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Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM)

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Abstract

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

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Data availability

All DNA sequences of adult parasites obtained from DNA barcodes, ND1 and COI in this study were deposited in the GenBank database (NCBI) as OP939413-OP939415 and OP928154-OP928157, respectively.

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Funding

This work was supported by Srinakharinwirot University (Research Grant No. 023/2564 and 575/2564). Additional funding was supported by Excellence Center in Veterinary Bioscience, Chiang Mai University, Chiang Mai, Thailand.

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, Naresuan University, Phitsanulok, 65000, Thailand

    Kittisak Buddhachat & Sirikhwan Sriuan

  2. Excellence Center in Veterinary Biosciences, Chiang Mai University, 50200, Chiang Mai, Thailand

    Kittisak Buddhachat

  3. Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand

    Sirapat Nak-on & Thapana Chontananarth

  4. Research and Innovation Unit for Diagnosis of Medical and Veterinary Important Parasites, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand

    Thapana Chontananarth

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  1. Kittisak Buddhachat
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  2. Sirikhwan Sriuan
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  3. Sirapat Nak-on
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  4. Thapana Chontananarth
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Contributions

Thapana Chontananarth (TC) is a major contributor and participates in all parts of the study. TC and Kittisak Buddhachat (KB) created the conceptualization for all experiments. TC and Sirapat Nak-on (SN) collected and identified the samples. Sirikhwan Sriuan (SS) and KB conducted the experiment in the laboratory, analyzed the data, created data visualization, and performed the statistical analysis. KB and TC were working together for writing the manuscript. All authors have read and accepted this manuscript.

Corresponding author

Correspondence to Thapana Chontananarth.

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Ethics approval

The animal ethic of this experiment was approved by Srinakharinwirot University under SWE-A-022–2563.

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Informed consent was obtained from all individual participants included in the study.

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Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.

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The authors declare no competing interests.

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Buddhachat, K., Sriuan, S., Nak-on, S. et al. Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM). Parasitol Res 122, 769–779 (2023). https://doi.org/10.1007/s00436-022-07769-0

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