Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans

Widespread resistance to chemotherapeutic agents is one of the biggest challenges facing human health and the agricultural industry, with resistance to all current anthelmintics now recorded and few new agents or vaccines available. Understanding the development of drug resistance in parasitic nematodes is critical to prolonging the efficacy of current anthelmintics, developing markers for monitoring drug resistance and is beneficial in the design of new chemotherapeutic agents or targets. This study describes the development of ivermectin-resistant strains of the model nematode Caenorhabditis elegans through step-wise exposure to increasing doses of ivermectin commencing with a non-toxic dose of 1 ng/ml. Resistant strains were developed that displayed a multidrug resistance phenotype with cross-resistance to the related drug moxidectin and to other anthelmintics, levamisole and pyrantel, but not albendazole. Resistance was associated with increased expression of the multidrug resistance proteins (MRPs) and P-glycoproteins. Resistance to ivermectin was reversible by the co-administration of MRP, P-glycoprotein and glutathione biosynthesis inhibitors, confirming the involvement of these proteins in resistance. In our model, resistance to low levels of ivermectin (相似文献   

Laboratory selection of a benzimidazole-resistant isolate of Trichostrongylus colubriformis for ivermectin resistance

In vivo ivermectin resistance was selected in an isolate of Trichostrongylus colubriformis (TcR) already known to be benzimidazole resistant. This was accomplished in sheep by using levels of ivermectin calculated to reduce the fecal egg output from each generation of T. colubriformis by congruent to 95%. The first indication of ivermectin resistance was observed with the F10. A dosage-titration trial comparing the parent TcR with the ivermectin-selected F21 demonstrated that the latter was congruent to 20 times more resistant to oral ivermectin therapy in experimentally infected sheep than was the parent isolate. Treatment of the F16 generation with 50 mg/kg of thiabendazole resulted in only 54% egg reduction and confirmed that benzimidazole resistance was stable.     

The sensory amphidial structures of Caenorhabditis elegans are involved in macrocyclic lactone uptake and anthelmintic resistance

Parasitic nematodes represent formidable pathogens of humans, livestock and crop plants. Control of these parasites is almost exclusively dependent on a small group of anthelmintic drugs, the most important of which belong to the macrocyclic lactone class. The extensive use of these drugs to control the ubiquitous trichostrongylid parasites of grazing livestock has resulted in the emergence of both single and multi-drug resistance. The expectation is that this resistance will eventually occur in the human parasites such as the common and debilitating soil transmitted nematodes and vector-borne filarial nematodes. While the modes of action of anthelmintics such as ivermectin, have been elucidated, notably in the model nematode Caenorhabditis elegans, the molecular nature of this resistance remains to be fully determined. Here we show that the anterior amphids play a key role in ivermectin uptake and mutations in these sensory structures result in ivermectin resistance in C. elegans. Random genetic mutant screens, detailed analysis of existing amphid mutants and lipophilic dye uptake indicate that the non-motile ciliated amphid neurons are a major route of ivermectin ingress; the majority of the mutants characterised in this study are predicted to be involved in intraflagellar transport. In addition to a role in ivermectin resistance, a subset of the amphid mutants are resistant to the non-related benzimidazole class of anthelmintics, raising the potential link to a multi-drug resistance mechanism. The amphid structures are present in all nematodes and are clearly defined in a drug-sensitive strain of Haemonchus contortus. It is predicted that amphidial drug uptake and intraflagellar transport may prove to be significant in the development of single and multi-drug resistance in the nematode pathogens of veterinary and human importance.     

Haemonchus contortus: effects of glutamate, ivermectin, and moxidectin on inulin uptake activity in unselected and ivermectin-selected adults.

Using [(3)H]inulin uptake as a measure of pharyngeal pumping activity, we have investigated and compared the effects of glutamate, ivermectin, and moxidectin on inulin uptake in susceptible and ivermectin-selected Haemonchus contortus. Inulin uptake is inhibited by glutamate, ivermectin, and moxidectin, at biologically relevant concentrations. Glutamate influences the responses to both ivermectin and moxidectin, suggesting that these three substances share a common mechanism of action. The effects of ivermectin on inulin uptake, but not moxidectin, are significantly altered as a result of selection with ivermectin. These results suggest that ivermectin and moxidectin may differ, to some extent, in their mode of action responses or mechanisms of resistance.     

Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.     

Screening for a new generation of anthelminthic compounds.In vitro selection of the nematodeCaenorhabditis elegans for ivermectin resistance

Development of resistance to ivermectin in vitro is reported for the first time. Two strains of Caenorhabditis elegans D5 and D6 able to grow on agar plates after treating with up to 3 mg ivermectin per L were selected. The parent strain N2 was extremely sensitive to ivermectin, its growth being inhibited by treatment with 0.1-0.2 mg IVM per L.     

Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.     

Influence of permethrin, diazinon and ivermectin treatments on insecticide resistance in the horn fly (Diptera: Muscidae)

The history of insecticide resistance in the horn fly, Haematobia irritans, and the relationship between the characteristics of horn fly biology and insecticide use on resistance development is discussed. Colonies of susceptible horn flies were selected for resistance with six insecticide treatment regimens: continuous single use of permethrin, diazinon and ivermectin: permethrin-diazinon (1:2) mixture; and permethrin-diazinon and permethrin-ivermectin rotation (4-month cycle). Under laboratory conditions, resistance developed during generations 21, 31 and 30 to permethrin, diazinon and ivermectin, respectively. The magnitude of resistance ranged from < 3-fold with ivermectin to 1470-fold with permethrin. Field studies demonstrated that use of a single class of insecticidal ear tag during the horn-fly season resulted in product failure within 3-4 years for pyrethroids and organophosphates, respectively. In laboratory studies, use of alternating insecticides or a mixture of insecticides delayed the onset of resistance for up to 12 generations and reduced the magnitude of pyrethroid resistance. In field studies, yearly alternated use of pyrethroids and organophosphates did not slow or reverse pyrethroid resistance (Barros et al., unpublished data), while a 2-year alternated use with organophosphates resulted in partial reversion of pyrethroid resistance. When pyrethroid and organophosphate ear tags were used in a mosaic strategy at two different locations, efficacy of products did not change during a 3-year period.     

Resistance of field isolates of Trichostrongylus colubriformis and Ostertagia circumcincta to ivermectin.

Twelve Romney lambs and 10 Angora goats were infected with 7000 infective third-stage larvae (89% Trichostrongylus, 11% Ostertagia) collected from goats suspected of harbouring ivermectin-resistant nematodes. On 28 days p.i., the lambs and goats were divided into treatment and control groups of six and five animals, respectively. The animals in the treatment groups were treated with ivermectin (0.2 mg/kg) and necropsied 35 days p.i. Faecal egg counts were estimated on days 28 and 35 p.i. and larval development assays (LDAs) were conducted on 22, 24, 26, 28, 30, 32, 34 and 35 days p.i. The ivermectin treatment reduced Trichostronglus colubriformis burdens by 39% and 13% and Ostertagia circumcincta by 33% and 0% in lambs and goats, respectively. When compared with a susceptible strain, the LDAs indicated a resistance factor before treatment in lambs for T. colubriformis of 2.6 and 1.5 with ivermectin and avermectin B2, respectively, which rose to 3.4 and 2.0 after treatment. The LD50 values of the two control groups were relatively constant throughout the experiment. Prior to ivermectin treatment the LD50 values of the treated groups were similar (P > 0.05) to the control groups but following ivermectin treatment their LD50 values increased steadily until the animals were killed on 35 days p.i. The LD50 values for ivermectin and avermectin B2 of sheep were always slightly higher and significantly different (P < 0.01) than those of goats indicating a host effect on this parameter. The greater reduction in worm counts in goats suggests a difference in the efficacy of ivermectin between lambs and goats. This is the first confirmed report of ivermectin resistance in a field strain of T. colubriformis.     

Genetic selection of low fertile Onchocerca volvulus by ivermectin treatment

Background

Onchocerca volvulus is the causative agent of onchocerciasis, or “river blindness”. Ivermectin has been used for mass treatment of onchocerciasis for up to 18 years, and recently there have been reports of poor parasitological responses to the drug. Should ivermectin resistance be developing, it would have a genetic basis. We monitored genetic changes in parasites obtained from the same patients before use of ivermectin and following different levels of ivermectin exposure.

Methods and Findings

O. volvulus adult worms were obtained from 73 patients before exposure to ivermectin and in the same patients following three years of annual or three-monthly treatment at 150 µg/kg or 800 µg/kg. Genotype frequencies were determined in β-tubulin, a gene previously found to be linked to ivermectin selection and resistance in parasitic nematodes. Such frequencies were also determined in two other genes, heat shock protein 60 and acidic ribosomal protein, not known to be linked to ivermectin effects. In addition, we investigated the relationship between β-tubulin genotype and female parasite fertility. We found a significant selection for β-tubulin heterozygotes in female worms. There was no significant selection for the two other genes. Quarterly ivermectin treatment over three years reduced the frequency of the β-tubulin “aa” homozygotes from 68.6% to 25.6%, while the “ab” heterozygotes increased from 20.9% to 69.2% in the female parasites. The female worms that were homozygous at the β-tubulin locus were more fertile than the heterozygous female worms before treatment (67% versus 37%; p = 0.003) and twelve months after the last dose of ivermectin in the groups treated annually (60% versus 17%; p<0.001). Differences in fertility between heterozygous and homozygous worms were less apparent three months after the last treatment in the groups treated three-monthly.

Conclusions

The results indicate that ivermectin is causing genetic selection on O. volvulus. This genetic selection is associated with a lower reproductive rate in the female parasites. We hypothesize that this genetic selection indicates that a population of O. volvulus, which is more tolerant to ivermectin, is being selected. This selection could have implications for the development of ivermectin resistance in O. volvulus and for the ongoing onchocerciasis control programmes.     

Molecular characterisation of a pH-gated chloride channel from <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis>

Reports of ivermectin resistance in scabies mites raise concerns regarding the sustainability of mass intervention programs for scabies worldwide and for the treatment of crusted scabies. Ligand gated ion channels (LGICs) are the primary targets of ivermectin in invertebrates. We report the molecular characterisation of SsCl—a novel LGIC from Sarcoptes scabiei var. hominis. While SsCl shows sequence similarity to other LGICs, phylogenetic analysis does not suggest strong homology to conventional glutamate, histamine or GABA gated channels. Instead, it is most similar to Drosophila pH-sensitive and group 1 clades. When expressed in Xenopus oocytes, SsCl forms a homomeric, pH-gated chloride channel that is irreversibly activated by ivermectin. These results provide the first confirmation that this group of LGIC exists in arachnids, and suggest that SsCl may be an in vivo target of ivermectin in S. scabiei.     

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.     

Chemical mutagenesis of the parasitic nematode Strongyloides ratti to isolate ivermectin resistant mutants

We describe a strategy for the mutagenesis of the free-living adult generation of Strongyloides ratti and selection of worms carrying new mutations in the subsequent F2 generation of infective larvae. We demonstrate that this strategy is successful via the selection of infective larvae that are resistant to the anthelmintic ivermectin at a concentration of 10 ng/ml. The majority of these larvae were unable to give rise to patent infections when used to infect parasite naive rats, implying that the majority of the ivermectin resistance mutations confer pleiotropic defects on parasitic, but not on free-living, development.     

Changes in anthelmintic resistance status of Haemonchus contortus and Trichostrongylus colubriformis exposed to different anthelmintic selection pressures in grazing sheep

This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of alternation between them, on existing levels of anthelmintic resistance in the nematode parasites Haemonchus contortus and Trichostrongylus colubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabendazole was shown to select positively against levamisole resistance in T. colubriformis resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between anthelmintic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.     

Study of the nematode putative GABA type-A receptor subunits: evidence for modulation by ivermectin

Two alleles of the HG1 gene, which encodes a putative GABA receptor alpha/gamma subunit, were isolated from Haemonchus contortus. These two alleles were shown previously to be associated with ivermectin susceptibility (HG1A) and resistance (HG1E), respectively. Sequence analysis indicates that they differ in four amino acids. To explore the functional properties of the two alleles, a full-length cDNA encoding the beta subunit, a key functional component of the GABA receptor, was isolated from Caenorhabditis elegans (gab-1, corresponding to the GenBank locus ZC482.1) and coexpressed in Xenopus oocytes with the HG1 alleles. When gab-1 was coexpressed with either the HG1A allele or the HG1E allele in Xenopus oocytes, gamma-aminobutyric acid (GABA)-responsive channels with different sensitivity to the agonist were formed. The effects of ivermectin on the hetero-oligomeric receptors were determined. Application of ivermectin alone had no effect on the receptors. However, when coapplied with 10 micro m GABA, ivermectin potentiated the GABA-evoked current of the GAB-1/HG1A receptor, but attenuated the GABA response of the GAB-1/HG1E receptor. We demonstrated that the coexpressed HG1 and GAB-1 receptors are GABA-responsive, and provide evidence for the possible involvement of GABA receptors in the mechanism of ivermectin resistance.     

Human Onchocerciasis: Modelling the Potential Long-term Consequences of a Vaccination Programme

Background

Currently, the predominant onchocerciasis control strategy in Africa is annual mass drug administration (MDA) with ivermectin. However, there is a consensus among the global health community, supported by mathematical modelling, that onchocerciasis in Africa will not be eliminated within proposed time frameworks in all endemic foci with only annual MDA, and novel and alternative strategies are urgently needed. Furthermore, use of MDA with ivermectin is already compromised in large areas of central Africa co-endemic with Loa loa, and there are areas where suboptimal or atypical responses to ivermectin have been documented. An onchocerciasis vaccine would be highly advantageous in these areas.

Methodology/Principal Findings

We used a previously developed onchocerciasis transmission model (EPIONCHO) to investigate the impact of vaccination in areas where loiasis and onchocerciasis are co-endemic and ivermectin is contraindicated. We also explore the potential influence of a vaccination programme on infection resurgence in areas where local elimination has been successfully achieved. Based on the age range included in the Expanded Programme on Immunization (EPI), the vaccine was assumed to target 1 to 5 year olds. Our modelling results indicate that the deployment of an onchocerciasis vaccine would have a beneficial impact in onchocerciasis–loiasis co-endemic areas, markedly reducing microfilarial load in the young (under 20 yr) age groups.

Conclusions/Significance

An onchocerciasis prophylactic vaccine would reduce the onchocerciasis disease burden in populations where ivermectin cannot be administered safely. Moreover, a vaccine could substantially decrease the chance of re-emergence of Onchocerca volvulus infection in areas where it is deemed that MDA with ivermectin can be stopped. Therefore, a vaccine would protect the substantial investments made by present and past onchocerciasis control programmes, decreasing the chance of disease recrudescence and offering an important additional tool to mitigate the potentially devastating impact of emerging ivermectin resistance.     

Further studies with a strain of Ostertagia ostertagi resistant to morantel tartrate.

Two experiments with a morantel resistant strain of Ostertagia ostertagi were carried out. In the first experiment five groups of five calves were infected with 60,000 larvae of this resistant strain. Calves of one group remained untreated, calves of the other groups were treated with morantel tartrate, oxfendazole, levamisole or ivermectin in the recommended doses. It was demonstrated that there was side resistance to levamisole, but not to oxfendazole or ivermectin. Compared with the untreated controls the reduction percentages of the worm burdens were 45.3 (morantel tartrate), 99.7 (oxfendazole), 83.5 (levamisole) and 100 (ivermectin). In the second experiment a comparison was made between the effect of levamisole against a morantel susceptible strain of O. ostertagi and the resistant strain. Two groups of five calves were infected with the susceptible strain and two groups with the resistant one. One group of each pair remained untreated, the other was treated with levamisole. The reduction percentages of the worm burdens were 99.6 (susceptible strain) and 63.6 (resistant strain). This result confirms the efficacy of levamisole to susceptible O. ostertagi and the side resistance of the morantel resistant strain.     

Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.     

The impact of ivermectin on onchocerciasis in villages co-endemic for lymphatic filariasis in an area of onchocerciasis recrudescence in Burkina Faso

In Burkina Faso, onchocerciasis was no longer a public health problem when the WHO Onchocerciasis Control Programme in West Africa closed at the end in 2002. However, epidemiological surveillance carried out from November 2010 to February of 2011, showed a recrudescence of infection in the Cascades Region. This finding was made at a time when ivermectin, a drug recommended for the treatment of both onchocerciasis and lymphatic filariasis, had been distributed in this area since 2004 for the elimination of lymphatic filariasis. It was surprising that ivermectin distributed for treating lymphatic filariasis had not prevented the recrudescence of onchocerciasis. Faced with this situation, the aim of our study was to evaluate the effectiveness of ivermectin on the onchocerciasis parasite. The percentage reduction in microfilarial load after treatment with ivermectin was used as a proxy measure for assessing possible resistance. A cohort study was carried out with 130 individuals who had tested positive for microfilariae of Onchocerca volvulus in 2010 using microscopic examination of skin-snip biopsies from five endemic villages. Subjects were followed from July 2011 to June 2012. The microfilarial load of each individual was enumerated by skin-snip biopsy in 2010, prior to the first ivermectin treatment against onchocerciasis under community guidelines. All individuals received two ivermectin treatments six months apart. In 2012, the microfilarial loads were determined again, six months after the second round of ivermectin and the reductions in parasite loads were calculated to measure the impact of the drug. The percentage reduction of the microfilarial loads ranged from 87% to 98% in the villages. In all villages, there was a statistically significant difference between the average microfilarial loads in 2010 and 2012. The level of reduction of microfilarial loads suggests that ivermectin is effective against the recrudescent population of O. volvulus in Cascades Region of Burkina Faso. Further investigations would be necessary to determine the causes of the recrudescence of onchocerciasis. (For French language abstract, see S1 Alternative Language Abstract—Translation of the Abstract into French by the authors.)     

Evaluation of acaricidal resistance status of Rhipicephalus microplus ticks from the hilly state (Uttarakhand) of India and evaluation of efficacy of a natural formulation for the management of resistant ticks

Experimental and Applied Acarology - The resistance status against deltamethrin, cypermethrin, coumaphos and ivermectin was assessed of Rhipicephalus microplus from five districts of Uttarakhand,...