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Molecular detection of Theileria annulata infection in cattle by conventional PCR and quantitative real time PCR in India.

ABSTRACT: Theileria annulata (T. annulata) is a tick-borne apicomplexan parasite that affects bovine. It is endemic in many tropical and subtropics areas, including Odisha, India. The objective of this study is to identify T. annulata infection in the peripheral blood of cattle as a biological sample by conventional PCR (cPCR) and quantitative PCR (qPCR). The phylogenetic analysis was done using the T. annulata merozoite surface antigen (Tams 1) gene. Out of 552 samples of examined blood smears by microscopy, 454 (82.24%) animals were positive for Theileria species. Out of 454 samples, 96 samples were further examined by both cPCR and qPCR, 52 samples (54.16%) were found positive for T. annulata in both PCR methodologies. Phylogenetic analysis revealed that T. annulata Odisha isolate was closely related to T. annulata Uttarakhand, India isolate (KM061799) and Hyderabad, India isolate (MK034702) with Nucleotide sequence identity 95.36%, 95.25%, respectively. This is the first study to detect T. annulata by qPCR in Odisha and supported that both PCR techniques were equally effective for the detection of Tams 1 gene of T. annulata in cattle's blood.

SUBMITTER: Selim AM

PROVIDER: S-EPMC7921255 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Molecular detection of Theileria annulata infection in cattle by conventional PCR and quantitative real time PCR in India.

Selim Ahmed Magdy AM Das Manoranjan M Senapati Santosh Kumar SK Jena Geeta Rani GR Mishra Chinmoy C Nath Indramani I Senapati Shantibhusan S Sethi Manisha M

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20200925 1

Theileria annulata (T. annulata) is a tick-borne apicomplexan parasite that affects bovine. It is endemic in many tropical and subtropics areas, including Odisha, India. The objective of this study is to identify T. annulata infection in the peripheral blood of cattle as a biological sample by conventional PCR (cPCR) and quantitative PCR (qPCR). The phylogenetic analysis was done using the T. annulata merozoite surface antigen (Tams 1) gene. Out of 552 samples ...[more]

PMID: 33746389

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Project description:Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Project description:Bovine tropical theileriosis is a major haemoprotozoan disease associated with high rates of morbidity and mortality particularly in exotic and crossbred cattle. It is one of the major constraints for of the livestock development programmes in India and southern Asia. Indigenous cattle (Bos indicus) are less affected by this disease than exotic and crossbred cattle. Genetic basis of resistance to tropical theileriosis in indigenous cattle is not well studied. Recent studies gives an idea that differentially genes expressed in exotic and indigenous breeds play an important role in breed specific resistance to tropical theileriosis. The present study was designed to visualize the global gene expression profiling in PBMCs derived from indigenous (Tharparkar) and crossbred cattle with in vitro infection of T. annulata. T. annulata Parbhani strain, originally isolated from Maharashtra (India) and maintained as cryopreserved stabilates of ground-up tick tissue sporozoite (GUTS) of infected H. anatolicum anatolicum was used as infective material. Two separate microarray experiments were carried out using separately each for crossbred and Tharparkar cattle. The crossbred cattle showed 1082 differentially expressed genes (DEGs). Out of total DEGs, 597 genes were downregulated and 485 were upregulated. Their fold change varies from 2283.93 to -4816.02. Tharparkar cattle showed 875 differentially expressed genes. Out of total DEGs in Tharparkar cattle, 451 genes were downregulated and 424 genes were upregulated. Their fold change varies from 94.93 to -19.20. A subset of genes was validated by quantitative RT-PCR and results correlated well with data obtained from the microarrays indicating that the microarray results gave an accurate report of transcript level. Functional annotation study of differentially expressed genes has confirmed their involvement in various pathways including response to oxidative stress, immune system regulation, cell proliferation, cytoskeletal changes, kinases activity and apoptosis. Gene network analysis of these differentially expressed genes provided an effective way to understand the interaction among them. It is therefore, hypothesised that the dissimilar susceptibility to tropical theileriosis exhibited by indigenous and crossbred cattle is due to breed-specific differences in the interaction of infected cells with other immune cells, which ultimately influences the immune response generated against T. annulata infection.

Dataset Information

Molecular detection of Theileria annulata infection in cattle by conventional PCR and quantitative real time PCR in India.

Publications

Molecular detection of <i>Theileria annulata</i> infection in cattle by conventional PCR and quantitative real time PCR in India.

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