Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines

Abstract

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10−6, 10−6 and 10−4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

Tick-Borne Pathogens Shape the Native Microbiome Within Tick Vectors

“…To detect the presence of pathogens and endosymbiont of interest, we utilized a PCR based approach to amplify the 18S rRNA gene of Theileria sp. [ 4 ], 16S rRNA gene of Anaplasma marginale [ 16 ], and GroEL gene of Wolbachia [ 17 ]. PCR positive DNAs were amplicon sequenced using both the forward and reverse primers and the partial sequences were subjected to NCBI BLAST program for further confirmation.…”

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

“…Serological tests have been developed to detect circulatory antibodies against the parasites, but these generally have poor sensitivity and specificity due to involving cross-reactions or nonspecific immune responses (Passos et al , 1998), and only detect previous exposure as opposed to current infection. Conventional PCR methods are useful in the detection of particular haemoprotozoan species for which the reagents and conditions have been developed, but can have limitations in the detection of other species and lack scalability (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). In contrast, the deep amplicon sequencing method is potentially providing more reliable and accurate in the automated high throughput analysis of all haemoprotozoan species.…”

“…Determination of sequence variations in the hyper-variable 18S rDNA cistron can discriminate between haemoprotozoan parasites (Gubbels et al , 1999) and overcome limitations of traditional gross parasitological methods for the diagnosis of haemoprotozoa at species level (Agudelo et al , 2013; Haanshuus et al , 2013; Lee et al , 2015; Lefterova et al , 2015; Mens et al , 2006; Rougemont et al , 2004; Steenkeste et al , 2009). Various PCR methods (reverse line blot (RLB)-PCR, quantitative (q)PCR, multiple PCR) have been described to amplify the 18S region for sequence determination, but these are low throughput, hence relatively expensive, and can be error-prone (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). These methods depend on the use of species-specific primers and probes, hence can only identify the tested species.…”

Molecular tools-advances, opportunities and prospects for the control of parasites of veterinary importance

“…Similarly, multiplex PCR developed for detection of Trypanosoma evansi and Theileria equi in equines (Sumbria et al 2016 ). Multiplex PCR is also applied for the simultaneous detection of natural infection of theileriosis, babesiosis and trypanosomosis in cattle (Sharma et al 2014 ; Kundave et al 2018 ). In canines, detection of Babesia canis vogeli, Babesia gibsoni and Ehrlichia canis could be carried out by multiplex PCR (Jain et al 2018 ).…”