice to control AAT. This study aimed to identify drug-resistant Trypanosoma congolense in Lambwe, Kenya, and assess if molecular test backed with mice tests is reliable in detecting drug sensitivity. METHODS: Blood samples were collected from cattle, in Lambwe, subjected to buffy coat extraction and Trypanosoma spp. detected under a microscope. Field and archived isolates were subjected to molecular characterization. Species-specific T. congolense and TcoAde2 genes were amplified using PCR to detect polymorphisms. Phylogenetic analysis were performed. Four T. congolense isolates were evaluated individually in 24 test mice per isolate. Test mice were then grouped (n=6) per treatement with diminazene, homidium, isometamidium, and controls. Mice were subsequently assessed for packed cell volume (PCV) and relapses using microscopy. RESULTS: Of 454 samples, microscopy detected 11 T. congolense spp, eight had TcoAde2 gene, six showed polymorphisms in molecular assay. Phylogenetic analysis grouped isolates into five. Two archived isolates were homidium resistant, one was also diminazene resistant in mice. Two additional isolates were sensitive to all the drugs. Interestingly, one sensitive isolate lacked polymorphisms, while the second lacked TcoAde2, indicating the gene is not involved in drug sensitivity. Decline in PCV was pronounced in relapsed isolates. CONCLUSION: T. congolense associated with homidium and diminazene resistance exist in Lambwe. The impact can be their spread and AAT increase. Polymorphisms are present in Lambwe strains. TcoAde2 is unlikely involved in drug sensitivity. Molecular combined with mice tests is reliable drug sensitivity test and can be applied to other genes. Decline in PCV in infected-treated host could suggest drug resistance.

Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, that threatens the conservation of small wild ruminants. The development of PPRV vaccines, diagnostics and therapeutics, greatly depends on in-depth genomic data. Yet, high guanine-cytosine (GC) content between matrix (M) and fusion (F) genes of PPRV poses difficulty for both primer design and nucleotide amplification. In turn, this has led into absence or low nucleotide sequence coverage in this region. This poses a risk of missing important part of the genome that could help to infer viral evolution. Here, an overlapping long-read primer-based amplification strategy was developed to amplify the GC-rich fragments between M-F gene junction using nexus gradient polymerase chain reaction (PCR). The resulting amplicons were sequenced by dideoxynucleotide cycle sequencing and compared with other PPRV nucleotide sequences available at GenBank. Our findings indicate clear PCR amplification products with expected size of the GC-rich fragments on agarose gel electrophoresis. The sequencing results of these fragments indicate 99.5 % nucleotide identity with PPRV strain KY628761. An extremely difficult PCR target of 67.4 % GC contents was successfully amplified and sequenced using this long-read primer approach. The long-read primer set may be used in tiling multiplex PCR for complete genome sequencing of PPRV.