Acaricide resistance status of deltamethrin and coumaphos in Hyalomma anatolicum ticks collected from different districts of Haryana.

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, ß-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Understanding the resistance mechanisms of Rhipicephalus microplus ticks to synthetic pyrethroids and organophosphates in south-west regions of Haryana, North India.

Chemical control of tick infestation on dairy farms in India strongly relies upon the use of synthetic pyrethroids (deltamethrin) and organophosphate (coumaphos) drugs. Therefore, the present manuscript aims to investigate the resistance status of Rhipicephalus microplus ticks against these acaricides. Fully engorged adult R. microplus ticks were randomly collected from 8 dairy farms in North India and evaluated for acaricide resistance by using the Larval Packet Test (LPT). Of these, ticks collected from one and three farms showed the emergence of Level I acaricide resistance against deltamethrin and coumaphos, respectively. Significant positive correlations were found in the enzymatic activity (α-esterase, ß-esterase, glutathione-S-transferase, and mono-oxygenase) of R. microplus tick resistant against coumaphos. Native electrophoretogram analysis showed six different types of esterase activity in R. microplus (EST-1b to EST-6b), and EST-5b activity was more predominantly expressed in resistant ticks. Further, inhibitor studies using various esterase inhibitors suggested that EST-5b is a putative acetylcholine-esterase (AchE), and increased expression of one of the AchE might be responsible for the emergence of acaricide resistance. Further, no mutations were detected in the carboxylesterase (G1120A) and domain II S4-5 linker region (C190A) of the sodium channel genes of resistant R. microplus ticks, indicating that increased expression of detoxification enzymes was the probable mechanism for the development of acaricide resistance in the resistant ticks.

Effects of duration of exposure, co-supplementation with iron and Nippostrongylus brasiliensis infection on accumulation of trichloroacetic acid-insoluble copper in rats given molybdate in drinking water.

Thiolation can convert molybdate (MoO4) into a series of thiomolybdates (MoSxO4-x) in the rumen, terminating in tetrathiomolybdate (MoS4), a potent antagonist of copper absorption and, if absorbed, donor of reactive sulphide in tissues. Systemic exposure to MoS4 increases trichloroacetic acid-insoluble copper (TCAI Cu) concentrations in the plasma of ruminants and induction of TCAI Cu in rats given MoO4 in drinking water would support the hypothesis that rats, like ruminants, can thiolate MoO4. Data on TCAI Cu are presented from two experiments involving MoO4 supplementation that had broader objectives. In experiment 1, plasma Cu concentrations (P Cu) tripled in female rats infected with Nippostrongylus brasiliensis after only 5 days exposure to drinking water containing 70 mg Mo L-1, due largely to an increase in TCAI Cu; activities of erythrocyte superoxide dismutase and plasma caeruloplasmin oxidase (CpOA) were unaffected. Exposure for 45-51 days did not raise P Cu further but TCA-soluble (TCAS) Cu concentrations increased temporarily 5 days post infection (dpi) and weakened the linear relationship between CpOA and TCAS Cu. In experiment 2, infected rats were given less MoO4 (10 mg Mo L-1), with or without iron (Fe, 300 mg L-1), for 67 days and killed 7 or 9 dpi. P Cu was again tripled by MoO4 but co-supplementation with Fe reduced TCAI Cu from 65 ± 8.9 to 36 ± 3.8 µmol L-l. Alone, Fe and MoO4 each reduced TCAS Cu in females and males when values were higher (7 and 9 dpi, respectively). Thiolation probably occurred in the large intestine but was inhibited by precipitation of sulphide as ferrous sulphide. Fe alone may have inhibited caeruloplasmin synthesis during the acute phase response to infection, which impacts thiomolybdate metabolism.

Effects of Molybdate and Tetrathiomolybdate Supplementation of Drinking Water on Immature Rats Infected with Nippostrongylus brasiliensis. 2. Copper Status and Tissue Molybdenum Accretion.

Molybdate (MoO4) and tetrathiomolybdate (MoS4) supplementation of rats via drinking water had opposite effects on the establishment of Nippostrongylus brasiliensis larvae but both induced hypercupraemia, temporarily inhibited activities of superoxide dismutase in liver and duodenum after infection and enlarged the femoral head. Effects of MoO4 and MoS4 on activities of caeruloplasmin oxidase (CpO) in plasma, erythrocyte superoxide dismutase (ESOD) and tissue copper (Cu) and molybdenum (Mo) were compared to test the hypothesis that species lacking a rumen can thiolate MoO4. Three groups of 18 immature Wistar rats were given Mo (70 mg/L as MoO4) or MoS4 (5 mg/L) via drinking water or remained untreated; all received a commercial, cubed diet and 12 from each group were infected with larvae of N. brasiliensis. Rats were killed 7-9 days later and liver, kidney, spleen, heart, muscle (quadriceps), brain and bone (femur) removed for Cu and Mo analysis. Plasma Cu was greatly increased by MoO4 and MoS4, without changing CpO activity, but the effect was more variable with MoO4 and accompanied by a smaller decrease in ESOD. Tissue Cu and Mo were increased by MoS4 in all tissues examined except brain and bone, correlating with plasma Cu and with each other; relationships were strongest in spleen, followed by kidney. MoO4 also increased soft tissue Cu and Mo but increases were generally smaller than those induced by MoS4 and correlations between the two elements and with plasma Cu generally weaker. Since hypercupraemia and correlated increases in liver and kidney Cu and Mo are characteristic of systemic thiomolybdate (TM) exposure, we conclude that MoO4 was partially thiolated to give a different TM profile from that produced by MoS4. The pathophysiological significance of systemic exposure to di- and tri-TM merits investigation in non-ruminants as agents of chelation therapy and in ruminants as agents of short-lived TM toxicity on Mo-rich pastures.

Effects of Chronic Exposure to Molybdate and Tetrathiomolybdate Supplementation of Water on Immature Rats Acutely Infected with Nippostrongylus brasiliensis. 1. Effects on Outcome of Infection and Host Health.

Low molybdate (MoO4) exposure via drinking water in mature rats infected with Nippostrongylus brasiliensis raised liver and plasma copper (Cu) concentrations. The possibility that anthelmintic effects were attributable to conversion of MoO4 to tetrathiomolybdate (MoS4) in a non-ruminant species was investigated by giving three groups of 18 immature rats drinking water containing 70 mg Mo l-1 as MoO4 (group A), 5 mg Mo l-1 as MoS4 (group B) or no supplement (group C), while receiving a commercial cubed diet. After 41 days, 12 rats from each group were inoculated subcutaneously with 2,000 L3-stage N. brasiliensis larvae. Subgroups were killed 7, 8 or 9 days post infection (dpi), when adult worms are normally expelled, and enzyme markers for the inflammatory response to infection were measured in plasma or liver. Male rats given MoS4 prior to infection grew more slowly than those given MoO4. Eight dpi, females given MoS4 had lost more bodyweight than those in group C, while those given MoO4 had gained weight. Mean worm counts at 7 dpi were 160, 65 and 250 ± 30.6 (SE), respectively, in groups C, A and B, and differed significantly from each other (P <0.05) but only rats given MoO4 remained infected 9 dpi (mean worm count 52 ± 16.4): Faecal egg counts followed a broadly similar pattern. Both Mo sources pre-empted increases in liver and duodenal superoxide dismutase activity, induced by infection 7 and 9 dpi, respectively, in group C and enlarged the femur: neither source prevented hypertrophy of the small intestine and a rise in serum mast cell protease concentration caused by infection. Since data for plasma Cu concentration and caeruloplasmin oxidase activity, reported separately, indicated MoO4 was thiolated in vivo, differences between Mo sources may be attributable to differences in the degree of thiolation, extent of thiomolybdate exposure and rates of thiomolybdate degradation at critical times in host or parasite development.

Comparative study of esterases in deltamethrin and diazinon resistant Rhipicephalus microplus and Hyalomma anatolicum ticks collected from the Trans-Gangetic plains of India.

A comparative analysis of esterases in susceptible and resistant ticks revealed six types of esterases (EST-1b, EST-2b, EST-3b, EST-4b, EST-5b and EST-6b) in Rhipicephalus microplus and four types (EST-1h, EST-2h, EST-3h, EST-4h) in Hyalomma anatolicum using α-naphthyl acetate substrate. Inhibition studies with eserine sulfate, p-chloromercuribenzoate, copper sulphate and phenylmethylsulfonyl fluoride revealed a marked variation in band intensity between susceptible and resistant ticks, with the latter being more intense. Qualitative expression of EST-4b along with an extra band of EST-5b and EST-6b were indicative of deltamethrin and diazinon resistance in R. microplus, whereas qualitative expression of EST-4h was probably responsible for diazinon resistance in H. anatolicum. The data suggest that increased esterase activity may represent a detoxification strategy leading to the development of resistance in these tick populations.

Sexual alteration in antioxidant response and esterase profile in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks.

The objective of the study was to reveal physiological link between sex specific engorgement pattern and evading mechanism of ticks against oxidative stress as well as acaricides. Quantitative determination of nitric oxide radical scavenging, superoxide dismutase activity and reduced glutathione (GSH) concentrations in salivary gland and gut extracts of male and female Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks established significant variation in antioxidant responses between two sexes of the ticks. Higher activity of these antioxidants and GSH depletion in females clearly indicate stronger antioxidant defense in female ticks which is to combat host mediated oxidative assault during feeding for greater engorgement and reproductive stress. The females are also better equipped with the mechanism of acaricide resistance as evidenced by higher expression of esterases than males in unfed whole tick extracts in current study.

Inhibition of bovine platelets aggregation in response to Hyalomma anatolicum salivary gland proteins/peptides.

AIM: Ticks are obligate ectoparasites that have an impact on wide range of vertebrates and also act as a potential vector for the transmission of tropical theileriosis, babesiosis, etc., causing significant loss to livestock production worldwide. While feeding, they introduce their saliva containing different bioactive molecules into the host. These molecules have the capability to counteract the host hemostatic mechanism to suck host blood successfully. Therefore, the study was aimed to isolate anti-platelet aggregating peptides from salivary gland extract (SGE) of Hyalomma anatolicum ticks, a commonly available tick in India. MATERIALS AND METHODS: Female H. anatolicum salivary glands were dissected out and SGE was prepared by homogenizing it in a suitable buffer under ice. Extract so obtained was fractionated by gel filtration chromatography using Sephacryl S-200 column. Total protein concentration in fractions was estimated and bovine platelets were isolated, stimulated with thrombin (positive control), treated with Gly-Pro-Arg-Pro amide (negative control) and with salivary gland fractions for identification of proteins/peptides having anti-platelet aggregating activities. RESULTS: Proteins/peptides present in various salivary gland fractions inhibited the bovine platelet aggregation and the percent inhibition ranged between 33% and 35.8%. CONCLUSION: The results suggests that the fractions of H. anatolicum salivary glands possess thrombin-induced anti-platelet aggregating activity and which could be further exploited for raising anti-tick vaccine and also for therapeutic purpose.

Acaricide resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Haryana and Rajasthan states of India.

Acaricide resistance status of Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum ticks against deltamethrin and diazinon was assessed in Hisar (Haryana) and its adjoining district Churu (Rajasthan) using adult immersion test (AIT) and larval packet test (LPT). The mortality slope, LC50, LC95, 95 % confidence limit and resistance factor of field ticks were determined. Results showed that R. (B.) microplus ticks collected from Tohana (Hisar) were found resistant to both the acaricides while the ticks of Agroha (Hisar) were found to be susceptible using AIT. Similar results were observed by using standard method of LPT. Again, H. anatolicum tick isolates of Tara Nagar (Churu) were found susceptible whereas Churu tick isolates were found to be resistant using AIT for both the acaricides. LPT indicated susceptible status of H. anatolicum ticks collected from Churu as well as Tara Nagar for both the acaricides. The study warrants the need for strategic use of available acaricides to overcome the development of acaricide resistance in ticks.

Partial characterization of a novel anti-inflammatory protein from salivary gland extract of Hyalomma anatolicum anatolicum 77Acari: Ixodidae) ticks.

AIM: Hyalomma anatolicum anatolicum ticks transmit Theileria annulata, causative agent of tropical theileriosis to cattle and buffaloes causing a major economic loss in terms of production and mortality in tropical countries. Ticks have evolved several immune evading strategies to circumvent hosts' rejection and achieve engorgement. Successful feeding of ticks relies on a pharmacy of chemicals located in their complex salivary glands and secreted saliva. These chemicals in saliva could inhibit host inflammatory responses through modulating cytokine secretion and detoxifying reactive oxygen species. Therefore, the present study was aimed to characterize anti-inflammatory peptides from salivary gland extract (SGE) of H. a. anatolicum ticks with a view that this information could be utilized in raising vaccines, designing synthetic peptides or peptidomimetics which can further be developed as novel therapeutics. MATERIALS AND METHODS: Salivary glands were dissected out from partially fed adult female H. a. anatolicum ticks and homogenized under the ice to prepare SGE. Gel filtration chromatography was performed using Sephadex G-50 column to fractionate the crude extract. Protein was estimated in each fraction and analyzed for identification of anti-inflammatory activity. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) was run for further characterization of protein in desired fractions. RESULTS: A novel 28 kDa protein was identified in H. a. anatolicum SGE with pronounced anti-inflammatory activity. CONCLUSION: Purification and partial characterization of H. a. anatolicum SGE by size-exclusion chromatography and SDS-PAGE depicted a 28 kDa protein with prominent anti-inflammatory activity.