Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

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.

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.

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

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.

RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge.

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (ß RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (ß/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (ß RBD HBsAg+SWE) or without HBsAg (ß RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only ß/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with ß RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC. IMPORTANCE Global COVID-19 vaccine distribution to low-income countries has been a major challenge of the pandemic. To address supply chain issues, RBD virus-like particle (VLP) vaccines that are cost-effective and capable of large-scale production were developed and evaluated for efficacy in preclinical mouse studies. We demonstrated that RBD-VLP vaccines protected K18-hACE2 mice against Alpha or Beta challenge similarly to Pfizer mRNA vaccination. Our findings showed that the VLP platform can be utilized to formulate immunogenic and efficacious COVID-19 vaccines.

SARS-CoV-2 receptor binding domain displayed on HBsAg virus-like particles elicits protective immunity in macaques.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain-hepatitis B surface antigen virus-like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>104) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log10) and nasal mucosa (~2.9 log10) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

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.

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.

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.

Acaricide resistance in Rhipicephalus microplus collected from selected districts of Madhya Pradesh, Uttar Pradesh and Punjab states of India.

The isolates of Rhipicephalus microplus collected from Madhya Pradesh (MP), Punjab (PJB) and Uttar Pradesh (UP) states of India were characterized using laboratory standardized adult immersion test (AIT) against macrocyclic lactone (ivermectin), synthetic pyrethroids (cypermethrin and deltamethrin), organophosphates (coumaphos and diazinon) and phenylpyrazole compounds (fipronil). Out of the six isolates tested, five isolates except MTH were resistant to deltamethrin and cypermethrin at level II with RF ranging from 16.4 to 24.02 and 7.05 to 13.2, respectively. In case of organophosphates, coumaphos was less effective showing resistance level II (RF 8.52-11.2) in all the six populations compared with diazinon to which three isolates (MHW, RWA and AGS) were resistant at level II. Except MTH, other five isolates were categorized at level I with RF ranging from 1.53 to 3.02 against ivermectin. The phenylpyrazole compound however was found effective, and none of the isolates could survive at a discriminating concentration. The possible strategy for the management of multi-acaricide-resistant ticks in the surveyed districts was discussed in the present study.

Characterization of acaricide resistance in Rhipicephalus microplus populations infesting cattle in northeastern India and assessment of local plant extracts for tick management.

In the present study, the tick isolates were collected from Assam state, of northeastern region (NER) and characterized using in vitro bioassay, biochemical and molecular assays. Comparing LC50 value of susceptible IVRI-I and larvae of field isolates, revealed that RF against deltamethrin was highest for Morigaon (MGN = 21.8) and lowest for Sonitpur (SNP = 3.3) isolate. The RF against cypermethrin was highest for Nagaon (NGO = 5.0) and lowest for Barpeta (BPT = 1.2) isolate. Against coumaphos, the highest RF of 4.5 was calculated for BPT (4.5) and lowest for NGO (1.3) isolate. While using adults based assay, highest RF of 24.68 against deltamethrin and lowest RF of 4.96 was determined for MGN and SNP isolate, respectively. In contrast to the results obtained using larvae, against cypermethrin, highest RF was recorded for Kamrup Metropolitan (KMP) while it was NGO isolate using larvae. In case of coumaphos, both larvae and adults of BPT isolate were also highly resistant and lowest RF was detected in SNP (2.30) isolate. All the isolates were susceptible to ivermectin. A significant correlation (p < 0.01) between deltamethrin resistance and higher expression of glutathioneS-transferase was observed while no correlation with esterase and monooxygenase enzymes activity was noted. For the development of possible ecofriendly control measure, different accessions of Argemone mexicana and Datura metel plant species were collected, extracted and screened against adult ticks. Two accessions, NEA-03 and NED-06 collected from Amlighat and Diphu (East Karbi Anglong) were more than 90 % effective. Further dose response study of these accessions determined the LC50 values of 4.86 and 3.96 %, respectively.The resistance status of the collected tick isolates was compared with the data generated from other regions having higher livestock population and possibility of exploitation of identified plant species for the development of natural antitick product is discussed.

Comparative impact of coumaphos, amitraz and plant extract of Ageratum conyzoides on the oogenesis of Rhipicephalus microplus.

The present experiment was conducted to evaluate and compare the impact of Ageratum conyzoides plant extract (ACE) with routinely used synthetic acaricides i.e., amitraz and coumaphos on the oogenesis of engorged adult females of Rhipicephalus microplus tick. On the day of dropping from the host, panoistic ovary of R. microplus appeared white in colour, horseshoe shaped, hollow tubular organ with immature oocytes predominantly in dorsal groove. Different developmental stages of oocytes (I-V) proceed simultaneously and asynchronously. Oocytes showed gradual increase in size, deep brown colored with accumulation of eggs in oviduct during 24-72 hours of development.At LC90 concentration a highly significant (p < 0.001) cessation of egg laying after exposure to amitraz and ACE while significant reduction (p < 0.01) of egg laying in coumaphos treated ticks was observed. Upon dissection of treated ticks, uterus and oviduct packed with eggs, which failed to pass out was observed. The histo-architectural alterations including presence of extensive vacuolation, alteration of oocyte morphology, deformation of chorion and disorganization of yolk granules were observed in the treated ovaries. Histochemically, low level of storage or synthesis of essential elements viz., proteins, polysaccharides and lipids in treated oocytes responsible for reduction of fertility and inhibition of progress of vitellogenesis was observed.

Identification and characterization of vaccine candidates against Hyalomma anatolicum-Vector of Crimean-Congo haemorrhagic fever virus.

Crimean-Congo haemorrhagic fever (CCHF) is a tick borne viral disease reported from different parts of the world. The distribution of the CCHF cases are linked with the distribution of the principal vector, Hyalomma anatolicum in the ecosystem. Presently, vector control is mainly dependent on repeated application of acaricides, results in partial efficacy and generated acaricide resistant tick strains. Amongst the different components of integrated management programme, immunization of hosts is considered as one of the sustainable component. To restrict CCHF virus spreading, use of anti-Hyalomma vaccines appears as a viable solution. Accordingly, present study was under taken to characterize and evaluate vaccine potential of two conserved molecules, ferritin2 (FER2) and tropomyosin (TPM). Silencing of the genes conferred a cumulative reduction (rejection + unable to engorge) of 61.3% in FER2 and 70.2% in TPM respectively. Furthermore, 44.2% and 72.7% reduction in engorgement weight, 63.6% and 94.9% reduction in egg masses in FER2 and TPM silenced ticks in comparison to LUC-control group was recorded. The recombinant protein, rHaFER2 was characterized as 35 kDa protein with pI of 5.84 and possesses iron binding domains. While rHaTPM is a 51kDa protein with pI of 4.94 having calcium binding domains. Immunization of cross-bred calves by rHaFER2 conferred 51.7% and 51.2% protection against larvae and adults of H. anatolicum challenge infestations. While rHaTPM conferred 63.7% and 66.4% protection against larvae and adults infestations, respectively. The results were comparable with the data generated by RNAi and it clearly showed the possibility for the development of anti-hyalomma vaccine to manage CCHF virus and Theileria annulata infection in human and animals.

Establishment of a multi-acaricide resistant reference tick strain (IVRI-V) of Rhipicephalus microplus.

Tick-borne diseases is a global threat and tick resistance to commonly used acaricides is a growing problem, thus calling for improved resistance monitoring tools. To aid in monitoring of resistance in field tick populations, a resistant colony of Rhipicephalus microplus was characterized with the aim to establish a reference multi-acaricide resistant tick strain. Using a standardized adult immersion test, the Lethal Concentration(LC)50 values for deltamethrin, cypermethrin, fenvalerate and diazinon against the laboratory selected resistant tick (LSRT) strain were determined as 306.7 ppm, 2776.9 ppm, 30262.1 ppm and 9458.7 ppm. Relative to the susceptible IVRI-I tick strain, the LSRT strain showed 4.78- and 5.84-fold increases in activity of esterases, a 6-fold increase for monooxygenases and a 2.24 fold increase for glutathione S-transferase. In the acetylcholinesterase 2 gene, 22 single nucleotide polymorphisms (SNPs) were identified in the LSRT strain. Four of these SNPs lead to amino acid substitutions and were consistently found in resistant field populations in India. A C190A mutation in the domain II S4-5 linker region of sodium channel gene resulting in a L64I amino acid substitution was recorded in the LSRT strain. Monitorable indicators for the maintenance of the strain, designated as the reference IVRI-V tick strain and representing the first established multi-acaricide resistant tick strain in India, were identified.

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of Rhipicephalus microplus tick collected from a northern state of India.

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9-58.6 and to one of the OP compounds, diazinon having RF of 3.5-13.7 but susceptible to coumaphos (RF < 1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.

Determination and validation of discriminating concentration of ivermectin against Rhipicephalus microplus.

Rhipicephalus microplus, the major cattle tick species of India is prevalent all over the country and causes huge economic loss directly or indirectly to the dairy industries. Chemical acaricides are playing an important role in managing tick infestations on livestock for many years and consequently, resistance to commonly used organophosphate (OP) and synthetic pyrethroid (SP) compounds has been reported. Subsequently, ivermectin (IVM) has been emerged as an alternative to manage OP and SP resistant ticks. However, with the increase of use during the last 5-8 years, there is a possibility of development of resistance and thus there is an urgent need to develop a robust resistance monitoring tool to safeguard the drug. Lethal concentrations for 50 and 95% mortality of treated ticks were determined to work out discriminating concentration (DC) in order to diagnose resistance in the field situation. The DC (2 x LC95) was determined as 93.54 ppm using an established reference susceptible IVRI-1 line of R. microplus adopting adult immersion test. For validation of DC, the resistance status was checked in seven tick isolates of R. microplus collected from northern and eastern regions of India. The RR50 and RR95 values of the field isolates against ivermectin were determined and were in the range of 1.56-8.25 and 1.93-27.58, respectively. All the collected isolates were found to have higher lethal concentration and resistance ratio in comparison to reference susceptible IVRI-1 tick line (LC50 = 21.68, LC95 = 46.77 ppm, RR = 1.0). Amongst the field isolates, the isolate collected from Fatehgarh Sahib district (FTG) of Punjab state showed highest RR50 of 8.25 indicating high level of resistance to IVM. The generated DC will be used for IVM resistance characterization of ticks infesting cattle in different parts of the country.

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus.

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6-99.4% and 98.2-99.7%) and among the tick species (77.3-99.3% and 85.1-99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5-89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2-53.4% and 72.0-74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p<0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.