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Genotyping of Deltamethrin Resistance in Rhipicephalus (Boophilus) microplus Population in Kerala, South India

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Abstract

Purpose

Genotyping of Rhipicephalus (Boophilus) microplus for polymorphisms in deltamethrin-resistant loci of sodium channel gene by allele-specific PCR (AS-PCR).

Methods

Adult R. (B.) microplus ticks were collected from naturally infested cattle in Kerala. The larval packet test (LPT) was performed with deltamethrin and dose response data were analysed by probit method. Adult and larval tick DNA were amplified by PCR and later sequenced to identify mutations, if any, in the domain II S4-5 linker and domain III S6 regions in para voltage-gated sodium channel gene, at loci that were previously documented to be associated with deltamethrin resistance. Allele-specific PCR was performed for the amplification of target gene locus (C190A and T2134C) to genotype 1000 larvae each, at these loci. Genotype frequency was statistically analysed by Chi-square test.

Results

Bioassay using LPT revealed that LC50 and LC95 values of all the R. (B.) microplus isolates in this study were more than double the reported values of reference susceptible strain. Sequence analysis of the PCR amplicons of domain II S4-5 linker of voltage-gated sodium channel gene revealed C190A mutation, A271G mutation as well as A–G mutation at 217th position. AS-PCR done to genotype C190A mutations revealed a frequency of 6%, 15% and 64%, respectively for homozygous-susceptible (SS), heterozygous (RS) and homozygous-resistant (RR) genotypes. In domain III S6 region of the gene, C2121T and A2102T mutations were observed. AS-PCR to genotype the previously reported T2134C mutation revealed 100% SS genotype in R. (B.) microplus isolates of Kerala.

Conclusions

Genotyping of R. (B.) microplus isolates of Kerala for target site mutations reportedly associated with deltamethrin resistance revealed a significantly high frequency of resistant genotypes at II S4–5 linker of voltage-gated sodium channel gene. This study forms the first report of such mutations in Kerala, south India and demands serious attention in the light of increased prevalence of tick-borne haemoparasitic infection.

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Availability of Data and Material

All the data supporting the conclusions of this article are available within the article. The sequences obtained in this study are available in GenBank under the accession numbers MT328784, MT328785, MT328786.

Code Availability

None.

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Acknowledgements

The authors gratefully acknowledge the technical support of Kerala Veterinary and Animal Sciences University, Pookode, Kerala, India for conducting the research.

Funding

No external grants were received.

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Authors and Affiliations

  1. School of Applied Animal Production and Biotechnology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, 680651, India

    Anand Amrutha & T. V. Aravindakshan

  2. Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, 680651, India

    Lakshmanan Bindu

  3. Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, 680651, India

    J. Siju

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  1. Anand Amrutha
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  2. Lakshmanan Bindu
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  3. J. Siju
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  4. T. V. Aravindakshan
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Contributions

AA performed the experiment. LB planned the research work and drafted the manuscript. JS provided support for genotyping studies. TVA critically reviewed the manuscript.

Corresponding author

Correspondence to Lakshmanan Bindu.

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The work has been done as per the guidelines of institutional ethical committee. The article does not contain any experimental studies on animals and the protocols followed using ticks are ethical.

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The ticks were collected from dogs upon pre-informed consent of clients.

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Amrutha, A., Bindu, L., Siju, J. et al. Genotyping of Deltamethrin Resistance in Rhipicephalus (Boophilus) microplus Population in Kerala, South India. Acta Parasit. 66, 1031–1038 (2021). https://doi.org/10.1007/s11686-021-00378-4

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