Sachin Kumar

Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agro-climatic regions of India

A cross sectional study was conducted to assess the prevalence of synthetic pyrethroids (SP) resistance in Rhipicephalus (Boophilus) microplus in India. Twenty-seven areas located in six agro-climatic regions were selected for the collection of engorged ticks using two stage stratified sampling procedure. Adult immersion test (AIT) and larval packet test (LPT) were optimized using laboratory reared susceptible line of R.(B.) microplus (IVRI-I) for determination of 95% lethal concentration (LC(95)) of deltamethrin (29.6 ppm in AIT and 35.5 ppm in LPT) and cypermethrin (349.1 ppm in AIT and 350.7 ppm in LPT). The AIT with a discriminating dose (2 × LC(95)) was used to detect deltamethrin and cypermethrin resistance in the field isolates of R.(B.) microplus. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The overall prevalence of SP-resistant R.(B.) microplus among the sampled farms was 66.6% (18/27). Out of these 18 areas, resistance to deltamethrin at level I was detected in 08 areas (resistance factor=2.0-4.9), at level II in 09 areas (RF=5.2-11.8), at level III in 01 area (RF=34.9) and at level IV in 01 area (RF=95.7). The resistance to cypermethrin was detected in 16 areas and level of resistance was detected at level I in 10 areas (RF=2.06-4.64) and at level II in 06 areas (RF=5.13-9.88). The middle-gangetic and trans-gangetic plains revealed higher density of resistant ticks where intensive cross bred cattle population are reared and the SP compounds are commonly used. The data generated on acaricide resistant status in ticks will help in formulating tick control strategy for the country.

Diazinon resistant status in Rhipicephalus (Boophilus) microplus collected from different agro-climatic regions of India

The resistance status of Rhipicephalus (Boophilus) microplus to Diazinon was evaluated in 20 locations situated at various agro-climatic regions of India. Adult immersion test (AIT) was optimized using laboratory reared acaricide susceptible IVRI-I strain of R. (B.) microplus and minimum effective concentration of Diazinon was determined as 635.2 ppm. The discriminating dose (DD) was worked out as 1270.4 ppm and was tested on female ticks collected from organized and unorganized farms located at different agro-climatic regions of India. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The average resistance factor (RF) of 6.1 (level II) was recorded in the ticks collected from the northern sub-temperate trans-gangetic plains while high average RF values of 26.65 (level III) was recorded in the ticks collected from tropical middle-gangetic plains. The tropical middle gangetic plain has a very high density of animal populations where farmers use Diazinon for tick control, for agricultural practices and for mosquito control. Due to the continuous use of OP compounds the environmental load of Diazinon has become high in the area. This is the first experimental data generated on Diazinon resistant status in ticks of India.

In vitro and in vivo efficacy of Acorus calamus extract against Rhipicephalus (Boophilus) microplus

To develop a environment friendly control measure against cattle tick, Rhipicephalus (Boophilus) microplus, medicinally important plants were identified and extracts were prepared. Twelve 95% ethanolic, thirteen 50% hydroethanolic and nine hot water extracts were prepared and tested against laboratory reared homogenous colony of R. (B.) microplus. Amongst the 34 extracts, 26 extracts showed no mortality within 72xa0h of application while 12.0u2009±u20094.9% to 35.0u2009±u20099.6% mortality of treated ticks was recorded in other extracts. Of the effective extracts, the extract prepared from rhizome of Acorus calamus proved highly efficacious and 100% final mortality within 14 DPT was recorded. The LC85 value of the extract was determined as 11.26. In vivo experiments confirmed the efficacy of the extract up to 42%, and repeat application was required after 7 DPT. The extract was found safe and no reaction was observed when animals were treated with 50% of the concentration, which was five times of the concentration used for in vivo studies. The possibility of using the extract for the control of cattle tick is discussed.

Acaricide resistance status in Indian isolates of Hyalomma anatolicum.

The multi host tick, Hyalomma anatolicum, is the commonest Hyalomma species in India and cattle serves as the main host of this species. A study to evaluate the acaricide resistance of H. anatolicum to deltamethrin, cypermethrin and diazinon was conducted in 20 areas located in three agro climatic regions known to have abundance of the species. Results obtained by the “larval packet test” (LPT) showed a low grade resistance (level-I, RFxa0<5) in the tick species to both deltamethrin and cypermethrin in 10 areas and higher grade resistance (level-II, RFxa0<25) to deltamethrin in one area, where intensive use of synthetic pyrethroids are practiced for tick control. Low grade resistance to diazinon (level I) was recorded in six areas where organophosphates compounds are extensively used for agricultural practices allowing increased exposure of the moulting instars of the ticks to these chemicals. Biochemical analysis of the samples suggested involvement of esterase and alterations of acetylcholinesterase in the resistance mechanisms.

Survey of pyrethroids resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: identification of C190A mutation in the domain II of the para-sodium channel gene.

Monitoring acaricide resistance and understanding the underlying mechanisms are critically important in developing strategies for resistance management and tick control. Eighteen isolates of Rhipicephalus (Boophilus) microplus collected from four agro-climatic regions of India were characterized and the resistant data were correlated with bioassay results, esterase enzyme activities and with the presence/absence of point mutation in the para-sodium channel gene. The adult immersion test was standardized to assess the level of resistance and resistant factors (RF) in the range of 1.2-95.7 were detected. Out of eighteen isolates, three were categorized as susceptible (RF<1.4), five isolates at level I (RF=1.5-<5), eight at level II (RF=5.1-<25), and one isolate each at level III (RF=26-<40) and level IV (RF=>41). The esterase enzyme ratio and survival% of tick isolates was observed significantly (p<0.001) correlated with correlation coefficient (r) in α- and β-esterase activity. The correlation of determination (R(2)) for α- and β-esterase activity indicated that 73.3% and 55.3% data points of field isolates were very close to the correlation lines. For detection of point mutation, three sites (mutation in domain IIS6, T2134A mutation in domain IIIS6 and C190A mutation in domain IIS4-5 linker) of sodium channel gene were amplified and sequenced. Comparative sequence analysis identified a cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region of para-sodium channel gene in six isolates and in reference deltamethrin resistant IVRI-IV line. The occurrence of mutation in the tick isolates having high resistance factor suggested that target site insensitivity and enhanced esterase activity is the possible mechanism of resistance to deltamethrin in the Indian isolates of R. (B.) microplus. These results also concluded that the mutation site in Indian tick isolates is similar to Australian and Brazilian tick isolates while it is different in tick isolates from Mexico and North America. This is the first report of occurrence of mutation in para-sodium channel gene of deltamethrin resistant Indian isolates of R. (B.) microplus.

Acaricidal properties of Ricinus communis leaf extracts against organophosphate and pyrethroids resistant Rhipicephalus (Boophilus) microplus

Indian cattle ticks have developed resistance to commonly used acaricides and an attempt has been made to formulate an ecofriendly herbal preparation for the control of acaricide resistant ticks. A 95% ethanolic extract of Ricinus communis was used to test the efficacy against reference acaricide resistant lines by in vitro assay. In in vitro assay, the extract significantly affects the mortality rate of ticks in dose-dependent manner ranging from 35.0 ± 5.0 to 95.0 ± 5.0% with an additional effect on reproductive physiology of ticks by inhibiting 36.4-63.1% of oviposition. The leaf extract was found effective in killing 48.0, 56.7 and 60.0% diazinon, deltamethrin and multi-acaricide resistant ticks, respectively. However, the cidal and oviposition limiting properties of the extract were separated when the extract was fractionated with hexane, chloroform, n-butanol and water. The HPTLC finger printing profile of R. communis leaf extract under λ(max.) - 254 showed presence of quercetin, gallic acid, flavone and kaempferol which seemed to have synergistic acaricidal action. In vivo experiment resulted in 59.9% efficacy on Ist challenge, however, following 2nd challenge the efficacy was reduced to 48.5%. The results indicated that the 95% ethanolic leaf extract of R. communis can be used effectively in integrated format for the control of acaricide resistant ticks.

Determination of discriminating dose and evaluation of amitraz resistance status in different field isolates of Rhipicephalus (Boophilus) microplus in India

Field tick isolates of Rhipicephalus (Boophilus) microplus were collected from eleven districts located in the northern and eastern states of India to access the resistance status to “Amitraz”. Adult immersion test was optimized using laboratory reared acaricide susceptible IVRI-I line and minimum effective concentration was determined as 487.7xa0ppm with 95xa0% confidence interval of 455.8–521.8. The discriminating concentration was determined as 975.4xa0ppm and was tested on female ticks collected by two stage stratified sampling from organized dairy farms and villages. Based on three variables, viz., mortality, egg masses and reproductive index, the resistance level was categorized. Resistance to amitraz was detected at level I in 3 isolates (RFxa0=xa01.56–5.0), at level II in 6 isolates (RFxa0=xa09.3–23.3) and at level III in 1 isolate (RFxa0=xa027.3) whereas one isolate was found susceptible. The highest resistance was found in the SKR isolate (RFxa0=xa027.3) and minimal resistance was detected in the N-24P isolate (RFxa0=xa01.56). These experimental data will help in designing tick control strategy which is suffering from acaricide failure and to overcome development of resistance in ticks.

Survey of acaricides resistance status of Rhipiciphalus (Boophilus) microplus collected from selected places of Bihar, an eastern state of India

Monitoring acaricide resistance in field ticks and use of suitable managemental practices are essential for controlling tick populations infesting animals. In the present study, the acaricide resistance status in Rhipicephalus (Boophilus) microplus ticks infesting cattle and buffaloes of five districts located in the eastern Indian state, Bihar were characterized using three data sets (AIT, Biochemical assays and gene sequences). Adult immersion test (AIT) was adopted using seven field isolates and their resistance factor (RF) was determined. Six isolates (DNP, MUZ, BEG, VSH, DRB and SUL) were found resistant to both deltamethrin and diazinon and except VSH all were resistant to cypermethrin. One isolate (PTN) was susceptible with a RF below 1.5. To understand the possible mode of resistance development, targeted enzymes and gene sequences of the para sodium channel and achetylcholinesterase 2 (AChE2) were analyzed. The esterase, monooxygenase and glutathione-S-transferase (GST) activity of reference susceptible IVRI-I line was determined as 2.47 ± 0.007 nmol/min/mg protein, 0.089 ± 0.0016 nmol/mg of protein and 0.0439 ± 0.0003 nmol/mg/min respectively, which increased significantly in the resistant field isolates. However, except esterases, the fold increase of monooxygenase (1.14-2.27 times) and GST (0.82-1.53 times) activities were not very high. A cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region was detected only in one isolate (SUL) having RF of 34.9 and in the reference deltamethrin resistant line (IVRI-IV). However, the T2134A mutation was not detected in domain IIIS6 transmembrane segment of resistant isolates and also in reference IVRI-IV line despite of varying degree of resistance. The flumethrin specific G215T and the recently identified T170C mutations were also absent in domain II sequences under study. Four novel amino acid substitutions in AChE2 gene of field isolates and in organophosphate (OP) resistant reference IVRI-III line were identified which can possibly have a role in resistance development.

Determination and establishment of discriminating concentrations of malathion, coumaphos, fenvalerate and fipronil for monitoring acaricide resistance in ticks infesting animals

Discriminating concentrations (DCs) of malathion, coumaphos, fenvalerate and fipronil were determined to monitor acaricide resistance in field conditions. The LC99 values with 95% confidence interval for malathion, coumaphos, fenvalerate and fipronil were 5126.8 (5011.5-5240.7), 131.0 (120.4-142.5), 2257.5 (2198.1-2318.4) and 6.2 (5.87-6.55), respectively. The narrow confidence intervals in LC50 and LC99 of adult immersion test (AIT) and larval packet test (LPT) affirming the homogeneity of IVRI-I line. Variation in LPT based LC50 and LC99 values of malathion (55.9ppm) and coumpahos (28.4ppm) compared to those obtained in AIT indicating that larvae were more susceptible to these chemicals. The DCs for malathion, coumaphos, fenvalerate and fipronil against adults were determined as 10253.6, 262.0, 4515.0 and 12.4ppm while against larvae the values were 111.8, 56.8, 4014.0 and 9.6ppm, respectively. The working efficiency of DCs was successfully tested in field tick isolates. Establishment of country specific DCs of commonly used insecticides for monitoring of resistance in field ticks is emphasized for establishing tick control strategies.

Identification of potential plant extracts for anti-tick activity against acaricide resistant cattle ticks, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

AbstractnTo develop an eco-friendly tick control method, seven plant extracts were prepared using 50 and 95xa0% ethanol and evaluated for acaricidal activity against cattle tick, Rhipicephalus (Boophilus) microplus. The adult immersion test was adopted for testing different extracts. Based on 72xa0h screening criterion, 95xa0% ethanolic extracts of Datura metel fruits and Argemone mexicana whole plant were found effective showing more than 50xa0% mortality of treated ticks. The 95xa0% ethanolic extracts of D. metel fruits and A. mexicana whole plant exhibited acaricidal and reproductive inhibitory effects on treated ticks. The LC90 values of D. metel and A. mexicana extracts were determined as 7.13 and 11.3xa0%, respectively. However, although both the extracts were found efficacious against deltamethrin-resistant IVRI-4 and multi-acaricide resistant IVRI-5 lines of R. (B.) microplus, they caused less mortality than treated ticks of the reference IVRI-I line. Phytochemical studies indicated the presence of alkaloids and glucosides in D. metel fruits and alkaloids, terpenoids, flavonoids and phenolics in A. mexicana whole plant extracts. The results indicated that these botanicals may play an important role in reducing the use of chemicals for tick control and possibly to manage resistant tick population in environment friendly manner.