Biological Parameters of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Fed on Rabbits,Sheep, and Cattle

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.     

Red deer (Cervus elaphus) as a host for the cattle tick Rhipicephalus microplus (Acari: Ixodidae) in Yucatan, Mexico

Rhipicephalus microplus is the most economically important cattle tick in the Mexican tropics. Wild ungulate species, including red deer (Cervus elaphus), are gaining popularity in diversified livestock ranching operations in Mexico. However, there is no information available on the susceptibility of red deer to infestation with the cattle tick, R. microplus, under hot, subhumid tropical conditions in Mexico. Biological data on R. microplus as an ectoparasite of cattle and red deer in a farm in the Mexican tropics are presented here. Ticks collected from red deer were identified as R. microplus (97 %) and Amblyomma cajennense (3 %), and tick species infesting cattle included R. microplus (95 %) and A. cajennense (5 %). Standard counts of R. microplus engorged females on red deer were 11 times higher than on cattle (428 ± 43 vs. 40 ± 18; p < 0.001). The reproductive efficiency index and larval hatching of R. microplus collected from cattle and red deer were similar (p > 0.05). Hemolymph samples of R. microplus collected from cattle were positive for Babesia spp. (10 %, 2/50) and all the samples from ticks infesting red deer were negative. Seventeen and ten percent of the blood samples from cattle and red deer were positive for Anaplasma marginale, respectively. The role of red deer as a host of R. microplus in Yucatan, Mexico and the importance of this host-parasite relationship relative to the epidemiology of R. microplus-borne diseases are discussed.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.     

Exploring the midgut proteome of partially fed female cattle tick (Rhipicephalus (Boophilus) microplus)

The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.     

Staphylococcus saprophyticus is a pathogen of the cattle tick Rhipicephalus (Boophilus) microplus

During experimental Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infestation on cattle, approximately 5% of engorged female ticks showed symptoms of bacterial infection. The affected ticks were unable to oviposit, secreted a distinctive yellow exudate through the genital orifice and eventually died. Microscopic analysis of tick exudate showed abundant clusters of Gram-positive cocci bacteria that were isolated and cultured on bacteriological medium. Biochemical phenotyping and 16S rRNA ribotyping analysis on cultured bacteria identified it as Staphylococcus saprophyticus. This species was also isolated from healthy tick larvae, indicating that S. saprophyticus is commonly found in ticks during different developmental stages. However, conspicuous symptoms are only found on fully engorged females. Cultured S. saprophyticus induced identical pathological symptoms when the bacteria were experimentally inoculated into healthy ticks, demonstrating it to be the causative agent of the R. microplus infectious lethal disease described in this work.     

Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes

Waladde S. M., Kemp D. H. and Rice M. J. 1979. Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes. International Journal for Parasitology9: 89–95. Newly moulted adult females of the cattle tick Boophilus microplus readily attach on a modified Baudruche membrane. Apparatus design permits the media offered below the membrane to be kept at 37°C and to be changed easily. Patterns of feeding activity were recorded within 2 h of placing the ticks on the membrane by monitoring the changes in electrical resistance between a tick and the medium with a high input impedance electric circuit. Differences in patterns of sucking and salivation were related to the chemical composition of media presented below the membrane. These observations suggest that the newly discovered cheliceral taste receptors of B. microplus are able to mediate changes in feeding patterns in response to stimulation by different chemical solutions in the feeding lesion. Incorporation of phosphorus-32 into the medium allows the volume ingested by ticks to be measured. The techniques described here facilitate the study of host factors that influence attachment, engorgement and detachment of the cattle tick.     

Bacteriological analysis of saliva from partially or fully engorged female adult <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> by next-generation sequencing

Tick-borne diseases are a major epidemiological problem worldwide. The aim of this study was to investigate the bacterial composition of saliva obtained from engorged adult Rhipicephalus microplus females. Saliva samples collected from partially or fully engorged adult female ticks were analysed using an ultra-high-throughput Illumina HiSeq 2500 sequencing system. To elucidate the possible routes of bacterial transmission, the bacterial flora from whole ticks were also investigated. Proteobacteria, Firmicutes, and Actinobacteria were the predominant phyla in all samples, and Acinetobacter, Rickettsia, Escherichia and Coxiella were the major genera. Microbial diversity in saliva samples from partially engorged ticks was more complex than that of samples from fully engorged individuals. The comparison of saliva and whole-tick samples suggests that bacteria in saliva also colonize the tick’s body. We believe that some bacterial genera, such as Dermacoccus, Achromia, SMB53, Sutterella, Providencia, Mycoplana, Oscillospira, and Agrobacterium, were found and reported in ticks for the first time. The Coxiella and Rickettsia detected in this study might be tick-borne pathogens, suggesting health risks associated with exposure to R. microplus in humans and animals. These findings may serve as the basis for developing strategies to control ticks and tick-borne diseases.     

Displacement of Rhipicephalus decoloratus by Rhipicephalus microplus (Acari: Ixodidae) in the Eastern Cape Province, South Africa

The objective of the study was to establish to what extent the native tick species Rhipicephalus decoloratus has been displaced by the invasive introduced tick, Rhipicephalus microplus at two communally grazed areas in the Eastern Cape Province, South Africa. To this end ticks were collected monthly from five cattle over a period of 2 years and from 10 drag-samples of the vegetation over a period of 1 year at each locality. Whereas 10 years previously only R. decoloratus and no R. microplus had been recorded in the vicinity of the two sites, R. microplus now comprised the bulk of collections at both. Furthermore, significantly more R. microplus were collected from cattle at both localities during the 2nd year of the survey than during the 1st. In addition to 83 instances of intraspecific coupling, there were 17 instances of R. microplus males coupled with R. decoloratus females. Collections made from cattle and goats on two farms close to the study sites revealed that R. microplus was present on both host species and that it significantly outnumbered R. decoloratus on one of the farms. Rhipicephalus decoloratus and R. microplus larvae as well as larvae exhibiting characteristics of both species were collected from the vegetation.     

Characterization of Ixophilin,A Thrombin Inhibitor from the Gut of Ixodes scapularis

Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the “anticoagulome” of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host.     

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.     

Two initial vaccinations with the Bm86-based Gavacplus vaccine against Rhipicephalus (Boophilus) microplus induce similar reproductive suppression to three initial vaccinations under production conditions

Background

The cattle tick, Rhipicephalus (Boophilus) microplus, affects livestock production in many regions of the world. Up to now, the widespread use of chemical acaricides has led to the selection of acaricide-resistant ticks and to environmental contamination. Gavac^plus is a subunit vaccine based on the recombinant Bm86 tick antigen expressed in yeast, capable to control infestations of R. microplus under controlled and production conditions. The vaccine constitutes the core element of broad control programs against this ectoparasite, in which acquired immunity in cattle to Bm86 is combined with a rational use of acaricides. At present, the conventional vaccine scheme consists of three doses that should be administered at weeks 0, 4 and 7, followed by a booster every six months.

Results

In this study we assayed a reduction in the number of the initial doses of Gavac^plus, evaluated the time course and the level of bovine anti-Bm86 antibodies elicited, and analyzed the vaccine effect on ticks engorging on immunized cattle under production conditions. Following three different immunization schemes, the bovines developed a strong and specific immune response characterized by elevated anti-Bm86 IgG titers. A reduction in the weight of engorging female ticks, in the weight of the eggs laid and also in R. microplus viable eggs percentage was obtained by using only two doses of Gavac^plus administered at weeks 0 and 4, followed by a booster six months later. This reduction did not differ from the results obtained on ticks engorging on cattle immunized at weeks 0, 4 and 7. It was also demonstrated that anti-Bm86 antibody titers over 1:640, measured in bovines immunized at weeks 0 and 4, were sufficient to affect weight and reproductive potential of female ticks as compared with ticks engorging on unvaccinated animals. In addition, no statistically significant differences were detected in the average weight of eggs laid by ticks engorged on immunized cattle that showed anti-Bm86 specific titers in the range of 1:640 to 1:81920.

Conclusion

The administration of two initial doses of Gavac^plus containing 100 μg of Bm86 antigen to non-immunized cattle under production conditions is sufficient to affect the weight and the reproductive capacity of R. microplus engorging females. According to these results, cattle herds' manipulation and vaccine costs could be potentially reduced with a positive impact on the implementation of integrated control programs against R. microplus.