Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Abstract

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

Challenges in Tick-Borne Pathogen Detection: The Case for Babesia spp. Identification in the Tick Vector

“…Thus, the microfluidic real-time PCR diagnostic system can supply broader capacities for the surveillance of potentially pathogenic microorganisms by targeting the main bacterial and protozoan genera involved in human and animal vector-borne diseases [142][143][144][145][146][147][148]. Most recently, a newly developed approach has been established and utilized in Pakistan to elucidate the composition of the piroplasm populations in cattle and buffalo [149]. By using a PCR-based Next Generation Sequencing (NGS)-informatic platform system with the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms as a target gene, the study revealed piroplasms in 68.9% of the samples analyzed, with higher overall occurrence for T. annulata (65.8%), followed by B. bovis (7.1%), B. bigemina (4.4%) and T. orientalis (0.5%), demonstrating the identification of mixed infections and the discovery of B. occultans in Pakistan.…”

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia

“…In addition, the amplification biases of primers and the ability to differentiate species varies between the hypervariable regions. As a result, studies on pathogen or parasite prevalence still largely use these semi-targeted approaches ( Ghafar et al., 2020 ; Wahab et al., 2020 ). A semi-targeted molecular approach using primers targeting conserved regions of the nuclear 18S rRNA locus provides a number of advantages over using generic eukaryote primers ( Bradley et al., 2016 ; Cannon et al., 2018 ).…”

Global prevalence and species diversity of tick-borne pathogens in buffaloes worldwide: a systematic review and meta-analysis

“…Of these studies, 107 were used for meta-analysis (Fig. 1 ), and nine were not included [ 42 , 69 , 91 , 103 , 104 , 108 , 116 , 119 , 123 ] for the following reasons: the piroplasm was not identified even to the genus ( n = 2), undefined mixed infections ( n = 4), and double testing of the samples to identify two separate species of the same genus ( n = 3). The country-wise distribution of these studies correlated with the number of reared buffaloes.…”