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

Selim, Ahmed Magdy; Das, Mamoni; Senapati, Santosh Kumar; Jena, Geeta Rani; Mishra, Chinmoy; Nath, I.; Senapati, Shantibhusan; Sethi, Manisha

doi:10.1007/s12639-020-01278-6

Cited by 6 publications

(6 citation statements)

References 23 publications

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“…Both T. annulata and T. orientalis were found in the cattle blood in our current investigation. Similar findings have been reported in India and Pakistan [ 38 , 39 , 40 , 41 ].…”

Section: Discussionsupporting

confidence: 90%

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Gompo¹,

Devkota²,

Kafle³

et al. 2023

Pathogens

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Background: Tick-borne protozoan parasites (TBPPs) cause significant problems for domestic animals’ health in Nepal. TBPPs are routinely diagnosed by labor-intensive blood smear microscopy. In Nepal, there are some reports of Babesia and Theileria in cattle, although species identification is rarely performed. Therefore, we performed conventional nested PCR (nPCR) followed by sequence analysis to identify TBPP species infecting cattle in Nepal. Methods: One hundred and six blood samples were collected from cattle in the Kathmandu Valley. Thin blood smears were prepared for microscopic examination. Parasite DNA was extracted from the blood, and nPCR and sequencing were performed to identify the TBPPs present. Results: Among the 106 samples, 45 (42.5%) were positive for piroplasm (Babesia spp. and Theileria spp.) via microscope observation and 56 (52.8%) samples were positive via nPCR. The obtained PCR products were used for direct sequencing, and we identified the species as B. bigemina, B. bovis, T. annulate and T. orientalis. Phylogenetic analyses showed that the B. bovis, B. bigemina and T. orientalis sequences from this study belonged to each species clade. On the other hand, T. annulate was divided into two clades in the analysis, and our T. annulate sequences were also divided in these two clades. The piroplasm-positive cattle showed lower hemoglobin and red blood cells than healthy cattle. Conclusions: To the best of our knowledge, this study is the first to apply molecular detection and species determination of TBPPs in cattle in Nepal. The results of this study may be used as a starting point for the development of successful TBPP surveillance and prevention programs in Nepal.

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“…Both T. annulata and T. orientalis were found in the cattle blood in our current investigation. Similar findings have been reported in India and Pakistan [ 38 , 39 , 40 , 41 ].…”

Section: Discussionsupporting

confidence: 90%

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Gompo¹,

Devkota²,

Kafle³

et al. 2023

Pathogens

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“…The merozoite surface antigen (Tams1) is highly specific for T. annulata and it does not show cross reactivity with other species of Theileria including T. parva, T. mutans, T. sergenti and T. buffali (d'Oliveira et al, 1995). The molecular diagnosis of T. annulata targeting Tams1 gene was widely exploited throughout the globe including India (Tuli et al, 2015;Kundave et al, 2018;Ganguly et al, 2020;Selim et al, 2021). The limitation of PCR Bovi-TheiDX PCR assay that gene was not mentioned and is laborious.…”

Section: Resultsmentioning

confidence: 99%

“…A number of genes of different species of Theileria have been partially explored include major surface protein (msp), ribosomal rRNA (18S rRNA), heat shock protein (hsp), merozoite piroplasm surface antigen (Tams) and lactate dehydrogenase. The polymorphism is more in Tams1 gene and variety of sequences of this gene were published and employed in the molecular diagnosis of Theileria species (Santos et al, 2013;Kundave et al, 2018;Ganguly et al, 2020;Selim et al, 2021)…”

Section: Introductionmentioning

confidence: 99%

Comparative Performance and Evaluation of Two Molecular Assays and Conventional Detection of Theileria annulata in Bovines

Ntesang

Kaur

Arora

et al. 2022

IJAR

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Background: Theileria annulata is predominant and of utmost economical importance tick borne pathogen of bovines in the region which is routinely diagnosed based on the microscopic examination of Romanowsky stained thin blood smears. Present study was intended to evaluate and analyze the detection efficacy of a commercial polymerase chain reaction kit based assay in comparison to conventional PCR assay and classical microscopy for detection of T. annulata from blood samples of bovines from Punjab state. Methods: In this comparative study 360 bovine blood samples from various districts of agro-climatic zones in Punjab were first screened for T. annulata by Giemsa-stained thin blood smear (GSTBS) examination. The same panel of blood samples was tested for T. annulata by a commercial PCR kit Bovi-TheiDX Theileria annulata (Genext Genomics) (PCR1) and established conventional PCR assay targeting merozoite piroplasm surface antigen (Tams1) gene of T. annulata (PCR2). Result: Out of 360 samples screened, positivity of T. annulata by GSTBS was found to be 12.5% (45/360) with a sensitivity of 37.20% and specificity of 96.00% when compared with commercial kit (PCR1), the difference was statistically significant (p less than .0001). The detection prevalence by PCR1 29.70% (107/360) and PCR2 assay 33.90% (122/360) showed significant (p less than .0001) difference. The conventional PCR targeting the Tams1 gene (PCR2) was found to be more sensitive (84.6%) (p less than 0.001) than PCR1 (69.7%).

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“…T. annulata is the most concerning Theileria species in Indian cattle and requires prompt diagnosis and treatment. Molecular techniques based on the PCR have been developed to detect and quantify Theileria species and the T. annulata parasite [ 11 , 12 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 36 , 37 , 38 ]. Although qPCR-based techniques have shown promise in detecting Theileria infections, absolute quantification of parasites remains challenging due to qPCR’s dependence on reference standard curves.…”

Section: Discussionmentioning

confidence: 99%

“…Despite its superior sensitivity and accuracy compared to previous molecular or serological approaches used in the field, there are still areas where the quantitative test may be improved. One key disadvantage of existing qPCR techniques is their reliance on the reference standard curve, which makes comparing qPCR findings between labs challenging [ 17 , 18 , 19 , 20 , 21 , 22 ]. Developing an assay with higher performance at low doses without the requirement for a reference standard curve should increase the detection of infection, hence facilitating case identification and disease monitoring.…”

Section: Introductionmentioning

confidence: 99%

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis

et al. 2023

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Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

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

Cited by 6 publications

References 23 publications

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Comparative Performance and Evaluation of Two Molecular Assays and Conventional Detection of Theileria annulata in Bovines

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis

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