Use of polymerase chain reaction: Restriction fragment length polymorphism to detect acaricidal resistance to synthetic pyrethroids in Boophilus microplus ticks of South India.

BACKGROUND: Boophilus microplus is an important ectoparasite of livestock. Apart from transmitting diseases, heavy tick burden can decrease production and damage hides. The synthetic pyrethroids which are advantageous over other acaricides for treatment of this infestation are now losing their efficacy due to development of resistant strains of ticks.
MATERIALS AND METHODS: Boophilus microplus ticks with a previous history of acaricidal treatment especially synthetic pyrethroids (SP) such as cypermethrin, deltamethrin and flumethrin were randomly collected from different pockets of four Southern States of India namely Tamil Nadu, Kerala, Karnataka, Andhra Pradesh, and Puducherry from cattle. Deoxyribonucleic acid extracted from pooled adult B. microplus tick from each State was subjected to polymerase chain reaction-restriction fragment length polymorphism to detect point mutation in carboxyl esterase gene.
RESULTS: A product size of 372 bp was obtained for cattle tick samples collected from all over Southern States of India.
CONCLUSIONS: B. microplus ticks found in Southern part of India are not resistant to commonly used SP.

INTRODUCTION

Boophilus microplus is considered to be the most important ectoparasite of livestock in the world. Heavy tick burdens on animals can decrease production and damage hides. The one-host tick B. microplus is an economically important ectoparasite of cattle involved in the transmission of the apicomplexan protozoa Babesia bigemina and Babesia bovis, the etiological agent of bovine babesiosis[1] and anaplasmosis (caused by Anaplasma marginale). Tick control mainly relies on the use of acaricides. Currently, synthetic pyrethroids (SP) such as deltamethrin, flumethrin, cypermethrin are commonly used acaricides in tick control. SP are advantages over other acaricides in many ways. They are easily absorbed and distributed. The prime target of action by SP is interference with the sodium channels of the arthropod nerve axons resulting in delayed repolarization and eventual paralysis. Efficacy of these acaricides have reduced due to the development of resistant strains of ticks.[2] There are several ways arthropods can become resistant to insecticides. One such mechanism is a mutation in the voltage gated sodium channel gene[3] or point mutations in esterase – encoding genes[4] or may be due to multiple mechanisms at the same time. Baffi et al.[5] identified point mutations in a fragment of a putative carboxyl esterase that causes pyrethroid resistance in strains of B. microplus from Brazil. In India, Shailaja,[6] studied the status of resistance in B. microplus ticks in seven agro climatic zones of Tamil Nadu. Detection of resistance to pyrethroids in ticks becomes imperative to avoid indiscriminate use of acaricides and expenditure incurred. In addition, knowledge about the resistance status of ticks is important to device an alternative tick control plan in case of resistance. Hence, the present study was envisaged to determine whether the specific mutations conferring resistance to pyrethroids due to point mutations in carboxyl esterase gene has occurred in B. microplus of South India using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP).

MATERIALS AND METHODS

Collection of ticks

B. microplus ticks were collected randomly from 25 to 50 infested cattle with a history of treatment with SP. The tick samples were collected from animals reported for recurring tick infestation in veterinary dispensaries and veterinary hospital of the four different states (Tamil Nadu, Andhra Pradesh, Karnataka, Kerala) and one Union Territory Puducherry.

Method of collection

Cattle treated with SP such as cypermethrin, deltamethrin, and flumethrin were selected irrespective of the number of such treatments. The adult ticks collected from the body of the infested cattle were kept alive in specimen vials using a porous cloth as a cover to enable respiration and ticks, which were collected from far off places, were preserved in 70% alcohol until further processing. The vials were individually labeled with a date, place of collection, and with the recent application of acaricide.

Molecular techniques to evaluate acaricide resistance

Genomic deoxyribonucleic acid (DNA) extraction

Tick samples (20 mg/tube – around 3 to 4 ticks) were snap frozen with liquid nitrogen and taken in micro centrifuge tubes. The samples were ground with the micro pestle to make it as a pulp. DNA extraction was carried out using the Genomic DNA Extraction kit (RBC Bioscience Corp., Taiwan [YGT 50]) based on manufacture's protocol. The purified DNA was stored in elution buffer at −20°C for future use.

PCR

PCR reaction was carried out using the DNA polymerase kit (Sigma, D4545, USA) in Eppendorf Master Cycler® ep, Germany with the cyclic conditions as per the protocol with the minor modifications.

Detection of mutation in carboxyl esterase gene

A PCR-RFLP was done to detect Asp → Asn substitution in carboxyl esterase gene of B. microplus ticks. The primers sequences were used from the publications of Guerrero et al.[7] The PCR product was analyzed by submarine gel electrophoresis as described by Sambrook et al.[8] with minor modifications and products were visualized using UV transilluminator and photographed (BioRad).

RESULTS AND DISCUSSION

Tick resistance to acaricides is a global problem and threat to the livestock and allied industries. Resistance to various acaricides has been reported in the past. Target-site insensitivity was the major mechanism of resistance to SP in B. microplus field strains, involving the presence of a sodium channel mutation. However, esterase-based mechanisms, other mutations or combinations of mechanisms can also occur.[910] Amongst ixodid ticks, molecular detection of acaricidal resistance to SP has been extensively studied in B. microplus. PCR-RFLP has been used to identify the presence of point mutation in B. microplus[4] in carboxyl esterase gene where a G → A substitution at nucleotide 1120 occurs, which creates an Eco RI site in the mutant strain. This can be detected by Eco RI digestion of the amplification products. The wild strain produces a product size of 372 bp, which remains unaltered even after digesting it with Eco RI while a mutant strain will be fragmented into 300 and 72 bp due to a single amino acid substitution. The present study was carried out to detect mutation if any in carboxyl esterase gene using PCR-RFLP. A product size of 372 bp was obtained after digesting the amplified PCR product of all the samples subjected for the reaction [Figure 1]. From the present study, it is thus inferred that B. microplus ticks found in Southern part of India are not resistant to commonly used SP. Susceptibility to SP was evaluated in Mexican B. microplus tick populations.[10] We also tested samples from other locations in the state and did not find mutations in carboxyl esterase gene as well as mutations in sodium channel gene (data not shown). Potentially, there is a different resistance mechanism involved, such as esterase detoxification as detected in Mexican ticks by Jamroz et al.[9] Alternatively, there may be a different sodium channel gene mutation in these ticks. Jonsson et al.[11] reported on a new sodium channel gene mutation that confers pyrethroid resistance in Australian ticks. The reinfestation of animals with ticks irrespective of treatment may be due to incorrect dosage of acaricides or other managemental factors. However, further research need to be conducted on suspected resistant populations in order to confirm the resistance and study the mechanistic basis of this resistance, before strong conclusions can be drawn regarding the state of acaricide resistance in India.

Figure 1

From left: Lane 1: TN -Tamil Nadu, Lane 2: KR-Kerala, Lane 3: KA-Karnataka, Lane 4: AP-Andhra Pradesh, Lane 5: PY-Puducherry, Lane 6: ML-Molecular ladder