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.     

Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.     

Development of enzyme linked immunosorbent assays to measure Bm86 antigen of Boophilus microplus (cattle tick) and to detect anti-Bm86 antibodies in serum samples

The immunization of cattle with the Boophilus microplus Bm86 antigen has been successful for the control of cattle tick infestations. To monitor the Bm86 production process and to measure the anti-Bm86 antibody titers in vaccinated cattle, mAb-based ELISA were developed and validated. The development of both immunological methods is essential to obtain a product with high quality and immunogenic properties and to monitor the immunological protection induced in vaccinated cattle against B. microplus.     

Polymorphism of the bm86 Gene in South American Strains of the Cattle Tick Boophilus microplus

Thirty Boophilus microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the bm86 and bm95 gene. A fragment of cDNA of 794 base pairs of the parasite larvae, included between nucleotides 278–1071s, was amplified and cloned on the pGEM-T vector. Two random clones were sequenced for each population and the nucleotides 278–1071 and predicted amino acid sequences compared with the bm86 and bm95 genes. Variations from 1.76 to 3.65% were detected in the nucleotides sequence when compared with the homologous sequence of the bm86 gene and a 3.4–6.08% in the homologous amino acid sequence of the Bm86 protein. When the sequences obtained were compared with the bm95 gene, variations from 0.50 to 3.15% were detected. Variations from 1.14 to 4.56% were detected for the Bm95 protein homologous sequences in the deduced amino acid sequence. Only five of the 30 strains analyzed presented two different types of alleles expressed and the two alleles of the Alegre population and allele 1 of the Betim population were the most divergent of all those analyzed.     

Effect of Soil Texture and Moisture on the Activity of Entomopathogenic Nematodes Against Female Boophilus annulatus Ticks

Soil texture, chemistry and moisture have a profound effect upon the activity and persistence of entomopathogenic nematodes (EPNs). Whereas nematodes’ natural habitat is within the soil, ticks and other arthropod pests prefer to stay on the soil surface and under stones or leaf litter; they spend much of their life cycle in the humid environment of the soil upper layer, therefore consideration of the effect of the soil environment on nematode activity is a pre-requisite for the sucessful use of EPNs against arthropod pests. In the present study we investigated the effects of soil type, and humidity on various nematode strains and on their effectiveness against ticks. Many infective juveniles (IJs) of Steinernema carpocapsae and S. riobrave were found in the uppermost soil layer whereas the heterorhabditid strains were almost absent from the upper 6 cm of the soil profile. The IJs of S. feltiae, and the S. carpocapsae strain S-20, exhibited an intermediate behavior. It was found that the activity of IJs of S. carpocapsae in the soil upper layer (1 cm depth) was strongly affected by soil type: the greatest number of IJs were recorded from sandy loam soil; less were found in the lighter soils – ‘Marine sand’ and ‘Calcareous sandstone’ – and only very few were recovered from heavy soils. Strikingly, even when the soil moisture was low and the number of nematodes found in the upper layer correspondingly low, tick mortality remained high. The results demonstrate: (a) the possible use of the nematodes as an anti-tick agent; (b) the importance of knowing the exact interaction of nematodes with the immediate environment of the pest, in order to optimize the pest-control activity of the nematode.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

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.     

The Susceptibility of Boophilus annulatus (Ixodidae) Ticks to Entomopathogenic Fungi

The pathogenicity of five species of entomopathogenic fungi (Deuteromycetes, species: Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, Paecilomyces fumosoroseus and Verticillium lecanii ) to the various developmental stages of Boophilus annulatus ticks was compared under laboratory conditions. M. anisopliae and B. bassiana strains were most virulent to engorged females and caused 85-100% mortality within 7-10 days post-inoculation (PI). The highest mortality of engorged females caused by other fungi reached only 25-60%. All tested fungi prevented or reduced the egg laying capability of the ticks several days before their death. Females surviving after treatment with the most virulent M. anisopliae strain (Ma-7) reached only 7-8% of their egg laying capacity as compared with the control. Other fungi caused a reduction of the weight of laid eggs by 35.4-80.8% as compared with untreated females. Only M. anisopliae and B. bassiana strains caused 70-98% mortality of the treated eggs. Unfed larvae of Boophilus annulatus were sensitive to M. anisopliae and M. flavoviride strains. The Ma-7 strain was most virulent to unfed larvae, with a mortality rate of 80.4% at a concentration of 1 ×107 spores ml -1 and 100% mortality at a concentration of 1 ×108 spores ml -1 .     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Influence of acaricide resistance on cattle-fever tick (Boophilus spp.) infestations in semi-arid thornshrublands: a simulation approach

Cattle-fever tick (Boophilus microplus and B. annulatus) populations that develop acaricide resistance become more difficult to control or eradicate. We used a simulation model to assess the direct and indirect effects of interactions among season, habitat type, grazing strategy, and acaricide resistance on the ability to eradicate Boophilus infestations in semi-arid thornshrublands of Texas, USA. Season of infestation appeared to have the strongest effect, with infestations begun on 27 September (autumn) tending to die out sooner than those begun on 1 March (spring) and to remain undetected. Habitat type had the next strongest effect, with infestations surviving much longer as canopy cover increased from uncanopied buffelgrass (Cenchrus ciliaris) habitats to mesquite (Prosopis glandulosa)-canopied grass habitats. Acaricide resistance had a moderate effect; as expected, highly resistant tick populations survived longer than those with no acaricide resistance. The importance of grazing strategy varied with changes in habitat type: as canopy cover increased, infestation duration increased faster under continuous grazing than under rotational grazing strategies. Importance of grazing strategy also varied with acaricide resistance: detected tick populations with no and slight acaricide resistance subjected to acaricide treatments tended to survive longer under rotational grazing than continuous grazing, due to reduced contact with a treated host. Populations with moderate and high resistance behaved more like untreated populations, tending to survive longer under continuous, rather than rotational, grazing, because they experienced less mortality on a treated host. Assuming acaricide treatments at 2-week intervals and maintenance of cattle in infested pastures, results indicate that, for each habitat type, infesting ticks have a threshold of acaricide resistance below which one can eradicate them faster with continuous grazing than with rotational grazing. As canopy cover increases, this threshold appears to shift from high resistance (in grass) to slight resistance (in mesquite).     

Synthetic vaccine (SBm7462) against the cattle tick Rhipicephalus (Boophilus) microplus: preservation of immunogenic determinants in different strains from South America

The synthetic vaccine SBm7462 is based on three immunogenic epitopes (4822, 4823 and 4824) contained within protein Bm86 derived from the Australian Yeerongpilly strain of the tick Rhipicephalus (Boophilus) microplus. Twenty strains of the tick originating from Brazil, Argentina, Colombia and Uruguay were analysed in order to identify differences compared with sequences present in components of vaccine SBm7462. For each parasite population, three cDNA fragments containing the nucleotides coding for the epitopes 4822, 4824 and 4823 were sequenced, and the amino acid sequences were deduced and compared with those of the homologous bm86 gene. The results indicate that the epitope sequences of vaccine SBm7462 are conserved in the South American populations of the tick. The conservation of such sequences is very important for the immunological response of different populations of R. (B.) microplus.     

Experimental infestation of llamas (Lama glama) with Boophilus microplus (Acari: Ixodidae)

Two llamas (Lama glama) were infested with approximately 20,000 Boophilus microplus larvae each and maintained under controlled conditions to collect the detached adult ticks. A total of 72 engorged and semi-engorged females were recovered with a mean weight of 163.0±70.6mg. Sixty-three (87.5%) of these ticks oviposited and 60 (95.2%) of the egg masses produced larvae. Six of the heaviest females laid 18,838 eggs from which 16,545 (87.8%) hatched. These results show that llamas are able to maintain a population of B. microplus under experimental conditions. Further studies are required to determine the role of llamas as alternative hosts for B. microplus under field conditions.     

In vitro Detection of Acaricide Resistance in Boophilus microplus (Acari: Ixodidae)

The cattle tick Boophilus microplus causes significant economic losses in cattle in the tropical areas of Central and South America. Acaricides are widely used to control tick infestations, with the consequent selection of tolerant and resistant subpopulations. Detoxifying enzymes, and esterases in particular, have been associated with organophosphate resistance in Mexican isolates of B. microplus ticks. Here, we present results of the biochemical detection of esterases and oxidases in 20 isolates of larvae and adult ticks and the effect of esterase and oxidase inhibitors on larvae and adult stages of the tick. Our results indicate the presence of significant differences both in vivo and in vitro between esterase and oxidase activities of OP-susceptible and OP-resistant isolates, even when samples were collected in the same area. The importance of such differences in regionalized integrated pest management and in the US Tick Eradication Program is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microsatellite Loci of the Cattle Tick Boophilus Microplus (Acari: Ixodidae)

This brief communication reports the identification of microsatellite loci in the economically important tick species Boophilus microplus. The data are potentially useful in distinguishing different strains of B. microplus. Eight polymorphic loci were isolated in larvae, male and female adults analysed individually from 12 field isolates and laboratory strains from Australia (n = 8), Brazil, Mexico, Papua New Guinea and Zimbabwe. Nucleotide sequencing of alleles at these microsatellite loci revealed that non-repeat bases interrupted dinucleotide and tetranucleotide repeats in some loci. Loci with non-repeat bases interrupting them were shorter compared with loci that were not interrupted. Thus the presence of non-repeat bases in a repeated sequence seems to constrain the evolution of additional repeats by slip-strand misparing at these loci.     

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac TM

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac^TM has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac^TM in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995–2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.     

The Distribution and Ecological Preferences of Boophilus microplus (Acari: Ixodidae) in Mexico

This paper focuses on the distribution of the cattle tick, Boophilus microplus, in Mexico. The study is aimed to understand the climate factors responsible of the recorded distribution that can statistically define the suitable habitat for the tick. Sites where the tick is recorded display significantly higher values of some climate variables in comparison with those where the tick is absent, namely mean monthly temperature (T) and atmospheric water vapour (W), yearly accumulated T, W and rainfall (R) (p < 0.001 for every variable), with smaller significance for the yearly sum of T/R and T/W ratios (p < 0.05). Interestingly, variables involving the Normalized Derived Vegetation Index (NDVI) do not shown statistical differences between the sites where the tick is present or absent. The best set of habitat-defining variables was integrated into a framework to assess the habitat suitability for the tick in Mexico. We used a point-to-point similarity metric to assign a classification value to a candidate site based on the proximity in environmental space of the most similar record site. A combination of 7 yearly and monthly values for temperature, rainfall and water vapour variables captured the tick distribution. Model performance, as tested with a separate set of distribution tests and defined by the AUC value, was 0.89. Causes of errors as detected with a visual comparison of both known and predicted distribution of the tick may be attributed to the use of a medium resolution, unable to capture locally important features of tick distribution, and to incomplete collections in some parts of the country.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

Therapeutic and persistent efficacy of a single injection treatment of ivermectin and moxidectin against Boophilus microplus (Acari: Ixodidae) on infested cattle

The effectiveness of a single treatment with either ivermectin or moxidectin was determined by administering a single subcutaneous injection of each endectocide at 200 g per kg body weight to cattle infested with all parasitic developmental stages (adults, nymphs, and larvae) of Boophilus microplus (Canestrini). The percentage reduction in the number of females that reached repletion following treatment (outright kill) was 94.8 and 91.1% for ivermectin and moxidectin, respectively. In addition, the reproductive capacity of the females that did survive to repletion was reduced by >99%, regardless of the endectocide. Based on these two factors, the therapeutic level of control obtained against ticks on the cattle at the time of treatment was 99.0 and 99.1% for ivermectin and moxidectin, respectively. Engorged females recovered from either group of treated cattle weighed 3-times less than untreated females, and the egg masses produced by treated females weighed 5–8-times less than egg masses produced by untreated females. Partitioning of data into three separate 7-d post-treatment intervals allowed for an estimation of the efficacy of each endectocide against each individual parasitic development stage (adult, nymph, and larva). Results indicated that both endectocides were 99.7% effective against ticks that were in either the adult or nymphal stage at the time of treatment. However, the level of control against ticks in the larval stage of development at treatment was significantly lower at 97.9 and 98.4% for ivermectin and moxidectin, respectively. Analysis of the persistent (residual) activity of the two endectocides indicated that neither material provided total protection against larval re-infestation for even 1-wk following treatment. Against larvae infested 1–4 wk following treatment, the level of control with moxidectin ranged from 92.4% (1 wk) to 19.5% (4 wk). These control levels were higher at each weekly interval than for ivermectin, which ranged from 82.4% (1 wk) to 0.0% (4 wk). The potential for the use of these injectable endectocide formulations in the US Boophilus Eradication Program is discussed.This paper reports the results of research only. Mention of a commercial or proprietary product in this paper does not constitute an endorsement by the U.S. Department of Agriculture. In conducting the research described in this report, the investigators adhered to protocol approved by the USDA-ARS Animal Welfare Committee. The protocol is on file at the USDA-ARS, Knipling-Bushland U.S. Livestock Insects Laboratory, Tick Research Unit, Kerrville, TX. The U.S. Governments right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.This revised version was published online in May 2005 with a corrected cover date.     

The novel protein BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis

The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68 kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.