Genetic Diversity of Rhipicephalus (Boophilus) microplus for a Global Scenario: A Comprehensive Review.

Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks.

Effect of ivermectin, amitraz and fipronil on midgut epithelium and digestive enzyme profile in Rhipicephalus microplus ticks (Acari: Ixodidae).

Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 µg/mL and 92.67 ± 3.72 µg/mL, respectively, as compared to the control group (150.0 ± 3.80 µg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 µmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 µmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.

Evolution of tick vaccinology.

Ticks represent a major concern for society worldwide. Ticks are also difficult to control, and vaccines represent the most efficacious, safe, economically feasible and environmentally sustainable intervention. The evolution of tick vaccinology has been driven by multiple challenges such as (1) Ticks are difficult to control, (2) Vaccines control tick infestations by reducing ectoparasite fitness and reproduction, (3) Vaccine efficacy against multiple tick species, (4) Impact of tick strain genetic diversity on vaccine efficacy, (5) Antigen combination to improve vaccine efficacy, (6) Vaccine formulations and delivery platforms and (7) Combination of vaccines with transgenesis and paratransgenesis. Tick vaccine antigens evolved from organ protein extracts to recombinant proteins to chimera designed by vaccinomics and quantum vaccinomics. Future directions will advance in these areas together with other novel technologies such as multiomics, AI and Big Data, mRNA vaccines, microbiota-driven probiotics and vaccines, and combination of vaccines with other interventions in collaboration with regions with high incidence of tick infestations and tick-borne diseases for a personalized medicine approach.

Perception of Ticks and Tick-Borne Diseases Worldwide.

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges.

Acaricides resistance in Rhipicephalus microplus and expression profile of ABC-transporter genes in the sampled populations.

Currently, livestock owners manage tick infestations using chemicals, but the method is increasingly losing effectiveness as resistant tick populations have established in the field conditions. Thus, to develop effective tick management strategies, monitoring of resistance in most predominant tick species, Rhipicephalus microplus was targeted. The ticks were collected from eleven districts of Madhya Pradesh and one district of Punjab and tested against deltamethrin (DLM), cypermethrin (CYP), coumaphos (CMP), ivermectin (IVM) and fipronil (FIP), through adult immersion and larval packet tests. The field isolates were highly resistant to DLM [Resistance factor (RF) = 3.98-38.84]. Against CYP, resistance was observed in BWN (Barwani; RF = 2.81) and MND (Mandsaur; RF = 3.23) isolates. Surprisingly, most of the isolates were susceptible to CMP (0.34-1.58). Emerging level of resistance against IVM (1.05-4.98) and FIP (0.40-2.18) was also observed in all the isolates. Significantly elevated production of esterases (p < 0.01) was 90% correlated with RF of DLM while no positive correlation between production of monooxygenase and Glutathione S-transferase with RF to DLM was noted. Multiple sequence analysis of S4-5 linker region of the sodium channel gene of all the isolates revealed a point mutation at 190th position (C190A) which is associated with DLM resistance. Treatment of resistant LDH (Ludhiana) isolate with IVM resulted in upregulation of RmABCC2 gene and insignificant upregulation of RmABCC1 and RmABCB10 genes indicating the probability of linking IVM resistance with over-expression of RmABCC2 gene. The possible tick management strategies are discussed.

Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean-Congo Hemorrhagic Fever Virus.

Hyalomma anatolicum is the principal vector for Theileria annulata, T. equi, and T. Lestoquardi in animals and the Crimean-Congo hemorrhagic fever virus in humans. Due to the gradual loss of efficacy of the available acaricides against field tick populations, the development of phytoacaricides and vaccines has been considered the two most critical components of the integrated tick management strategies. In the present study, in order to induce both cellular and humoral immune responses in the host against H. anatolicum, two multi-epitopic peptides (MEPs), i.e., VT1 and VT2, were designed. The immune-stimulating potential of the constructs was determined by in silicoinvestigation on allergenicity (non-allergen, antigenic (0.46 and 1.0046)), physicochemical properties (instability index 27.18 and 35.46), as well as the interaction of constructs with TLRs by docking and molecular dynamics analysis. The immunization efficacy of the MEPs mixed with 8% MontanideTM gel 01 PR against H. anatolicum larvae was determined as 93.3% and 96.9% in VT1- and VT2-immunized rabbits, respectively. Against adults, the efficacy was 89.9% and 86.4% in VT1- and VT2-immunized rabbits, respectively. A significant (p < 0.001) reduction in the anti-inflammatory cytokine (IL-4) and significantly higher IgG response was observed in a VT1-immunized group of rabbits as compared with the response observed in the control group. However, in the case of the VT2-immunized rabbits, an elevated anti-VT2 IgG and pro-inflammatory cytokine (IL-2) (>30 fold) along with a decreased level of anti-inflammatory cytokine IL-4 (0.75 times) was noted. The efficacy of MEP and its potential immune stimulatory responses indicate that it might be useful for tick management.

Co-Immunization Efficacy of Recombinant Antigens against Rhipicephalus microplus and Hyalomma anatolicumTick Infestations.

The immunoprophylactic management of ticks is the most effective option to control tick infestations and counter spread the acaricide resistance problem worldwide. Several researchers reported an inconsistent efficacy of the single antigen-based immunization of hosts against different tick species. In the present study, to develop a multi-target immunization protocol, proteins from Rhipicephalus microplus BM86 and Hyalomma anatolicum subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the BM86, SUB, and TPM coding genes amongst Indian tick isolates of targeted species were 95.6-99.8%, 98.7-99.6%, and 98.9-99.9%, respectively, while at the predicted amino acid level, the identities were 93.2 to 99.5, 97.6 to 99.4, and 98.2 to 99.3%. The targeted genes were expressed in the eukaryotic expression system, pKLAC2-Kluyveromyces lactis, and 100 µg each of purified recombinant protein (Bm86-89 kDa, SUB-21 kDa, and TPM-36 kDa) mixed with adjuvant was injected individually through the intramuscular route at different sites of the body on days 0, 30, and 60 to immunize cross-bred cattle. Post-immunization, a statistically significant (p < 0.001) antibody response (IgG, IgG1, and IgG2) in comparison to the control, starting from 15 to 140 days, against each antigen was recorded. Following multi-antigen immunization, the animals were challenged twice with the larvae of R. microplus and H. anatolicum and theadults of H. anatolicum, and a significant vaccine efficacy of 87.2% and 86.2% against H. anatolicum larvae and adults, respectively, and 86.7% against R. microplus was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.

Evaluation of cytotoxic effects of amitraz and fipronil on digestive, reproductive and neural processes of engorged Rhipicephalus microplus (Acari: Ixodidae) female.

Fipronil and amitraz are potentially toxic compounds used for controlling ticks infesting pet and livestock. The use of fipronil on large animals was limited because of its high costs while amitraz is still persisting in the market since its introduction over four decades ago. Though resistance in ticks against these pesticides has been reported worldwide since 2000, the toxicity of these chemicals at cellular level in ticks is still poorly understood. The present study aimed to examine the gross and cellular impact of fipronil and amitraz on the gut, ovaries and synganglion of engorged Rhipicephalus microplus females. Fipronil and amitraz treated tick groups showed formation of a large number of vacuoles of different size throughout the cytoplasm of generative cells whereas sessile, residual and detached digestive cells were very low in numbers. The treatment of ticks resulted in the formation of vacuolations at periphery of all oocytes. Ultra-thin sections of the synganglion revealed severe rupture of neural lamella and perineurium with apoptosis of neural cells after fipronil treatment whereas in the amitraz treated ticks, severe destruction of neuropile region and extensive vacuolation of type I and II cells of cortical region as compared to the unexposed ticks were noted.

Molecular detection of pathogens in the ova and unfed larvae of Rhipicephalus annulatus and Haemaphysalis bispinosa ticks infesting domestic cattle of south India.

This study aimed to investigate the presence of pathogens in the engorged ticks infesting domestic cattle, their ova, and unfed larvae. The engorged female ticks infesting domestic cattle of Wayanad district of Kerala, south India were collected and kept for oviposition. The dead females after the complete oviposition, their egg masses, and unfed larvae were screened for the presence of various pathogens by specific PCRs. The presence of Babesia bigemina, Anaplasma marginale, A. phagocytophilum, and Rickettsia spp. similar to R. raoultii was confirmed in Rhipicephalus annulatus ticks, their egg masses, and unfed larvae. Theileria orientalis was detected in Rh. annulatus females, but not in their egg masses or progenies. The presence of A. phagocytophilum and Rickettsia spp. similar to R. raoultii was confirmed in Haemaphysalis bispinosa ticks, their egg masses, and unfed larvae too. The presence of coinfections of B. bigemina with A. phagocytophilum and A. marginale were detected in Rh. annulatus ticks and their progenies.

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.

The resistance status against deltamethrin, cypermethrin, coumaphos and ivermectin was assessed of Rhipicephalus microplus from five districts of Uttarakhand, through adult immersion test and larval packet test. The field isolates were highly resistant to deltamethrin (median resistance ratio [RR50] = 9.10-29.13-fold) followed by cypermethrin (2.23-3.55). Surprisingly the isolates were susceptible to coumaphos (0.34-3.17). Emerging resistance against ivermectin (1.55-3.27) was also observed in all the isolates. Elevated levels of esterases (enzyme ratio = 2.93-5.84-fold), glutathione S-transferases (5.10-10.06) and monooxygenases (1.68-4.02) in resistant fields isolates were highly correlated (47.4-86.0%) with the resistant factor (RR50) determined by bioassay. All the isolates except Uttarkashi possess mutation at the 190th position in domain II of the sodium channel gene. As a mitigation strategy an Ageratum conyzoides-based characterized natural formulation was tested against all the isolates and was found effective at the concentration of 10.1-11.5%. The possibility of using the natural formulation for the management of multi-acaricide resistant ticks is discussed.

Comparative susceptibility of Rhipicephalus microplus collected from the northern state of India to coumaphos, malathion, deltamethrin, ivermectin, and fipronil.

The chemical-based tick management method is gradually losing its clutch due to the establishment of resistant ticks. For development of region-specific tick management strategies, the present study was aimed to evaluate the comparative resistance profile of Rhipicephalus microplus isolates collected from seven districts of Uttar Pradesh, a northern state of India. Comparative analysis of the dose-response data using adult immersion test (AIT) against coumaphos, malathion, deltamethrin, ivermectin, and fipronil revealed that all the isolates were resistant to discriminating concentration of deltamethrin having LC50 of 295.12-436.52 ppm with a resistance ratio of 22.02-32.58. An emerging low level of ivermectin resistance (resistance ratio, RR50 = 1.03-2.26) with LC50 in the range of 22.39-48.98 ppm was found across the isolates. The coumaphos was highly effective against all except Amethi (AMT) isolate. Similarly, malathion was efficacious against most of the isolates except Pratapgarh (PRT) and Sultanpur (SUL) isolates showing LC50 of 5128.61 and 5623.41 ppm, respectively. All the isolates were responsive to fipronil. Comparative detoxifying enzymes profiles revealed a significant correlation between the increased activity of esterase and deltamethrin resistance. The GST activity was 51.2% correlated with RR50 of malathion while esterase activity was significantly correlated (68.9%) with RR50 of coumaphos. No correlation between the ivermectin resistance and enzyme activity was established. Multiple sequence analysis of S4-5 linker region of the sodium channel gene of all the isolates revealed a point mutation at 190th position (C190A) which is associated with deltamethrin resistance. The possible tick management strategies in this part of the country are discussed.

Analysis of Genetic Diversity in Indian Isolates of Rhipicephalus microplus Based on Bm86 Gene Sequence.

The control of cattle tick, Rhipicephalus microplus, is focused on repeated use of acaricides. However, due to growing acaricide resistance and residues problem, immunization of animals along with limited use of effective acaricides is considered a suitable option for the control of tick infestations. To date, more than fifty vaccine candidates have been identified and tested worldwide, but two vaccines were developed using the extensively studied candidate, Bm86. The main reason for limited vaccine commercialization in other countries is genetic diversity in the Bm86 gene leading to considerable variation in vaccine efficacy. India, with 193.46 million cattle population distributed in 28 states and 9 union territories, is suffering from multiple tick infestation dominated by R. microplus. As R. microplus has developed multi-acaricide resistance, an efficacious vaccine may provide a sustainable intervention for tick control. Preliminary experiments revealed that the presently available commercial vaccine based on the BM86 gene is not efficacious against Indian strain. In concert with the principle of reverse vaccinology, genetic polymorphism of the Bm86 gene within Indian isolates of R. microplus was studied. A 578 bp conserved nucleotide sequences of Bm86 from 65 R. microplus isolates collected from 9 Indian states was sequenced and revealed 95.6-99.8% and 93.2-99.5% identity in nucleotides and amino acids sequences, respectively. The identities of nucleotides and deduced amino acids were 94.7-99.8% and 91.8-99.5%, respectively, between full-length sequence (orf) of the Bm86 gene of IVRI-I strain and published sequences of vaccine strains. Six nucleotides deletion were observed in Indian Bm86 sequences. Four B-cell epitopes (D519-K554, H563-Q587, C598-T606, T609-K623), which are present in the conserved region of the IVRI-I Bm86 sequence, were selected. The results confirm that the use of available commercial Bm86 vaccines is not a suitable option against Indian isolates of R. microplus. A country-specific multi-epitope Bm86 vaccine consisting of four specific B-cell epitopes along with candidate molecules, subolesin and tropomyosin in chimeric/co-immunization format may provide a sustainable option for implementation in an integrated tick management system.

Current status and future prospects of multi-antigen tick vaccine.

Ticks are blood sucking ectoparasite that transmit several pathogens to humans and animals. Tick management focusing on use of chemicals has several drawbacks including development of multi-acaricide resistant tick populations. To minimize the use of chemicals on animals and on the environment, immunization of natural hosts is considered a viable component of Integrated Tick Management System. Most of the tick vaccine trials are focused on single antigen immunization directed against homologous challenge. From commercial point of view, vaccination against one given tick species is not a feasible option. In this context, multi-antigen vaccines comprising of candidate antigens of multiple tick species or both ticks and tick-borne pathogens have commercial potential. Different strategies are considered for the development of multi-antigen tick and/or tick-borne pathogen vaccines. Further, the efficacy of vaccine can be improved by adopting the 'omics' tools and techniques in selection of novel antigens and efficient delivery like Lipid Nano Particle (LNP)-mRNA vaccines, viral vector vaccine, live vector vaccine etc. into the host. The subject has been reviewed to address the current status of multi antigen tick vaccines and formulations of the future strategies for the control of TTBDs of human and animals.

Association between overexpression of cytochrome P450 genes and deltamethrin resistance in Rhipicephalus microplus.

Cytochrome P450 monooxygenases mediated metabolic detoxification has been recognized as one of the mechanisms involved in resistance to pyrethroids, which is a class of pesticides that includes acaricides such as deltamethrin. Several cytochrome P450 (CYP) genes were identified in arthropod pests which are upregulated in response to exposure to pesticides used as acaricides. However, to date, limited information is available with respect to CYP genes and their response to acaricide exposure in ticks. We cloned and sequenced four CYP genes, the CYP41, CYP3006G8, CYP319A1 and CYP4W1 from reference susceptible IVRI-I strain of Rhipicephalus microplus. The expression pattern of the genes was investigated using qPCR in reference susceptible IVRI-I, pyrethroid-resistant IVRI-IV and multi-acaricide resistant IVRI-V strains. The effect of a single exposure of deltamethrin, at a concentration of 2600 µg/mL and 299.7 µg/mL on IVRI-IV and IVRI-V strains, respectively, on the expression of the four CYP genes was evaluated. In IVRI-IV strain, the CYP41 gene was highly overexpressed (FC 8.72) while CYP3006G8 was underexpressed with FC of 0.06. All the four genes were overexpressed in IVRI-V strain. After exposure to deltamethrin, the CYP3006G8 transcript levels were significantly upregulated at all time intervals in both resistant strains with the highest FC of 11.62 at 12 h in IVRI-IV and 13.38 at 3 h in IVRI-V. Our results suggest that the constitutive overexpression of CYP41 and deltamethrin induced upregulation of CYP3006G8 contribute to the development of pyrethroid resistance, specifically deltamethrin, in these two reference strains.

Development and Validation of a Reversed Phase High Performance Liquid Chromatography-Photodiode Array Detection Method for Simultaneous Identification and Quantification of Coumarin, Precocene-I, ß-Caryophyllene Oxide, α-Humulene, and ß-Caryophyllene in Ageratum Conyzoides Extracts and Essential Oils from Plants.

BACKGROUND: Ageratum conyzoides is an aromatic plant. It is considered as an invasive and cosmopolite weed, widely spread in tropical and subtropical regions. Phytochemicals such as benzopyrenes, flavonoids, and terpenoids are reported from A. conyzoides. OBJECTIVE: Development and validation of a reversed-phase HPLC-photodiode array (PDA) detection method for simultaneous identification and quantification of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene in extracts of A. conyzoides and essential oils was carried out. METHODS: Separation of analytes was achieved on a RP-18 (250 mm × 4.6 mm, 5 µm) column using a solvent system comprising of a mixture of acetonitrile and water with 0.05% trifluoroacetic acid in gradient elution mode at ambient temperature with flow rate of 1 mL/min. RESULTS: The retention time of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 4.38, 12.86, 20.10, 33.34, and 35.11 min, respectively. Limits of detection for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene were 2.5, 2.5, 2.5, 0.025, and 2.5 µg/mL, respectively. Similarly, LOQ were 10, 10, 10, 0.10, and 10 µg/mL for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß- caryophyllene, respectively. Repeatabilities (RSD, %) values for intraday and interday precision for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 0.765-2.086 and 0.886-2.128; 0.879-1.672 and 0.979-1.825; 0.696-2.418 and 0.768-2.592; 1.728-2.362 and 1.965-2.378; 1.615-2.897 and 1.658-2.906, respectively. CONCLUSIONS: The separation of five analytes was achieved within 50 min. The developed and validated HPLC-PDA method was successfully applied for identification and quantification of above five analytes in A. conyzoides extracts and essential oils. The method could be used for meeting the characterization criteria of phytoformulations.

Acaricidal Activity of Artemisia nilagirica Leaves Against Rhipicephalus (Boophilus) annulatus Ticks.

Since time immemorial, human beings have used various parts of plants in either prevention or treatment of ailments. Plants are rich sources of secondary metabolites such as alkaloids, steroids, terpenoids, flavonoids, and phenolic compounds with a high structural diversity. Many plants/herbs with specific biological activities such as antitumor, antioxidant, anti-inflammatory, antifungal, sedative, and acaricidal activity have been reported. Artemisia nilagirica (C. B. Clarke) Pamp. (Compositae) is a plant traditionally used for insect control in the southern part of India. Previous studies have demonstrated the activity of Artemisia species against pests. The present study thus evaluates the acaricidal activity of crude ethanolic extract of A. nilagirica leaves and its fractions against Rhipicephalus (Boophilus) annulatus. Ticks are ectoparasites that transmit several protozoal, viral, and rickettsial diseases. In south India, R. (B.) annulatus is the commonly observed tick species. Control of these acarine parasites that adversely affect milk and meat production is a tough task. Chemical acaricides such as organophosphates, synthetic pyrethroids, amitraz, and ivermectin are commonly used in tick control. The high cost, environmental hazards, and development of acaricidal resistance are some of the drawbacks of these chemical acaricides. Plant-based formulations are one of the promising approaches for the control of ectoparasites. Previously, extracts from various medicinal/aromatic plants were reported for acaricidal activity from our laboratory, such as Tetrastigma leucostaphylum (Dennst.) Alston, Chassalia curviflora (Wall.) Thwaites, Jatropha curcas L., and Ageratum conyzoides Hieron. Biochemical quantification, fluorescence analysis, and primary phytochemical analysis are already reported for the ethanolic extract and its fractions of areal parts of A. nilagirica. Phytochemical characterization of ethanolic extract of A. nilagirica from Kerala, India was shown to have the presence of terpenoids, flavonoids, steroids, saponins, fixed oils and fats, tannins, and glycosides.

Standardization of tick specific biochemical tools for estimation of esterases, monooxygenases and glutathione S-transferase for characterization of acaricide resistance.

Protocols to determine metabolic resistance in ticks were mainly derived from reports published using mosquitoes and agriculturally important insects without prior standardization. In the present study, biochemical assays were standardized to quantify acaricide metabolizing enzymes in tick homogenates. Three variables viz., age, number of larvae and reaction time were optimized using reference susceptible IVRI-I and deltamethrin resistant IVRI-IV (Resistance Factor = 194) tick strains. The optimum conditions for estimation of general esterases were 10-15 day old 40 larvae with 15 mins reaction time, 15-20 day old 40 larvae with 20 mins reaction time for Glutathione S- transferase, while 10-15 day old 80 larvae with 5 mins reaction time for monooxygenase. The standardized protocols were further validated in multi acaricide resistant strain (IVRI-V) and in nine field isolates having variable resistant factors to different acaricides. In all the nine heterogeneous field isolates, a significant correlation (p < .05) between resistance to synthetic pyrethroids and over-expression of esterases and monooxygenase was noticed. Similarly, esterases and GST activities were significantly correlated with resistance to organophosphates. The details of the assay protocol are explained for adoption in different laboratories.

Molecular Investigation of the Status of Ticks on Infected Cattle for Coxiella burnetii in India.

PURPOSE: In Indian subcontinent, the epidemiological studies on the status of ticks in the transmission of Coxiella burnetii have not been explored comprehensively. The objective of the present study was to investigate the status of ticks for C. burnetii among coxiellosis positive cattle. METHODS: The present study was carried out in three locations of the northern states of India. A total of 1648 tick samples were collected from the tick infested cattle (n = 146) that were tested positive for coxiellosis by indirect serum-ELISA assay and/or the trans-PCR assay. The tick samples were screened using the trans-PCR assay targeting species-specific IS1111 transposase gene of C. burnetii. The sequencing of PCR products was planned to differentiate C. burnetii and Coxiella-like bacteria (CLB). RESULTS: The collected ticks were identified as Rhipicephalus microplus (n = 1049), Hyalomma anatolicum (n = 416), and Hyalomma spp. (n = 183). On molecular investigation, none of the collected tick samples were found to be positive for the IS1111 gene. CONCLUSION: The findings of the present study ruled out the involvement of ticks in circulation of the pathogen within the cattle population that were screened. However, extensive epidemiological studies are needed to conclusively rule out or establish the role of ticks as a competent vector for C. burnetii transmission in cattle and other hosts.

Emergence of fipronil resistant Rhipicephalus microplus populations in Indian states.

The intensive usage of chemical acaricides for the control of the cattle tick Rhipicephalus microplus has resulted in the development and establishment of multi-acaricide resistant populations. Fipronil, a phenylpyrazole insecticide, is currently marketed in India for the management of this important veterinary tick species. Here, we tested Indian isolates of R. microplus which have developed multi-acaricide resistance, for their susceptibility to fipronil. Twenty-five field isolates from five agro-climatic zones of the country were collected and tested by adult immersion test (AIT) and larval packet test (LPT). Sixteen isolates with resistance factor (RF) in the range of 1.56-10.9 were detected using LPT, whereas only 11 isolates with RF ranging from 1.05 to 4.1 were detected using AIT. A significant variation of RF between both tests was found, which raises doubt about the suitability of larva-based assays in screening of fipronil resistance. The data indicated possible cross-resistance between groups of acaricides in fipronil-resistant tick populations.

Menace of acaricide resistance in cattle tick, Rhipicephalus microplus in India: Status and possible mitigation strategies.

Livestock in general and dairy sector in particular plays a vital role in the Indian economy and in the socio-economic development of millions of people. Rhipicephalus microplus, a one-host and the most prevalent cattle tick in various agro climatic zones of India. This tick species is responsible for severe economic losses to livestock owners both through the direct effects of blood sucking, injection of toxins and indirectly by transmitting pathogens. Due to inadvertent and indiscriminate use of chemicals to kill ticks, field tick population has developed resistance to almost all chemicals that are used to manage them. The intention of this review is to provide the current status of resistance in ticks, the possible mechanisms of resistance operating in the tick population, factors contributing to the development of resistance and the managemental strategies.