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.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

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.     

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.     

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.     

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.     

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.     

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.     

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.     

Anti-tick effects of Melinis minutiflora and Andropogon gayanus grasses on plots experimentally infested with Boophilus microplus larvae

Our objective in this trial was to investigate the anti-tick effects of molasses grass (Melinis minutiflora) and gamba grass (Andropogon gayanus) against Boophilus microplus, using buffel grass (Cenchrus ciliaris) as a control. During a 3-year-period fieldwork was carried out in the tropical, subhumid climate at Progreso, Municipality of Jiutepec, Morelos, Mexico. Experimental plots were established for three treatments with six replicates under a random factorial design in order to evaluate each of the four seasons. Additional work was developed for two more consecutive autumn seasons to evaluate the anti-tick effects of these grasses. Once the plots were established they were infested with 5000 B. microplus larvae and sampled by dragging flannel cloths. The anti-tick effects were measured by counting the number of larvae recovered from each of the experimental plots. Both grasses, M. minutiflora and A. gayanus, demonstrated anti-tick effects; however, M. minutiflora grass had the greatest anti-tick effects through the seasons studied and in the three consecutive autumns evaluated.     

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.     

A thrombin inhibitor from the gut of <Emphasis Type="Italic">Boophilus microplus</Emphasis> ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin–Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.     

Serine Proteinase Inhibitors from Eggs and Larvae of Tick Boophilus microplus: Purification and Biochemical Characterization

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as E1, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCl buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant K _i determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTIs) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The K _i for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.     

Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus

Thirty-one isolates of Metarhizium anisopliae were bioassayed against the cattle tick (Boophilus microplus). More than half of the isolates showed a high degree of virulence to ticks. Radial growth curves for growth between 20 degrees C and 40 degrees C were obtained for all isolates. This information together with information on virulence will be important for the selection of isolates suitable to kill ticks on the surface of cattle. A biopesticide for cattle ticks must kill ticks rapidly at temperatures within the upper end of most isolates' growth curves. It was also found that the time taken to achieve 100% tick mortality in vitro using a virulent isolate could be halved by applying conidia in a 10% oil emulsion. Scanning electron microscopy and light microscopy were used to investigate and compare the germination and penetration of conidia formulated in aqueous and oil formulations. It was found that conidia in both formulations were able to germinate and produce appressoria on the surface of ticks in less than 11h. Marked weakness within 26h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.     

A comparison of the immunosuppressive effects of salivary gland extracts from two laboratory strains of Boophilus microplus

This study addresses three questions related to the immune response of cattle to tick salivary gland extracts. Firstly, is there a difference in the inhibition of proliferation of Concanavalin A (ConA) stimulated bovine lymphocytes induced by salivary gland extracts of the N and Y strains of Boophilus microplus? Second, is there a difference in the development rate of the Y and N tick strains? Third, does the host affect the inhibitory effect of salivary gland extract on the proliferation of ConA stimulated lymphocytes from the two tick strains? Salivary gland extract of the Y strain inhibited in vitro proliferation of lymphocytes stimulated by ConA significantly more than that of the N strain, when each strain was raised on different animals. A difference in the development rate was observed between the tick strains when raised on the same animal, with female ticks of the Y strain developing faster and reaching a greater fully engorged weight than ticks of the N strain. The difference in their rate of development did not appear to contribute to a difference in inhibitory effects of the salivary gland extracts and there was no difference between the inhibitory effects of salivary gland extracts from both strains. However, when Y strain ticks were raised on different animals, there was a significant difference in the inhibition of lymphocyte proliferation between the two salivary gland extracts. Therefore, it was concluded that there is no difference between the inhibitory effects of the two tick strains and that the host has an influence on salivary gland extract composition of B. microplus and its inhibitive properties.     

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.     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.