Factors related to cattle infestation level and resistance to acaricides in Boophilus microplus tick populations in New Caledonia

Boophilus microplus, even in the absence of babesiosis, is a major disease of the cattle in New Caledonia where the particular farming system associates continental European breeds and a tropical climate tempered by the Pacific Ocean. In order to have a better understanding of the factors involved in cattle tick infestation, to decrease the possible wastage and use of chemicals and to increase the lifetime of the acaricides, the veterinary authorities investigated the conditions of the chemical treatments. A survey among 148 cattle farms of the whole of New Caledonia was carried out and factors that explain the development of tick resistance and cattle infestation have been determined. From this survey, three typologies for the main characteristics of the farms have been set up, the technical practices of the farmers and the tick control measures applied by the farmers, respectively. Some variables are significantly associated with the tick resistance to deltamethrin but their contribution to the explanation model is always moderate: farms in the south, with a positive resistance gradient from east to west, absence of bush fire and membership to a cattle farmers organization. The more the farmers have intensified their breeding-male castration, weaning, heifer separation, drenching, etc.-and pasture-high stock rate, mowing, extra feeding of the cows, many paddocks, etc.-techniques, the higher was the probability for the ticks in their farm to be resistant to deltamethrin. The technical details of the acaricide treatment had a low contribution to the explanation model. However, the use of a spray generated more resistance than a dip. Furthermore, there is a negative resistance gradient when the farmers increased the treatment interval average. Considering infestation, none of the variables from the three typologies were associated with the two infestation variables (1: semi-engorged tick females and 2: other ticks) at the herd level. However, the seven studied variables-the three typologies, breed, age, body condition score and breeding status-affected significantly the two infestation variables at the cow level, but their predictive ability remained very low (R(2)<3.5%). This result-individual effect more important than herd effect on the infestation-is confirmed by the importance of the variance of the intra-farm factors (99%) when compared with inter-farm factors (1%). Cows of Charolais breed, in poor body condition, old, pregnant or lactating, and those of the farms with irrational and high pressure control of ticks are the most infested.     

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.     

Synthetic pyrethroid resistance in field samples in the cattle tick (Boophilus microplus)

First evidence of resistance to synthetic pyrethroids in the cattle tick in Australia, other than the pre-existing cross-resistance in DDT-resistant ticks, has emerged. Three new strains have been investigated and characterised. The first, detected in central Queensland, showed increased ability to detoxify pyrethroids resulting in a marginal loss of efficacy of all pyrethroid tickicides tested, except flumethrin. Subsequently two more strains have been detected, with serious implications for chemical tick control. The first, from southern Queensland, has a high level of specific resistance to flumethrin. The second, from central Queensland, has a broad spectrum resistance encompassing all pyrethroids currently in use. The toxicology and biochemistry of these new resistant strains have been investigated and alternative tickicides evaluated. The significance of these latest developments is discussed.     

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.     

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).     

Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin)

Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 degrees C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks.     

Acaricidal activity of Palicourea marcgravii, a species from the Amazon forest, on cattle tick Rhipicephalus (Boophilus) microplus

Leaves of Palicourea marcgravii were extracted successively with hexane, ethyl acetate and ethanol in order to evaluate their acaricidal activity on larvae and adult stages of the cattle tick Rhipicephalus (Boophilus) microplus. The ethyl acetate extract showed the highest bioactivity of the tested extracts, which contained 0.12% monofluoroacetic acid. On engorged female, the ethyl acetate extract showed a lethal concentration 50% - LC(50)=30.08 mg ml(-1), inhibitory concentration 50% - IC(50)=5.79 mg ml(-1) and lethal time 50% - LT(50)=4.72 days; 100% reproduction was controlled at concentrations of 50 mg ml(-1) and on larvae the ethyl acetate extract showed a LC(50)=2.46 mg ml(-1). No alkaloids were detected in any of the extracts. This is the first report on the acaricidal activity of P. marcgravii extracts against R. microplus as well as the acaricidal properties of a plant species containing monofluoroacetic acid.     

Resistance of Holstein-Friesian cows to infestation by the cattle tick (Boophilus microplus)

In two experiments, the milk production of 59 Holstein-Friesian cows in mid lactation was measured over 1 week before artificial infestation with 2,500 or 5,000 Boophilus microplus larvae. Host resistance, the proportion of female ticks applied but not engorging was estimated from weekly counts of engorging female ticks of 4.5-8. 0mm long. Mean host resistance was 79 and 67% in two experiments. Host resistance was not significantly related to milk yield before infestation, to early pregnancy, or to parity. Culling the 10% of cattle with the least resistance to ticks would result in removal of 19-21% of ticks in a herd. The results suggest that selection to improve the existing low levels of resistance to B. microplus might be undertaken without compromising milk production. However, low levels of resistance among the cattle studied and difficulties in assessment of resistance are likely to limit the usefulness of selection within the Holstein-Friesian breed.     

First report of fipronil resistance in Rhipicephalus (Boophilus) microplus of Mexico

Five strains of Rhipicephalus (Boophilus) microplus collected from Tamaulipas Mexico were tested for resistance against several classes of acaricides commonly used. All were resistant to fipronil. Four of five were co-resistant to permethrin and coumaphos in addition to being resistant to fipronil. One strain, El Zamora was found multi-resistant to permethrin, coumaphos, fipronil, and amitraz. Selection with fipronil for 3 generations produced a resistance ratio of 8.3 and 9.4 at the LC₅₀ and the LC₉₉ estimates, respectively. Permethrin resistance in El Zamora was possibly linked to elevated esterase (CZEST9) and could be a contributing factor of resistance to fipronil. The implications of resistance for the control of the southern cattle tick in the future are discussed.     

Possible risk factors on Queensland dairy farms for acaricide resistance in cattle tick (Boophilus microplus)

A case control study was carried out within a cross-sectional survey designed to investigate the management by Queensland dairy farmers of the cattle tick Boophilus microplus. Although 199 farmers were surveyed, data on acaricide resistance were only obtained from 66 farms. Multiple models were used to predict the probability of acaricide resistance associated with 30 putative risk factors. The region of the state in which the farm was located and the frequency of acaricide application were consistently associated with acaricide resistance. The risk of resistance to all synthetic pyrethroids (Parkhurst strain) was highest in Central Queensland and increased when more than five applications of acaricide were made in the previous year, when spray races were used and when buffalo fly treatments with a synthetic pyrethroid were applied frequently. The probability of resistance to amitraz (Ulam strain) was highest in Central Queensland, increased when more than five applications of acaricide were made in the previous year, and decreased on farms when a hand-spray apparatus was used to apply acaricides to cattle. The probability of resistance to flumethrin (Lamington strain) was highest in the Wide Bay-Burnett region.     

Progress in the epidemiology and diagnosis of amitraz resistance in the cattle tick Boophilus microplus

Amitraz is a rapidly acting acaricide that has been in use for the control of cattle ticks for more than 30 years. Resistance against amitraz was first reported in Boophilus microplus in Australia in 1980 but has been slow to spread in comparison to resistance against synthetic pyrethroids. The most recent estimate of prevalence of amitraz resistance in Australia is 10.8%. In Mexico, the development and distribution of amitraz appears to have been more rapid and the prevalence has been estimated to be 19.4% in Yucatan state. In New Caledonia, about 10% of properties were confirmed to have amitraz resistance. There is little reliable information on the prevalence of amitraz resistance in southern Africa. Risk factors have been identified, but the small sample sizes in the studies that have attempted to identify risk factors using survey data suggest caution in their interpretation. Regional variation in prevalence has been reported, as has a positive relationship with frequency of acaricide application. There is evidence to suggest that in Australia, amitraz resistance might have emerged on a small number of properties and been disseminated by cattle movements. There is also some evidence to suggest that amitraz resistance can diminish in the field when selection pressure is not applied. The mode of inheritance of amitraz resistance is uncertain and it has been suggested that it is a polygenic trait. The mechanism of amitraz resistance is unknown. Two possibilities have been proposed: octopamine receptor and monoamine oxidase. There is some equivocal support for both possibilities. The larval packet test bioassay is the most reliable method of diagnosing amitraz resistance in B. microplus, and this test has been modified by Miller to provide more accuracy and repeatability. Molecular tests are in development but will not eliminate the need for the bioassay.     

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.

     

Acaricidal activity of an oleoresinous extract from Copaifera reticulata (Leguminosae: Caesalpinioideae) against larvae of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The acaricidal activity of oleoresinous extract (oleoresin) from the copaiba tree, Copaifera reticulata was investigated. Oleoresin was dissolved with dimethylsulfoxide (DMSO) and mixed with distilled water at a rate of 0.4 ml of DMSO to 24.6 ml water. Rhipicephalus (Boophilus) microplus larvae were exposed to filter paper envelopes impregnated with different oleoresin concentrations. Four envelopes were impregnated with each tested solution. Thirty larvae between 14 and 21 days old were fasted and placed in each envelope. Bioassays were performed at 27 degrees+/-1 degrees C, RH>or=80% and a photoperiod of 12:12 h. Larval mortality was observed 24 h after treatment. Four replicates for each concentration were evaluated to determine lethal concentrations (LC). Oleoresin LC50 and LC99 values were 1579 and 3491 ppm, respectively. The potential use of oleoresin for control of R. (B.) microplus is discussed. The results of this study reinforce the importance of the preservation of C. reticulata in its natural Biome, as a potentially auto-sustainable medicinal resource among the flora of Amazonian forest.     

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.