18S rRNA Gene-Based Piroplasmid PCR: An Assay for Rapid and Precise Molecular Screening of Theileria and Babesia Species in Animals

Abstract

PurposeThe parasites of genera such as Babesia and Theileria are called piroplasmids due to the pear-shaped morphology of the multiplying parasite stages in the blood of the vertebrate host. Because of the enormous number of parasite species and the challenges of multiplex PCR, initial screening of samples using piroplasmid-specific PCR may be a more cost-effective and efficient technique to identify parasite species, especially during epidemiological studies. Accordingly, 18S rRNA PCR was standardized and optimized on common piroplasmids of different animals like cattle, buffaloes, sheep, goats, dogs, horses, and leopards. Methods Bloods samples from 1250 animals were collected from different animals in Junagadh district of Gujarat, India. 18S rRNA PCR was standardized and optimized as a primary method for molecular screening of piroplasms in domestic and wild animals. The method was checked for its analytical sensitivity and specificity. Parasite species-specific PCR and sequencing was used to validate the test. Moreover, in-silico restriction enzyme (RE) analysis was also done to assess its applicability in PCR-RFLP. Results Piroplasm infections were recorded in 63.3% of animals in Junagadh. The 18S rRNA PCR detected the piroplasmid DNA in as low as 39 picograms (pg) of whole blood genomic DNA isolated from microscopically Theileria positive blood samples and no reactivity was recorded from common but unrelated haemoparasites viz., Trypanosoma evansi, Hepatozoon spp., Anaplasma spp., and Ehrlichia canis was observed. The 18S rRNA PCR assay findings were confirmed by speciesspecific PCR and sequencing. Analysis of different sequences generated using 18S rRNA PCR revealed that the amplicon size of Babesia spp. is nearly 400 bp (393-408 bp) whereas Theileria spp. were more than 400 bp (418-424 bp). The percentage of sequence divergence among Babesia and Theileria spp. was 7.3-12.2% and 0.7-12.2%, respectively. In-silico restriction enzyme (RE) analysis reveals the presence of at least one site for a commercially available RE in 18S rRNA fragments of every parasite, which can differentiate it from its congeners. Conclusions The presented universal oligonucleotide-based PCR assay provides a highly sensitive, specific, cost-effective, and rapid diagnostic tool for the initial screening of piroplasmids infecting domestic and wild animals and is potentially helpful for large-scale epidemiological studies.

Agrárias: Pesquisa e Inovação nas Ciências que Alimentam o Mundo IX

“…The extraction of the genomic DNA of Babesia spp was carried out by means of the column purification procedure using a commercial kit. For the PCR test, we proceeded to amplify the variable portion of the gene that codes for the 18S small subunit ribosomal RNA (ssrRNA), using the oligonucleotides PIRO-A and PIRO-B (Olmeda et al 1997;Carret et al, 1999) which can be used to amplify different species of the Piroplasmids Babesia (Carret, 1999) and Theileria (Kumar et al, 2022). PCR reactions were performed in 0.2 ml PCR tubes, with a final volume of 25 µl, to which 12.5 µl of Master mix (Taq polymerase, dNTPs, Magnesium Chloride), 5.5 µl of nuclease-free water, 5 µl of purified DNA (containing 100 ng), and 2 µl of a mixture of the sense oligonucleotides PIRO-A (5'-AATACCCAATCCTGACACAGGG-3')…”

“…Diagnosis of the disease can be made by using Figueroa et al, 1996). In most of these methods, however, one PCR test per species is required and sometimes two PCR tests per species if DNA re-amplification is performed with the nested PCR method (Martínez et al, 2021;Kumar et al, 2022).…”

USE OF A PCR-RFLP MOLECULAR TEST FOR THE DIFFERENTIATION OF Babesia bovis AND Babesia bigemina IN THE DIAGNOSIS OF BOVINE BABESIOSIS

“…In The extraction of the genomic DNA of Babesia spp was carried out by means of the column purification procedure using a commercial kit. For the PCR test, we proceeded to amplify the variable portion of the gene that codes for the 18S small subunit ribosomal RNA (ssrRNA), using the oligonucleotides PIRO-A and PIRO-B (Olmeda et al 1997;Carret et al, 1999) which can be used to amplify different species of the Piroplasmids Babesia (Carret, 1999) and Theileria (Kumar et al, 2022). PCR reactions were performed in 0.2 ml PCR tubes, with a final volume of 25 µl, to which 12.5 µl of Master mix (Taq polymerase, dNTPs, Magnesium Chloride), 5.5 µl of nuclease-free water, 5 µl of purified DNA (containing 100 ng), and 2 µl of a mixture of the sense oligonucleotides PIRO-A (5'-AATACCCAATCCTGACACAGGG-3')…”

“…199 Figueroa et al, 1996). In most of these methods, however, one PCR test per species is required and sometimes two PCR tests per species if DNA re-amplification is performed with the nested PCR method (Martínez et al, 2021;Kumar et al, 2022).…”

Metabarcoding using nanopore long‐read sequencing for the unbiased characterization of apicomplexan haemoparasites

“…Nonetheless, the sensitivity of microscopy is low and morphological identification of closely related species, for example, within the 'large' and 'small' canine Babesia, may be difficult-to-impossible to achieve (Müller et al, 2010;Salih et al, 2015;Troskie et al, 2019). Serodiagnostic methods circumvent some of these issues, however, they also have drawbacks, such as an inability to discern past from current infections (Kaewkong et al, 2014;Kumar et al, 2022;Salih et al, 2015).…”