Association between ecological factors and the presence of Rhipicephalus (Boophilus) microplus larvae in Puerto Rico

A prevalence study was conducted to survey tick larvae populations in Puerto Rico (PR), compare the number of infested sites with Rhipicephalus (Boophilus) microplus larvae between the wet and dry season, and assess the associations of ecologic factors on the presence of R. microplus larvae. Ninety-six sites were selected using a GIS-based sampling method. Each site was sampled twice; the first sampling was performed during the dry season (March 4-18, 2007) and the second sampling during the wet season (August 13-26, 2007). Sites were sampled using a tick drag with a 1-m(2) white flannel cloth along a 50-m straight course. Only 2 tick species were identified. In the dry season, 15 sites (0.16, 95?% CI?=?0.09-0.24) were identified with R. microplus larvae (n?=?606) and 9 sites (0.09, 95?% CI?=?0.04-0.17) with Dermacentor (Anocentor) nitens larvae (n?=?779), whereas in the wet season 5 sites (0.05, 95?% CI?=?0.02-0.12) were identified with R. microplus (n?=?94), and 5 sites (0.05?%, 95?% CI = 0.02-0.12) with D. nitens (n?=?275). Difference in the number of infested sites with R. microplus was significant (P =?0.031) between the 2 seasons. Factors associated with the presence of R. microplus larvae in PR were wind speed of >4.0?km/h (OR?=?0.07, 95?% CI?=?0.01-0.63), more than 25?% bushes and shrubs on the site (OR?=?11, 95?% CI?=?1.6-71), and presence of cattle on the site (OR?=?26, 95?% CI?=?3.4-188).     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

Detecting Resistance to Organophosphates and Carbamates in the Cattle Tick Boophilus Microplus, with a Propoxur-based Biochemical Test

Rapid and sensitive detection of resistance to insecticides in arthropods is needed. In the cattle tick, Boophilus microplus, resistance to a variety of acaricides is widespread. The most commonly used assay for resistance, the larval packet test, takes at least two, but generally six weeks for a one-host tick like B. microplus to complete and may take up to three months to complete for three-host ticks. Here we describe a test for resistance to organophosphate acaricides that can be used on larvae and adult ticks which takes less than 24 hours. The test measures the difference in acetylcholinesterase (AChE) activity in homogenates of ticks in the presence and absence of propoxur, a carbamate acaricide. We found clear discrimination of organophosphate-susceptible and organophosphate-resistant adults with 100 M propoxur. AChE from susceptible ticks had almost no activity at this concentration of propoxur whereas AChE from resistant ticks had 67% of its potential activity. AChE from heterozygote ticks could also be distinguished from AChE from homozygous-susceptible and homozygous-resistant ticks. This is the first biochemical test for resistance to an acaricide. Rapid, sensitive tests like ours will allow resistance to organophosphates to be detected soon after it develops in the field, thus, the spread of resistance might be slowed and the useful life of acaricides extended.     

In vitro efficacy of extracts of Melia azedarach against the tick Boophilus microplus

The efficacy of ripe fruit extracts of Melia azedarach L. (Rutales: Meliaceae) was evaluated against the tick, Boophilus microplus (Canestrini) (Acari: Ixodidae). Ripe fruits of M. azedarach dried and powdered were extracted by Soxhlet apparatus successively using hexane, CHCl3 and 96% aqueous ethanol. Larvae and engorged females were immersed in decreasing concentrations from 0.25% to 0.015% of each extract. The mortality of larvae was evaluated 24, 72 and 168 h after treatment. The effectiveness of treatment against engorged females was assessed by measuring egg production. All tested extracts caused mortality of B. microplus larvae, with higher mortality rates observed in CHCl3 (100%) and hexanic extract (98%) than in ethanolic extract (50%) 168 h after treatment. The mortality was dependent on concentration and on time after treatment. Similarly hexanic and CHCl3 extracts showed higher effectiveness (varying from 14% to 100%) against B. microplus engorged females than ethanolic extract (varying from 0% to 46%). Melia azedarach extracts did not kill the adult females, but inhibited partially or totally egg production and embryogenesis. These results show that the less polar the extract of M. azedarach ripe fruits the more its effectiveness against larvae and engorged females of B. microplus. This plant may therefore be useful in the control of resistant B. microplus populations.     

Boophilus microplus: cellular responses to larval attachment and their relationship to host resistance.

The histology of early feeding lesions of the cattle tick B. microplus has been studied using 32P labelled larvae to standardize the duration of attachment. Critical studies were made on 3-h lesions in six separate experiments on different groups of British breed animals. Each group consisted of three animals--one previously unexposed to ticks, one of high resistance and one of low resistance. The degree of mast cell disruption, eosinophil concentration and degranulation, and the extent of epidermal vesiculation were all significantly greater at the site of attachment on highly resistant hosts. In previously unexposed animals there was no mobilization of eosinophils nor mast cell breakdown and no epidermal vesiculation. Possible immune mechanisms producing mast cell disruption and the infiltration and concentration of eosinophils are suggested, and the effect of eosinophil degranulation on larval attachment and feeding is discussed.     

Carbaryl resistance in Mexican strains of the southern cattle tick (Acari: Ixodidae)

Susceptibility to carbaryl in six Mexican strains of the southern cattle tick, Boophilus microplus (Canestrini), was evaluated with the Food and Agricultural Organization larval packet test. Tick strains from the cattle fever tick quarantine zone in Texas were more susceptible to carbaryl than to coumaphos or diazinon. Compared with the susceptible reference (Gonzalez) strain, Mexican tick strains demonstrated 10.9-59.5-fold resistance to carbaryl. Significant cross-resistance was found between carbaryl and the organophosphate acaricides coumaphos and diazinon. Bioassay results with synergists suggested that metabolic detoxification mechanisms did not play a major role in carbaryl resistance. Resistance to carbaryl was likely conferred by insensitive acetylcholinesterase. The implications of carbaryl resistance in tick eradication and control also are discussed.     

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus.

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. microplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed.     

Displacement of Boophilus decoloratus by Boophilus microplus in the Soutpansberg region, Limpopo Province, South Africa

Boophilus ticks (n = 25,987) were collected from cattle at 30 communal dip tanks and five commercial farms in the Soutpansberg region, Limpopo Province, South Africa, between May 1999 and December 2001. Only 6.6% were Boophilus decoloratus, while 93.4% were Boophilus microplus, a tick that had not been reported from this area previously. B. microplus was the most common Boophilus tick on communal dip tank cattle, while B. decoloratus was more commonly found on commercial farms. Where the two species occurred together, B. microplus tended to displace B. decoloratus. The displacement was almost complete at the communal dip tanks, while on the commercial farms the population change was still in progress at the end of the survey. The present study demonstrated that a postulated reproductive interference was insufficient in preventing B. microplus from spreading when the climatic conditions were favourable to this species, as the displacement in most of the areas appeared to be rapid and complete.     

Dermacentor variabilis: resistance to ticks acquired by mast cell-deficient and other strains of mice

The acquisition of resistance to ticks was monitored in mice of six different strains. Mice were subjected to repeated infestations with Dermacentor variabilis larvae, different skin sites being used for each successive infestation. In the third and fourth infestations, resistance was expressed in three strains of mice (WBB6F1-W/Wv, WBB6F1-+/+, and CFW), as demonstrated by significant reductions in percentages of larvae engorging and in mean weights of fed larvae. Both WBB6F1-W/Wv mice, which are mast cell-sufficient strain attained significantly higher levels of resistance. It is suggested that mast cells may play a relatively minor role in the mechanisms of resistance in this strain of mice. C57B1 mice also expressed tick resistance in their third and fourth infestations as measured by reduced percentages of engorged larvae, but not by reduced mean larval weights. Possibly, the mechanisms of tick resistance in this strain differ from those in other strains. Two other mouse strains (C3H-HeJ and C3H-HeSn) remained relatively susceptible to tick feeding throughout five infestations. In secondary infestations of all strains tested, no resistance was evident. Instead, enhanced feeding of larvae appeared to occur. A new objective measurement of tick resistance is the mean weights of detached, unengorged larvae taken from resistant animals at the end of the infestation period. These were found to be consistently less than those from susceptible animals.     

Esterase profile of Rhipicephalus (Boophilus) microplus populations collected from Northern India exhibiting varied susceptibility to deltamethrin

Rhipicephalus (Boophilus) microplus is an economically important ectoparasite of cattle. Chemical acaricides remain the most practical method for control of these pests. During past two decades there have been increasing reports of resistance development against synthetic pyrethroids in tick populations of this species throughout the world. A study was conducted to determine the level of susceptibility of R. (B.) microplus to deltamethrin collected from different geographical locations of northern India. LPT bioassay results revealed LC(50) values of deltamethrin ranging from 0.035 to 0.00037?% A.I. Esterase profile of the tick larval extracts using native PAGE, revealed 5 bands of esterase activity designated EST-5 to EST-1A. Inhibitory tests recognized EST-1, EST-2 and EST-3 as Acetylcholinesterases (AchEs), EST-4 and EST-5 as Carboxylesterases (CaEs). The band intensity varied between tick populations of various locations, being more intense in case of the resistant populations. An extra band of esterase activity (EST-1A) was obtained in larval extracts of ticks from 3 locations. This increased esterase activity may be involved in the resistance development in these tick populations. Acaricide resistance is a multi-factorial phenomenon, thus other causes of increased resistance like sodium channel mutation and reduced drug penetration (e.g. cuticle thickening) and behavioural changes (e.g. avoiding the pesticides) are to be tested in future in order to confirm the basic cause of the resistance development in these acaricide resistant tick populations.     

Association of BoLA-DRB3.2 alleles with tick (Boophilus microplus) resistance in cattle

Losses caused by bovine tick burdens in tropical countries have a tremendous economic impact on production systems. Besides reducing production, this parasite can cause death in the most susceptible animals. The use of commercial acaricides has been the major method of control, but their misuse has led to tick resistance to many chemicals. More recently, vaccines have been used in some countries without solving the problem completely. An alternative could be the development of resistant animals and the use of genetic markers and candidate genes that could help with the enormous task of selecting resistant animals. The bovine lymphocyte antigen genes (BoLA) have been shown to be associated with some parasitic infestations and disease incidence. Thus, the objective of the present study was to determine the association of BoLA-DRB3.2 alleles with tick resistance in cattle. The study was conducted on 231 F2 (Gyr x Holstein) animals that were artificially infested with 10,000 tick larvae. Log of tick count +1 was used as the dependent variable in a mixed animal model with allele substitution effects in addition to fixed effects of year and season at tick count, sex of calves, age of animal at tick count, hair type (short-straight, short-curl, long-straight, and long-curl), coat color (white, >75% white, 50- 75% white, and 25-50% white), and additive genetic, permanent environmental and residual effects as random. Females showed fewer ticks than males. Animals with short-straight hair were more resistant to tick infestation than animals with long-curl hair, and animals with whiter coat color also had fewer ticks. An association between BoLA alleles and lower tick number was found for alleles DRB3.2 *18, *20 and *27 at the 5% significance level. Also, one allele (DRB3.2*16) showed an association at the 10% level. Allele *27 was the most frequent in the population (30.7%), followed by alleles *16 (10.8%), *20 (8.7%) and *18 (2.4%). These results suggest that BoLA-DRB3.2 alleles could be used to help in the selection of animals resistant to tick infestation. However, further studies involving a larger population of cattle in combination with other BoLA genes may help to understand the mechanisms of resistance to parasites.     

Experimental immunisation of crossbred cattle with glycoproteins isolated from the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus

In India, control of tick infestations is a major problem facing the owners of highly productive crossbred cattle. To develop suitable immunoprotective measures against the multitick infestations, specific problem encountered by the Indian farmers, an attempt has been made to identify candidate protein molecules in the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus. Employing two steps affinity chromatography method, glycoproteins of 34 and 29 kDa were isolated from H. a. anatolicum and B. microplus, respectively. The additive effect of the isolated glycoproteins was tested by the immunisation of crossbred male calves at a dose of 200 microg/animal of each glycoprotein, three divided dosages. Following challenge infestations by the stages of two species of ticks, a significant rejection of (73.6%; P < 0.01) of larvae and adults (75.0%; P < 0.01) of H. a. anatolicum and adults (89.8%; P < 0.01) of B. microplus were noted. The direct effect of immunisation on the stages of the challenged ticks (%DT) was calculated as 69%, 52% against larvae and adults of H. a. anatolicum and 60% against adults of B. microplus. A direct corelation between protection and anti-glycoprotein antibody titre was detected. The effect of the isolated glycoproteins on limiting the tick infestation is discussed in detail.     

BmiGI: a database of cDNAs expressed in Boophilus microplus, the tropical/southern cattle tick

We used an expressed sequence tag approach to initiate a study of the genome of the southern cattle tick, Boophilus microplus. A normalized cDNA library was synthesized from pooled RNA purified from tick larvae which had been subjected to different treatments, including acaricide exposure, heat shock, cold shock, host odor, and infection with Babesia bovis. For the acaricide exposure experiments, we used several strains of ticks, which varied in their levels of susceptibility to pyrethroid, organophosphate and amitraz. We also included RNA purified from samples of eggs, nymphs and adult ticks and dissected tick organs. Plasmid DNA was prepared from 11,520 cDNA clones and both 5' and 3' sequencing performed on each clone. The sequence data was used to search public protein databases and a B. microplus gene index was constructed, consisting of 8270 unique sequences whose associated putative functional assignments, when available, can be viewed at the TIGR website (http://www.tigr.org/tdb/tgi). A number of novel sequences were identified which possessed significant sequence similarity to genes, which might be involved in resistance to acaricides.     

Testing the interspecific body size principle in ungulates: the smaller they come, the harder they groom

Tick removal grooming may be centrally regulated by an internal timing mechanism operating to remove ticks before they attach and engorge (programmed grooming model) and/or evoked by cutaneous stimulation from tick bites (stimulus-driven model). The programmed grooming model predicts that organismic and environmental factors that impact the cost-benefit ratio of grooming (e.g. body size and habitat) will influence the rate of tick removal grooming. The body size principle predicts that smaller-sized animals, because of their greater surface-to-mass ratio, should engage in more frequent tick removal grooming than larger-bodied animals in order to compensate for higher costs of tick infestation. The body size principle may be tested intraspecifically between young and adult animals, or interspecifically among species of contrasting body sizes. To rigorously test the interspecific body size prediction, we observed the programmed grooming (oral and scratch grooming) of 25 species (or subspecies) of bovids at a tick-free zoological park in which stimulus-driven grooming was ruled out. Multiple correlation analysis revealed highly significant negative correlations between species-typical mass and mean species grooming rates when habitat was controlled for in the model. Species-typical habitat type (classified along a gradient from most open to most closed) was positively correlated with mean oral grooming rate, indicating that species tended to groom at a higher rate in woodland and forest habitats (where typical tick density would be high) compared with more open environments. Species mass accounted for up to two-thirds of the variation in grooming rate across species, whereas habitat accounted for ca. 20% of variation in oral grooming. Similar results were obtained when the analysis was expanded to include 36 species/subspecies of six different families. The body size principle can therefore account for a large proportion of species-typical differences in programmed grooming rate among ungulates. However, to understand the tick defence adaptations of very large mammals that rarely or never engage in oral or scratch grooming (e.g. elephants, giraffes, rhinoceros), alternative tick defence strategies must be considered, such as thick skin, wallowing, rubbing and tolerance of oxpeckers and other tick-eating birds. Copyright 2000 The Association for the Study of Animal Behaviour.     

Effect ofDermacentor albipictus (Acari: Ixodidae) on blood composition,weight gain and hair coat of moose,Alces alces

The physiological effects of the winter tick,Dermacentor albipictus, on moose,Alces alces, were investigated. Blood composition, weight gain, food intake and change in the hair coat of moose calves, four infested withD. albipictus larvae, and eight uninfested, were monitored. Infested moose groomed extensively, apparently in response to feeding nymphal and adult ticks, and developed alopecia. Other clinical signs included: chronic weight loss, anemia, hypoalbumenemia, hypophosphatemia, and transient decreases in serum asparate transaminase and calcium during the period of nymphal and adult female tick engorgement. Infested animals did not become anorexic. Two moose with severe hair loss had increases in gamma globulin shortly after the onset of female tick engorgement. Results suggest that alopecia is associated with tick resistance. Animals that groom and develop hair loss likely carry fewer ticks and therefore suffer less severely from blood loss.     

Sexually and Developmentally Dimorphic Grooming: A Comparative Survey of the Ungulata

Grooming is effective in removing fitness‐compromising ectoparasites, such as ticks. Tick‐removal grooming is regulated both by a central control mechanism (programmed grooming model) and by cutaneous stimulation from tick bites (stimulus‐driven model). The programmed grooming model predicts that organismic factors that impact the cost‐benefit ratio of grooming (e.g., sex and body size) will influence the rate of grooming. The ‘vigilance principle’ predicts that breeding males of sexually dimorphic species will groom less than females to maintain high levels of vigilance for rival males and oestrus females. The intraspecific body size principle predicts that juveniles will engage in more frequent grooming than larger‐bodied adults to compensate for the higher cost of tick infestation for smaller animals. To examine the generalizability of these predictions, we surveyed the grooming rate of 53 species/subspecies of ungulates (primarily Bovidae and Cervidae) in tick‐free zoological parks in which stimulus‐driven grooming was controlled for. Matched‐pair analysis supported both predictions. Males delivered fewer oral grooming episodes per hour than females in 85% of sexually dimorphic species (n = 40), but only 56% of monomorphic species (n = 11) exhibited sexually dimorphic grooming, which was not different from random. Juvenile oral episodes per hour exceeded that of adult females in 74% of surveyed species (n = 36). As predicted by the body size principle, the grooming rate of juveniles declined as juveniles grew larger and less vulnerable to tick infestation. In separate analyses of Bovidae and Cervidae to control for common ancestry, both families supported sexually dimorphic grooming, but developmentally dimorphic grooming was supported only for Bovidae. Our results indicate that sexually dimorphic grooming is widespread in the ungulate species surveyed, suggesting that programmed grooming evolved at least as early as the common ancestor to the Artiodactyla.     

Immunology of interactions between ticks and laboratory animals

There is good reason to believe that the resistance to ixodid ticks acquired by guinea pigs, rabbits and mice is immunologically mediated. One proposed mechanism for this resistance, which may well be common to all these laboratory animals, involves cutaneous hypersensitivity reactions. Basophils accumulate at tick attachment sites in the skin of resistant animals and degranulate in response to tick salivary antigens, releasing histamine and other mediators. The mediators may directly cause ticks to cease salivating and feeding and then to detach, or they may induce reflex grooming reactions by the host, leading to the removal of ticks from the itching skin.There are gaps in the evidence supporting this hypothesis, and it is likely that other modes of tick resistance remain to be described. However, it should be recognized that, although there have been a few details added to the story in the last fifty years, William Trager's original classic observations and conclusions still stand as the core of the current dogma.