Molecular characterization of deltamethrin resistance in Rhipicephalus microplus ticks infesting cattle in Nagpur district of Maharashtra using allele-specific PCR

Introduction

Rhipicephalus microplus is one of the major tick species infesting livestock and poses serious problems in livestock management and disease control. It is a common ectoparasite of cattle and other farm animals, causing considerable economic losses due to reduced milk and meat production, irritation, and damage to the skin. It acts as a vector for tick-borne diseases such as Babesiosis and Anaplasmosis which may cause high morbidity and sometimes mortality in affected animals resulting in reduced health and productivity. (Gosh and Nagar, 2014) [6] . Ticks cause harm to livestock through multiple effects, including toxicity, blood loss due to feeding, irritation, damage to hides, and subsequent weight loss (Furman and Loomis, 1984) [3] . Tick control using of chemicals is the most commonly used method, however frequent and indiscriminate use and lower dosing of acaricides is responsible for development of acaricidal resistance (Sagar et al. 2020) [18] . Amongst the synthetic pyrethroids, deltamethrin is the most widely used acaricide for tick control in India. (Kumar et al. 2020) [9] . Several studies across India have reported the development of deltamethrin resistance in Rhipicephalus microplus, with varying resistance levels observed in Indian states such as in Haryana. Gujarat, Rajasthan, Jammu & Kashmir, Kerala and Telangana. Synthetic pyrethroid acts by targeting the voltage-gated sodium channels of nerve cells in ticks, leading to paralysis and death. Allele-specific polymerase chain reaction (AS-PCR) is used to detect resistance to synthetic pyrethroids by identifying specific point mutations in target genes, thereby confirming resistance in Rhipicephalus microplus. (Morgan, 2009) [14] . The information regarding the resistance status of deltamethrin in cattle ticks from the Nagpur region of Maharashtra would be valuable for developing appropriate alternative strategies to manage acaricidal resistance in ticks.

were sterilized using 1% sodium hypochlorite solution. The identification of ticks was done based on standard morphological keys as per Walker et al., (2003) [22] ; Soulsby, (1982) [19] and Geevarghese and Mishra, (2011) [4] . After identification the engorged female ticks were treated with 25 ppm deltamethrin for 2 minutes and allowed for egg laying in a BOD incubator at 27 ± 1 °C and 80-85% relative humidity. About14 days-old larvae after hatching were then stored in 70% alcohol at room temperature for genomic DNA extraction.

Genomic DNA extraction

The preserved larvae of R. microplus ticks were washed in normal saline, dried and weighed to get a weight of 25mg. The weighed larvae were transferred in sterile zip lock pouches and crushed in mortar and pestle to make it homogenized. Properly crushed larvae were then transferred to 2 ml sterile Eppendorf tube by rinsing the larval side of the pouch with normal saline. Then genomic DNA extraction of crushed larvae was carried out using HIMEDIA HiPurA TM Multi-Sample DNA Purification Kit as per manufacturer protocol with some modification. The quality and quantity of obtained DNA was assessed by agarose gel electrophoresis as well as byNanodrop 2000C spectrophotometer and then stored at-20 0 C for further processing.

Amplification of genomic DNA template by Allele-Specific PCR Allele-specific PCR was performed by using template DNA obtained from larvae of R. microplus ticks by using primers and PCR conditions as described by Rineshkumar (2010) [16] and Amrutha et al. (2021) [1] . The primer details are mentioned in Table 1

Susceptible reaction

For susceptible reaction, ASS-Forward Allele-specific susceptibleprimer was used as forward primer and ASC-Reverse Allele-specific common primer was used as reverse primer.

Resistant reaction

ASR-Forward Allele-specific resistant primer was used as forward primer and ASC-Reverse Allele-specific common primer was used as reverse primer in resistant reaction.

Control reaction

R1-Forward Non-specific control primer was used as forward primer and ASC-Reverse Allele-specific common primer was used as reverse primer in control reaction. The PCR product of 5 µl was loaded in each well along with the DNA ladder (100 bp) in 1.5% agarose gel and samples were run at 90 volts for 45 min. PCR product was sent for sequencing. After sequencing of PCR product, the data obtained from sequencing was analysed in megalign software for detection absence/presence of mutation in gene.

Results and Discussion

The [20,15] . The sequence is submitted to BankIT NCBI and published with Accession no.-PP357617.

Discussion

The detection of resistance-associated mutations in the voltage-gated sodium channel (VGSC) gene clearly indicates that target-site insensitivity plays a major role in the development of synthetic pyrethroid resistance in these tick populations. A higher amount of resistance occurs in ticks as ticks have higher reproductive potential leading to the inheritance of resistant factors resulting in a greater number of resistant ticks. By knowing the mechanism of resistance development rapid and accurate diagnostic tools for early detection of resistance can be developed (FAO, 2004) [2] . AS-PCR enables the detection of specific point mutations in genes associated with resistance, thereby confirming synthetic pyrethroid resistance in Rhipicephalus microplus. (Morgan, 2009) [14] . The findings of the present study are consistent with earlier studies conducted in different parts of India. Deltamethrin resistance in six agro climatic zones of India in Indian tick populations and associated it with target-site insensitivity, including mutations in the sodium channel gene was reported by Vatsya et al. (2011) [21] . Amrutha et al. (2021) [1] reported multiple mutations (e.g., C190A, A271G) in the voltagegated sodium channel gene using AS-PCR, confirming their association with resistance. Rineshkumar, (2010) [16] revealed the deltamethrin resistance in Sultanpur and Bareilly isolates of R. microplus ticks with allele frequencies of 88% and 20% respectively. Similarly, Kumar et al. (2013) [8] Identified C190A mutation in domain II of the sodium channel gene, confirming its role in deltamethrin resistance., Gosh et al. (2015) [5] from Bihar, Kumar et [15] from Uttar Pradesh, Upadhya et al. (2020) [20] from Assam, Kumar et al. (2021) from Haryana detected the presence of mutation in S4-5 linker region of sodium channel gene in R. microplus ticks by performing PCR. The studies from abroad, Guerrero et al.

(2001) [7] from Mexico; Rodriguez-Vivas et al. (2012) [17] from Mexico; Lovis et al. (2012) [13] from America, Australia and Africa also reported the presence of a mutation in sodium channel gene in synthetic pyrethroidresistant R. (Boophilus) microplus ticks. The detection of resistance in the Nagpur region has significant practical implications. Since R. microplus is a major vector of diseases like babesiosis and anaplasmosis, ineffective tick control can lead to increased disease incidence, reduced animal productivity, and economic losses for farmers. Therefore, regular monitoring of resistance is crucial for effective disease and tick management.

Conclusion

The present study clearly indicated that Rhipicephalus microplus ticks from all selected talukas of Nagpur district of Maharashtra found to develop homozygous resistance to deltamethrin confirmed by molecular analysis. Mutation in the sodium channel gene, where cytosine was replaced by adenine, was also identified and is likely responsible for this resistance. The findings revealed that this drug will not be further effective for tick control in this region. Therefore, it is needed to restrict the indiscriminate use of deltamethrin and further monitoting for acaricidal resistance status in ticks is necessary. The adaptation of an alternative and integrated tick control approaches are important to deal the problem and to prevent further spread of resistance. Overall, the study provides the baseline information on the status of deltamethrin resistance in the Nagpur region, which can be implemented in planning effective and sustainable tick control strategies such as integrated tick management and the use of biological or ethnoveterinary methods for sustainable tick control.

Acknowledgement

AcknowledgmentsThe authors are grateful to associate dean, Nagpur Veterinary College of Maharashtra Animal and Fishery Sciences University Nagpur, for providing necessary research facilities.

References

1. Amrutha A, Bindu L, Siju J, Aravindakshan TV. Genotyping of deltamethrin resistance in Rhipicephalus (Boophilus) microplus population in Kerala, South India. Acta Parasitol. 2021;66:1031-1038.
2. FAO. Resistance management and integrated parasite control in ruminants: Guidelines. Module 1. Ticks: Acaricide resistance-diagnosis, management and prevention. Animal Production and Health Division; Rome; 2004. p. 25-77.
3. Furman DP, Loomis EC. The ticks of California (Acari: Ixodida). Bull Calif Insect Surv. 1984;25. University of California Press.
4. Geevarghese G, Mishra AC. Haematophagous ticks of India. Elsevier Publications; New Delhi; 2011.
5. Ghosh S, Kumar R, Nagar G, Kumar S, Sharma AK, Srivastava A, et al. Survey of acaricide resistance status of Rhipicephalus (Boophilus) microplus from Bihar, India. Ticks Tick Borne Dis. 2015;6(5):668-675.
6. Ghosh S, Nagar G. Problem of ticks and tick-borne diseases in India with emphasis on tick control research: A review. J Vector Borne Dis. 2014;51:259270.
7. Guerrero FD, Davey RB, Miller RJ. Use of allelespecific PCR assay to genotype pyrethroid resistant strains of Boophilus microplus. J Med Entomol. 2001;38(1):44-50.
8. Kumar R, Nagar G, Sharma AK, Kumar S, Ray DD, Chaudhuri P, et al. Survey of pyrethroid resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: Identification of C190A mutation. Acta Trop. 2013;125(2):237-245.
9. Kumar R, Sharma AK, Ghosh S. Menace of acaricide resistance in cattle tick Rhipicephalus microplus in India: Status and mitigation strategies. Vet Parasitol. 2020;278:108993.
10. Kumar R, Nagar G, Sharma AK, Kumar S, Ray DD, Chaudhuri P, et al. Detection of mutations in S4-S5 linker region of sodium channel gene in Rhipicephalus microplus. Ticks Tick Borne Dis. 2021;12(1):101-110.
11. Kumar S, Sharma AK, Nagar G, Rawat SS, Tiwari SS, Kumar R, et al. Characterization of acaricide resistance in tick isolates from Rajasthan, India. Indian J Anim Sci. 2016;86(1):14-23.
12. Lenka DR, Ravindran R, Jyothimol G, Udaykumar M, Reddy PMT, Sathish N, et al. Deltamethrin resistance in South Indian isolates of Rhipicephalus (Boophilus) microplus. Vet Parasitol Reg Stud Reports. 2016;5:3741.
13. Lovis L, Guerrero FD, Miller RJ, Bodine DM, Betschart B, Sager H. Distribution of sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus. Int J Parasitol Drugs Drug Resist. 2012;2:216-224.
14. Morgan JA, Corley SW, Jackson LA, Lew-Tabor AE, Moolhuijzen PM, Jonsson NN. Mutation in parasodium channel gene of cattle tick Rhipicephalus (Boophilus) microplus linked to pyrethroid resistance. Int J Parasitol. 2009;39(7):775-779.
15. Nagar G, Sharma AK, Kumar S, Saravanan BC, Kumar R, Gupta S, et al. Molecular mechanism of pyrethroid and organophosphate resistance in Rhipicephalus microplus. Exp Appl Acarol. 2018;75:319-331.
16. Rineshkumar. Characterization of resistance mechanism to pyrethroids in Rhipicephalus (Boophilus) microplus. PhD thesis. Indian Veterinary Research Institute; Izatnagar, Uttar Pradesh; 2010.
17. Rodríguez-Vivas RI, Hodgkinson JE, Rosado-Aguilar JA, Villegas-Pérez SL, Trees AJ. Prevalence of pyrethroid resistance phenotype and genotype in Rhipicephalus (Boophilus) microplus in Mexico. Vet Parasitol. 2012;184(2-4):221-229.
18. Sagar SV, Saini K, Sharma AK, Kumar S, Kumar R, Fular A, et al. Acaricide resistance in Rhipicephalus microplus from selected Indian states. Trop Anim Health Prod. 2020;52:611-618.
19. Soulsby EJL. Helminths, arthropods and protozoa of domesticated animals. 7th ed. Baillière Tindall; London; 1982.
20. Upadhaya D, Kumar B, Kumar S, Sharma AK, Fular A, Bisht N, et al. Characterization of acaricide resistance in Rhipicephalus microplus and evaluation of plant extracts. Vet Parasitol. 2020;277:109011.
21. Vatsya S, Yadav CL. Evaluation of acaricide resistance mechanisms in field populations of Rhipicephalus (Boophilus) microplus. Int J Acarol. 2011;37(5):405410.
22. Walker AR, Bouattour A, Camicas JL, Estrada-Pena A, Horak IG, Latif AA, et al. Ticks of domestic animals in Africa: A guide to identification of species. Bioscience Reports; Edinburgh; 2003.