Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas,India

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

     

Eprinomectin pour-on for control of Boophilus microplus (Canestrini) ticks (Acari: Ixodidae) on cattle

The efficacy of a commercial pour-on formulation of eprinomectin, a macrocyclic lactone, against experimental infestations of Boophilus microplus (Canestrini) ticks was evaluated in two trials involving 27 Bos taurus calves. The first trial was designed to evaluate the effects of a single treatment at a dose of 0.5 mg/kg of body weight against standard size B. microplus females (4.5-8.0 mm long). A significant reduction in tick numbers (P<0.05, Wilcoxon test) was observed between treated calves as compared to untreated ones from Day 3 (44% efficacy) after treatment to the end of the trial on Day 28 (96.9%), with a peak efficacy of 97.1% on Day 21. In the second trial the effect of eprinomectin on standard size tick numbers, engorgement weight and fertility of female ticks from calves with a single treatment dose of 1 mg/kg on Day 0 and calves treated twice at a dose of 0.5 mg/kg on Days 0 and 4 was evaluated. An efficacy >93% was obtained from Day 2 to Day 28 after treatment in calves treated twice at 0.5 mg/kg, and to the end of the trial (Day 35) in calves treated once with 1 mg/kg. The 1mg/kg treatment provided >98% residual efficacy for at least 7 days. During the first part of the second trial the efficacy of eprinomectin resulted from a dramatic adverse effect on engorgement weight and fertility of female ticks, with 100% control on Day 5 (dosage of 1 mg/kg) and on Days 6 and 7 (two doses of 0.5 mg/kg). Following Day 7, most of the effect was due to reduction in the number of standard size female ticks.     

Genetic incompatibility between Boophilus decoloratus (Koch, 1844) and Boophilus microplus (Canestrini, 1888) and hybrid sterility of Australian and South African Boophilus microplus (Acarina: Ixodidae)

Virgin females of both Boophilus decoloratus and Boophilus microplus, when mated with males of the other species, subsequently produced sterile eggs. Counts of spermiophore capsules in female seminal receptacles showed that the males of both species will mate with the females of both species and that B. microplus males show a slightly greater, but statistically insignificant, mating capacity than B. decoloratus males. South African B. microplus females, when mated with an Australian strain of B. microplus males, produced a 62% yield of viable hybrid progeny while the reciprocal cross produced only a 1,82% hatch of non-viable larvae. The hybrids were sterile when interbred and no hatch resulted when the Fl males were backcrossed with parent females. The reciprocal backcross of hybrid Fl females to parent males resulted in a low percentage hatch of non-viable larvae.     

Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies.     

Mortality, weight loss and anaemia in Bos taurus calves exposed to Boophilus microplus ticks in the tropics of Colombia

One hundred and sixteen pure-bred Normandy calves previously immunised against babesiosis and anaplasmosis were transported to the Caribbean Coast of Colombia where they divided into 2 equal groups and placed in separate pastures. One group sustained heavy infestation with Boophilus microplus ticks. The second group became lightly infested. The heavily infested calves suffered average losses in body weight of 38 kg and a 48% decrease in mean packed cell volume. Twenty-three (40%) died 16 to 39 days following arrival from severe ixodiasis and babesiosis. Mortality did not occur nor were significant weight losses observed in the group of lightly infested calves.     

Factors affecting the detachment rhythm of engorged Boophilus microplus female ticks (Acari: Ixodidae) from Charolais steers in New Caledonia

As in most parts of the world where the cattle tick Boophilus microplus is established, resistance of ticks to acaricides occurs in New Caledonia. In order to implement laboratory resistance tests on larvae, engorged females collected in suspected farms are necessary. Investigations on the detachment schedule of the engorged females were conducted to explain certain field situations such as the lack or scarcity of engorged females on highly infested cattle driven from the pasture to the pen in the morning. Three experiments on Charolais steers naturally infested on pastures showed that: (1) engorged female burdens at sunrise are similar whether the steers spend the night in pasture or in a pen; (2) compared with steers maintained in a pen, morning detachment of females increases when the steers stay on the pasture or move from the pasture to the pen; (3) detachment rhythm of engorged females on steers staying the morning in a pen, is not influenced by feeding activity or exposition of steers to sun; (4) detachment occurs earlier for females attached on anatomical sites exposed to sun, and earlier from these sites for the steers in pasture or walking than for steers in a pen.     

Relationship between the resistance of crossbred cattle to ticks, Boophilus microplus (Canestrini, 1887) and Hyalomma anatolicum anatolicum (Koch, 1844)

Studies were undertaken to determine the effect of repeated pure infestations with Boophilus microplus on susceptibility to subsequent pure infestations with Hyalomma anatolicum anatolicum, and the effects of pure infestations with both species of tick on susceptibility to a series of mixed infestations. Crossbred (Bos indicus X Bos taurus) calves were infested with Boophilus microplus (17 times), H. a. anatolicum (four times), followed by five mixed infestations of B. microplus and H. a. anatolicum. The decline in B. microplus engorgement from a mean yield of 274.4 +/- 60.3 ticks per host after the first exposure, to a mean yield of 9 +/- 4.6 per animal after the seventeenth exposure, was observed in animals exposed to only B. microplus. This might be due to acquired resistance. However, these animals were found to be as susceptible to H. a. anatolicum as animals which had never been exposed to ticks of either species. A decline in the yield of H. a. anatolicum from a mean yield of 92.1 +/- 10.7 after the first exposure to 54.7 +/- 11.3 after the fourth exposure, indicated that the cattle could also acquire resistance to repeated pure infestations with this species. After repeated pure infestations with both tick species, cattle reacted to five mixed infestations showing a high degree of resistance to B. microplus and low resistance to H. a. anatolicum (mean yield for B. microplus was only 10 +/- 8.1 ticks per host after the first mixed exposure and declined to 1.3 +/- 1.7 after the fifth, whereas the mean yield for H. a. anatolicum was 71.4 +/- 11.3 ticks per host following the first exposure and declined to 37.3 +/- 7.8 after the fifth). Host responses elicited to one species do not provide cross-resistance to the second species used in this study.     

Comparison of the reproductive biology between acaricide-resistant and acaricide-susceptible Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The reproductive fitness of Rhipicephalus (Boophilus) microplus (Canestrini) strains resistant to organophosphate (OP), pyrethroid (P), or formamidine (F) acaricides was compared to an acaricide-susceptible (SUS) strain to determine whether the acquisition of resistance affected reproductive fitness in the resistant strains. The SUS strain females had a 3.0 days preoviposition period, a 12.1 days oviposition period, a 22.5 days egg incubation period, a mean of 3670 eggs per female, and a mean percentage egg hatch of 78.1%, which were all remarkably similar to these same parameters reported for this species throughout the world. The reproductive biology of the P-resistant strain (PYR) and the F-resistant strain (FOR) were, for the most part, similar to those of the SUS strain. In the few instances where statistical differences did occur there was little evidence that the variation had any biological basis that could be attributed to a reduction in fitness related to resistance to P or F acaricides. Although the comparison of reproductive parameters of the OP-resistant strain (OPR) and the SUS strain identified statistical differences between the mean egg incubation and oviposition periods, the magnitude of the differences was not sufficient to conclude that the OPR strain was biologically less fit than the SUS strain. However, the OPR strain produced 30% fewer eggs (2562 eggs per female) than the SUS strain (3670 eggs per female) indicating the acquisition of resistance placed the OPR at a selective disadvantage relative to the SUS strain. This coupled with a lower, though non-significant, egg hatch was used to predict there would be a reduction of at least 34.1% in larval numbers available to potentially re-infest subsequent cattle than were available from the SUS strain. These data may aid the development of management strategies that can be used to control OP-resistant ticks.     

Immunoprotective potential of a Rhipicephalus (Boophilus) microplus metalloprotease

Ticks have serious impacts on animal and human health, causing significant economic losses in cattle breeding. Besides damage due to the hematophagous behavior, they transmit several pathogens. Low cost and environmental safety have made vaccines a promising alternative control method against tick infestation. Metalloproteases (MPs) have been shown to be essential for diverse biological functions in hematophagous organisms, inhibiting blood clotting, degrading extracellular matrix proteins, and inhibiting host tissue repair via anti-angiogenic activity. In this study, we analyzed the immunoprotective potential of a recombinant MP against Rhipicephalus (Boophilus) microplus infestation. First, a cDNA encoding R. microplus amino acids sequence with highly conserved regions of the metzincin (reprolysin) group of MP was identified (BrRm-MP4). After expression and purification, recombinant BrRm-MP4 was used as a vaccinal antigen against R. microplus infestation in cattle (Bos taurus taurus). All vaccinated bovines developed immune response to the antigen, resulting in increased antibody level throughout the immunization protocol. Immunization with rBrRm-MP4 reduced tick feeding success, decreasing the number of engorged females and their reproduction potential, representing a 60% overall protection. These results show that rBrRm-MP4 provides protection against tick infestation, placing it is a potential candidate for an anti-tick vaccine.     

Rhipicephalus (Boophilus) microplus embryo proteins as target for tick vaccine

Rhipicephalus (Boophilus) microplus is one of the most widely distributed tick in the world. The control of the parasite is based mainly on the use of chemical acaricides, which are produced from a limited set of molecules. These drugs induce selection of acaricide-resistant ticks, and are an important source of environmental pollution. An approach based on anti-tick vaccines may circumvent these obstacles. Characterization of the physiological function of tick molecules may be useful to develop new vaccines. Previously, we reported the ability of some tick proteins as inducers of protective immune response. Vaccination studies using tick proteins like native (nBYC), recombinant (rBYC) egg-yolk aspartic endopeptidase and cysteine endopeptidase (VTDCE) from R. microplus and glutathione S-transferase (Hl-GST) from Haemaphysalis longicornis demonstrated the immunogenicity and antigenicity of these proteins in bovines. Eventually, immunization with these proteins triggered a partial immune response against R. microplus infestation in cattle, manifested mainly as a reduction in egg fertility (7.7% and 13.9% for nBYC, 5.9% for rBYC; 4.7% for VTDCE, 7.9% for Hl-GST), and in the number of fully engorged ticks (18.2% for rBYC, 14.6% for VTDCE, 53% for Hl-GST). The data so far obtained suggest that these proteins have potential to be used as antigens in an anti-tick vaccine. Other proteins involved in tick embryogenesis also have this potential, like THAP and BmCl1, which are enzymes with key roles in vitellin and hemoglobin hydrolysis. Moreover, the identification of analogous proteins present in other tick species may bring information about the way to develop a vaccine against multiple tick species which can help to solve the problem faced by numerous countries where animals are parasitized by more than one tick species. The aim of the present review is to comprehensibly summarize the data obtained in the last few years by our collaborative research, discussing the efforts we have made to find antigens efficient enough for a cattle tick-controlling vaccine. This review discusses tick physiology studies aimed at the selection of possible targets, characterization of the selected proteins with emphasis on their biochemical and immunological aspects and results of vaccine trials on bovines.     

Diazinon resistant status in Rhipicephalus (Boophilus) microplus collected from different agro-climatic regions of India

The resistance status of Rhipicephalus (Boophilus) microplus to "Diazinon" was evaluated in 20 locations situated at various agro-climatic regions of India. Adult immersion test (AIT) was optimized using laboratory reared acaricide susceptible IVRI-I strain of R. (B.) microplus and minimum effective concentration of Diazinon was determined as 635.2 ppm. The discriminating dose (DD) was worked out as 1270.4 ppm and was tested on female ticks collected from organized and unorganized farms located at different agro-climatic regions of India. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The average resistance factor (RF) of 6.1 (level II) was recorded in the ticks collected from the northern sub-temperate trans-gangetic plains while high average RF values of 26.65 (level III) was recorded in the ticks collected from tropical middle-gangetic plains. The tropical middle gangetic plain has a very high density of animal populations where farmers use Diazinon for tick control, for agricultural practices and for mosquito control. Due to the continuous use of OP compounds the environmental load of Diazinon has become high in the area. This is the first experimental data generated on Diazinon resistant status in ticks of India.     

Accumulation of acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) populations from New Caledonia Island

Rhipicephalus (Boophilus) microplus has been pesticide-controlled for several decades in the pacific island of New Caledonia. Since 1996, pesticide-control has been based on either deltamethrin (Butox) or amitraz (Taktic) in herds harbouring deltamethrin-resistant ticks. In this island, the first R. microplus deltamethrin- and amitraz-resistances were detected in 1992 and 2003, respectively. Using LPT bioassays, we have undertaken to update data regarding the geographical distribution and the physiological diversity likely to be involved in these resistances. We confirmed that after 17 years of intensive use of deltamethrin, several resistances of moderate levels (<30-fold) have evolved and/or diffused in any part of the island. We also evidenced that amitraz-resistant phenotypes have recently evolved in diverse western tick populations, although none has reached fixation in any tick population yet. According to synergists bioassays, the physiological changes involved in amitraz-resistance in New Caledonia would involve target modification and detoxifying P450 cytochrom oxydase(s). It may also involve detoxifying esterase(s) although this later point will need confirmation on samples bearing higher frequency of resistant phenotypes. Results are discussed with regard to the local evolutionary dynamics of resistance.     

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.