TY - JOUR
T1 - A novel metabarcoded deep amplicon sequencing tool for disease surveillance and determining the species composition of Trypanosoma in cattle and other farm animals
AU - Yasein, Ghulam
AU - Zahid, Osama
AU - Minter, Emily
AU - Ashraf, Kamran
AU - Rashid, Imran
AU - Shabbir, Muhammad Zubair
AU - Betson, Martha
AU - Sargison, Neil
AU - Chaudhry, Umer
N1 - Funding Information:
The Roslin Institute uses facilities funded by Biotechnology and Biological Sciences Research Council (BBSRC), UK. Work at the University of Veterinary and Animal Science Pakistan uses facilities funded by the Higher Education Commission of Pakistan. The authors of this study would like to thank Professor Liam Morrison (Roslin Institute, University of Edinburgh, UK) for his great support and for providing positive control samples.
Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/6
Y1 - 2022/6
N2 - The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have developed strategies to control trypanosomiasis in humans and livestock in endemic areas. These require a better understanding of the distribution of different Trypanosoma species and improved predictions of where they might appear in the future, based on accurate diagnosis and robust surveillance systems. Here, we describe a metabarcoding deep amplicon sequencing method to identify and determine the Trypanosoma species in co-infecting communities. First, four morphological verified Trypanosoma species (T. brucei, T. congolense, T. vivax and T. theileri) were used to prepare test DNA pools derived from different numbers of parasites to evaluate the method’s detection threshold for each of the four species and to assess the accuracy of their proportional quantification. Having demonstrated the accurate determination of species composition in Trypanosoma communities, the method was applied to determine its detection threshold using blood samples collected from cattle with confirmed Trypanosoma infections based on a PCR assay. Each sample showed a different Trypanosoma species composition based on the proportion of MiSeq reads. Finally, we applied the assay to field samples to develop new insight into the species composition of Trypanosoma communities in cattle, camels, buffalo, horses, sheep, and goat in endemically infected regions of Pakistan. We confirmed that Trypanosoma evansi is the major species in Pakistan and for the first time showed the presence of Trypanosoma theileri. The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including evaluation of drug treatment responses, understanding of the emergence and spread of drug resistance, and description of species interactions during co-infections and determination of host and geographic distribution of trypanosomiasis in humans and livestock.
AB - The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have developed strategies to control trypanosomiasis in humans and livestock in endemic areas. These require a better understanding of the distribution of different Trypanosoma species and improved predictions of where they might appear in the future, based on accurate diagnosis and robust surveillance systems. Here, we describe a metabarcoding deep amplicon sequencing method to identify and determine the Trypanosoma species in co-infecting communities. First, four morphological verified Trypanosoma species (T. brucei, T. congolense, T. vivax and T. theileri) were used to prepare test DNA pools derived from different numbers of parasites to evaluate the method’s detection threshold for each of the four species and to assess the accuracy of their proportional quantification. Having demonstrated the accurate determination of species composition in Trypanosoma communities, the method was applied to determine its detection threshold using blood samples collected from cattle with confirmed Trypanosoma infections based on a PCR assay. Each sample showed a different Trypanosoma species composition based on the proportion of MiSeq reads. Finally, we applied the assay to field samples to develop new insight into the species composition of Trypanosoma communities in cattle, camels, buffalo, horses, sheep, and goat in endemically infected regions of Pakistan. We confirmed that Trypanosoma evansi is the major species in Pakistan and for the first time showed the presence of Trypanosoma theileri. The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including evaluation of drug treatment responses, understanding of the emergence and spread of drug resistance, and description of species interactions during co-infections and determination of host and geographic distribution of trypanosomiasis in humans and livestock.
KW - Trypanosomiasis
KW - trypanosoma
KW - metabarcoding
KW - deep amplicon sequencing
U2 - 10.1016/j.actatropica.2022.106416
DO - 10.1016/j.actatropica.2022.106416
M3 - Article
SN - 0001-706X
VL - 230
JO - Acta Tropica
JF - Acta Tropica
M1 - 106416
ER -