Genotyping amitraz resistance profiles in Rhipicephalus microplus Canestrini (Acari: Ixodidae) ticks from Punjab, India.

Acaricide resistance is one of the greatest threats to sustainable and effective control of vector ticks worldwide. The amitraz resistance status in cattle tick, Rhipicephalus microplus populations collected from 18 districts of Punjab in north-western India were characterized using bioassay and molecular assays. The modified larval packet test was used and the resistance factors (RF) against amitraz for the field populations were in the range of 0.36-4.85, indicating level I resistance status in ten populations. Characterization of a partial segment of the octopamine/tyramine (OCT/Tyr) receptor gene of R. microplus field populations from Punjab revealed a total of 18 nucleotide substitutions in the coding region out of which 5 were non-synonymous substitutions. Three of these non-synonymous substitutions (T8P, V15I and A20 T) were earlier reported in American and South African populations of R. microplus. Among the two single nucleotide polymorphisms (A22C-T8P; T65C-L22S) potentially linked to amitraz resistance in American, South African and Zimbabwean resistant populations, only the T8P substitution was recorded from the Barnala population. The PCR-RFLP assay using EciI restriction enzyme was used for genotyping of the larvae as homozygous resistant (RR), homozygous susceptible (SS) and heterozygous (SR). Genotyping of 514 larval DNA samples from 18 field populations revealed 92.8 % larval population as SR and the remaining 7.2 % as RR genotypes. The percentage of resistant alleles in the tick populations was 53.6 (range 50.0-57.2) indicating its moderate distribution in the region. The present study is the pioneer report establishing the hypothesis that amitraz-resistance is recessively inherited and heterozygous individuals show phenotypic susceptibility to the drug in the Indian tick populations.

In vitro assessment of synergistic combinations of essential oils against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), is one of the most important ectoparasite of cattle, responsible for causing severe economic losses in the tropical and subtropical regions of the world. The development of resistance to most of the commonly used chemical acaricides has stimulated the search for new herbal products as an eco-friendly tick control alternative. The aim of the present study was to evaluate the in vitro acaricidal activity of essential oils (EOs) of cloves (Syzygium aromaticum), bark of cinnamon (Cinnamomum zeylanicum) and leaves of lemon grass (Cymbopogon citratus) against unfed larvae of R. (B.)microplus by larval packet test. Also, various combinations were prepared by mixing of equal concentrations of any two and all three EOs and used to study the effects of interactions of mixtures against larvae of R. (B.) microplus. The lethal concentrations at 50% (LC50), 90% (LC90) and 99% (LC99) with 95% confidence limits (CL) were estimated by Probit analysis using PoloPlus. The computer software, CompuSyn, was used for determining the effects of interactions (synergistic, additive or antagonistic) of EO mixtures by calculation of Combination index (CI) and Dose-reduction index (DRI). Among the individual and mixture of two EOs, cinnamon EO and cinnamon EO + lemon grass EO combination showed highest acaricidal activity against R. (B.) microplus larvae. Further, the combination of cinnamon EO + lemon grass EO showed high synergism with CI value of 0.381, followed by cinnamon EO + clove EO showing moderate synergism whereas, clove EO + lemon grass EO showed only additive effect, with favorable dose reduction for each constituent drug in all three combinations. The combination of all three EOs (1:1:1) showed high synergism (CI value of 0.376) and favorable dose reduction (DRI index of 8.19, 25.64 and 4.64 for clove, cinnamon and lemon grass EOs, respectively) against R. (B.) microplus larvae.

Discrimination, molecular characterisation and phylogenetic comparison of porcine Eimeria spp. in India.

Infections with Eimeria spp. are common in pigs worldwide, occasionally affecting animals clinically after weaning or during the fattening period when diarrhoea and weight loss can be observed upon infection with the more pathogenic species. Molecular characterization of pathogens is valuable to accurately delimit species and development novel diagnostics, although sequences which define Eimeria species that infect pigs are scarce. Only three of the eight common species are currently represented in GenBank. In this study we describe the occurrence of Eimeria species in pigs sampled in Punjab, India; going on to use the samples to generate new species-specific 18S rDNA sequences for all of the previously uncharacterised species. Using these data we report the first phylogenetic analyses to include the eight Eimeria species that commonly infect the domestic pig (Sus scrofa domesticus). Consideration of phylogenetic trees produced using Maximum Likelihood, Neighbour Joining, Maximum Parsimony and Unweighted Pair Group Method with Arithmetic Mean methods indicate that the 18S rDNA sequences present lower levels of genetic diversity than Eimeria which infect avian species and are insufficient to infer stable phylogenies.

Multiple mutations in the acetylcholinesterase 3 gene associated with organophosphate resistance in Rhipicephalus (Boophilus) microplus ticks from Punjab, India.

The organophosphate (OP) resistance status in Rhipicephalus (Boophilus) microplus ticks collected from seventeen districts located in the northwestern Indian state, Punjab were characterized using three data sets (bioassay, biochemical and molecular assays). Adult immersion test (AIT) was adopted and the resistance factors (RF) for the field isolates were determined. Resistance to malathion was detected in 12 isolates among which 11 showed level I resistance status while level II status was recorded in one isolate (RF of 5.35). To understand the possible mechanism of resistance development, acetylcholinesterase (AChE) activity and gene sequences of the AChE3 were analyzed. A significantly (P<0.001) higher level of percent uninhibited AChE activity was recorded in all field isolates (36.36±0.46-43.77±1.21) in comparison to the susceptible population (29.39±0.40). The AChE activity was positively correlated with RF against malathion with a correlation coefficient (r) of 0.359. Analysis of nucleotides and their deduced amino acids sequences of partial AChE3 gene revealed the presence of six amino acid substitutions (I48L, I54V, V71A, I77M, S79P and R86Q). Three novel amino acid substitutions (V71A, I77M and S79P) in partial AChE3 gene were also identified in some of the isolates which may possibly have a role in OP resistance development. The PCR-RFLP assay with HaeIII revealed the presence of restriction site corresponding to R86Q mutation in all the field isolates along with an additional restriction site in seven field isolates corresponding to V71A mutation. The results of the study indicate the involvement of both insensitive AChE and higher percent uninhibited AChE activity as the possible mechanism in these field isolates.

First report of ivermectin resistance in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Punjab districts of India.

The larval immersion test (LIT) was used on the progenies of Rhipicephalus (Boophilus) microplus collected from four districts of the Punjab state, India to test the resistance to ivermectin. The regression graphs of probit mortality of larval ticks plotted against log values of increasing concentrations of ivermectin were utilized for the determination of slope of mortality, lethal concentration for 50%(LC50), 95%(LC95), resistance ratios (RR50, RR95) and the resistance levels (RL). Values of the coefficient of determination (R(2)) for LIT ranged from 0.900 to 0.978, and the RR50 and RR95 values against ivermectin ranged from 2.97 to 8.85 and 2.42 to 8.47, respectively, indicating resistance status in all field isolates. On the basis of RR values, three field isolates (BAT, GUR, HOS) showed level II, whereas PTK isolate showed presence of level I resistance status against ivermectin. This appears to be the first report of ivermectin resistance in R. (B.) microplus from Punjab, India.

Esterase mediated resistance against synthetic pyrethroids in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Punjab districts of India.

Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SP), in Rhipicephalus (Boophilus) microplus collected from thirteen districts of Punjab (India) was carried out using adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentrations of drugs were utilized for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index and percentage inhibition of oviposition) the resistance levels were categorized. Against cypermethrin RFs of 1.48-11.22 were recorded in 12 isolates whereas, one isolate was susceptible. Resistance factors against deltamethrin were 2.4-38.54 and all 13 isolates were found to be resistant. Quantitative analysis of general esterase activity (measured by the production of the metabolite naphthol) revealed a range of 3.34 ± 0.30-13.75 ± 1.33 and 1.31 ± 0.15-8.09 ± 0.68 µmol/min/mg protein for α and ß-esterase activity, respectively in different field isolates. Further, multiple pairwise comparisons of the mean values with susceptible strain (Tukey, P = 0.05) revealed significant elevated levels of both α-esterase and ß-esterase in nine tick isolates resistant to both deltamethrin and cypermethrin. The data generated on acaricide resistant status and esterase mediated mechanism in ticks will help in formulating tick control strategy for the region.

A comparative study on cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum from Punjab (India).

A study to evaluate cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Muktsar and Mansa districts of Punjab state, India, was conducted by using adult immersion test (AIT). The regression graphs of probit mortality of ticks plotted against log values of concentrations of cypermethrin was utilized for the determination of slope of mortality, lethal concentration for 50% (LC50), and the resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index, and percentage inhibition of oviposition), the resistance levels were categorized. Resistance to cypermethrin was categorized as level II and I in R. (B.) microplus collected from Muktsar and Mansa districts, respectively, whereas, H. anatolicum from both locations showed a susceptible status. The RF values of Muktsar and Mansa field samples of engorged R. (B.) microplus (5.48 and 2.18, respectively) were much higher as those of engorged H. anatolicum (1.12 and 0.82, respectively) indicating a lower level and slower rate of development of cypermethrin resistance in multi-host ticks. The data generated in the current study might be of immense help in formulating suitable control measures against ticks and tick-borne diseases of animals.

Molecular detection of Anaplasma marginale infection in carrier cattle.

Anaplasma marginale infections are responsible for widespread morbidity and mortality particularly in crossbred and exotic breeds of cattle in the tropics and subtropics. In the present study, a semi-nested PCR assay was utilized for the detection of A. marginale infection in carrier cattle from different areas of Punjab state, India. An amplicon of 458 bp specific for msp5 of A. marginale was detected in 45.2% of blood samples when subjected to primary PCR assay against the routine blood smear examination, which revealed inclusion bodies in only 12.5% of samples. Semi-nested PCR employing product of samples negative by primary PCR produced the amplicons of desired size (345 bp) in 51% (29/57) of samples indicating that nested PCR, when coupled with primary PCR, resulted in increased sensitivity in detection of A. marginale infection in carrier cattle from 45.2 to 73.1%. These results suggest higher threshold detection limits of the nested PCR over the conventional technique used for diagnosis of anaplasmosis which is ideally suited for diagnosis of carrier cases which act as nidus for the spread of the infection to the susceptible stock in endemic areas.