Characterization and phylogenetic analysis of multidrug-resistant protein - encoding genes in Trypanosoma evansi isolated from buffaloes in Ngawi district, Indonesia.

BACKGROUND AND AIM: Excessive use of trypanocidal drugs can lead to cases of drug resistance. Multiple cases of resistance have been widely reported for drugs such as isometamidium chloride and diminazene aceturate. These cases deserve serious attention, especially in Indonesia, where the first case was recorded and where the molecular basis of trypanocidal drug resistance has never been evaluated. This study aimed to analyze the multidrug resistance protein (MRP) gene in Trypanosoma evansi isolates, sampled from Indonesia, by focusing on the phylogenetic relationship between these isolates and other Trypanosoma spp. MATERIALS AND METHODS: A total of 88 blood samples were drawn from buffaloes in the Ngawi district, Indonesia. Animals infected with T. evansi were detected through the microhematocrit technique and Giemsa blood smear methods. Positive blood samples were used to inoculate in male mice (Mus musculus BALB-C strain) as an animal model for culturing the T. evansi. The genomic DNA of the blood taken from the T. evansi- infected mice was used for polymerase chain reaction amplification, sequencing, and phylogenetic analysis. RESULTS: Two genes were analyzed; the first gene detected for T. evansi corresponded to Trypanosoma brucei with a homology of 99% and the second gene to Trypanosoma brucei gambiense, with a homology of 100%. These two genes of the MRP from T. evansi showed clear similarity to the MRPE and MRPA genes of the T. brucei ssp. CONCLUSION: The MRP gene is conserved on the subspecies level of T. brucei. Only few point mutations were found between various sequences, which mean that the proteins have the same structure. This is important to treat the parasite with the appropriate drugs in the future.

Detection of immunogenic proteins from Anopheles sundaicus salivary glands in the human serum.

INTRODUCTION: The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes. METHODS: IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting. RESULTS: Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls. CONCLUSIONS: These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.