Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT. Author summaryAfrican animal trypanosomiasis (AAT) is an endemic disease in sub-Saharan Africa that hinders the development of livestock production on the continent. The control of the disease is based on chemotherapy, vector control and diagnosis. Misuse, as well as the continuous/regular use of a limited number of anti-trypanosomal drugs, is responsible for the appearance of increasingly drug-resistant strains of trypanosomes. In terms of serological diagnosis, the most efficient test at present suffers from a lack of reagent standardization. Unfortunately, even the most promising candidates fail due to low sensitivity in primately or chronically infected animals. Based on this observation it seems obvious that diagnosis must be revisited. In this study we evaluated the diagnostic potential of two Trypanosoma brucei proteins, TbLysoPLA and TbGK, in indirect ELISA for antibody detection. To provide a proof of concept that the judicious association of immunoreactive proteins could improve the sensitivity and specificity of tests based on recombinant antigens, we used these molecules alone and then in combination, associated or not with the BiP protein of T. congolense. The evaluation in serological diagnosis showed that the two proteins used separately had a poor performance. However, when used together with the BiP protein, they showed a sensitivity of 60% and a specificity between 87 and 96%, comparable to the reference tests. It shows for the first time that the performance of protein combinations is much better than that of the proteins tested individually for the diagnosis of TAA.

Automated summary

African animal trypanosomiasis (AAT) is a prevalent disease in sub-Saharan Africa, hindering livestock production development. This study evaluated the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei in indirect ELISA for antibody detection. The results showed that the combination of these proteins with the BiP protein of T. congolense improved sensitivity and specificity compared to individual testing, demonstrating the importance of protein combinations in the diagnosis of AAT.