CHARACTERISATION OF AMITRAZ RESISTANCE IN RHIPICEPHALUS SANGUINEUS
Loading...
Date
2023-01-25
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
COLLEGE OF VETERINARY AND ANIMALS SCIENCES MANNUTHY, THRISSUR , KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Abstract
Rhipicephalus sanguineus, a three-host tick distributed across the world is an obligate ectoparasite with its primary host as dogs vectoring various pathogenic agents causing canine disease conditions such as babesiosis, ehrlichiosis and Q-fever. Use of chemical acaricides ubiquitously for the control of ticks had led to development of acaricide resistance against most of these chemicals. Early diagnosis of acaricidal resistance is necessary to avoid widespread dispersal of resistance and the awareness of mechanisms imparting resistance can help in devising resistance diagnosis tools such as molecular markers.
The present study was conducted to assess the development of resistance to amitraz in brown dog ticks. Dog ticks were collected from naturally infested dogs, and the engorged female R. sanguineus s. l. were reared in laboratory conditions for egg laying and larval hatching. Adult immersion tests were performed to evaluate the effects of amitraz on mortality, reproductive index and percentage inhibition of oviposition. Non-linear regression analysis on mortality of ticks and log amitraz concentrations were done to calculate the LC50 of each isolate, that were utilized to calculate the resistance factors used to determine the resistance levels. The AIT results showed that resistance to amitraz at resistance levels II, III, IV is prevalent in different regions of Thrissur. And the negative slopes of reproductive index on log amitraz concentrations indicated that amitraz has a negative effect on egg laying.
Larval packet test (LPT) was done and discriminating dose (DD) was arrived. The LPT-DD with only a single concentration of amitraz at 1200 ppm (DD), were performed to determine the survivability percentage of larvae exposed to amitraz. Resistance status was ascribed based on the percentage survivability of exposed larvae. The LPT-DD revealed that 48.57 per cent of field isolates were resistant of which 72.22 per cent were highly resistant with up to 90 per cent survival rate at the DD. Larvae from six of the resistant tick isolates were utilised to perform full-dose response bioassays (modified LPT) for each isolate revealing that the LC50 of each resistant tick isolate was very much higher than the field recommended dose of amitraz (250 ppm). Two levels of resistance level II and III were detected in four and two isolates, respectively.
Molecular characterisation of resistance was done by PCR-RFLP, amplifying the exon 3 region of octopamine receptor gene of R. sanguineus s. l. followed by restriction digestion with the RE Tth111I to detect the mutation at position C75T. The results revealed that 97.5 per cent of larvae were homozygous susceptible (CC), while heterozygous susceptible (CT) genotypes were detected in 2.5 per cent larvae at position C75T. However, both susceptible and resistant populations were detected by bioassays in these isolates. The homozygous resistant genotype (TT) was not observed. Nucleotide sequence analysis was performed to identify mutations. Novel non-synonymous substitutions at positions T131C, A157G and G208A were observed in resistant tick isolates, which needs further studies to associate it with amitraz resistance.