The effect of the cattle tick on growth rates and reproductive rates of Bos indicus cross heifers in north Queensland

Tick infestation occurs over 1.3 x 106 km2 in northern Australia. It has been difficult to estimate the economic effects of ticks due to a lack of information on their effects on growth and reproduction (Anon 1975). 12th Biennial Conference. February 1978. Melbourne, Victoria

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.

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 °C and 40 °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 11 h. Marked weakness within 26 h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.

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

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

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Activation of several key components of the epidermal differentiation pathway in cattle following infestation with the cattle tick, Rhipicephalus (Boophilus) microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus, and the diseases it transmits pose a persistent threat to tropical beef production. Genetic selection of host resistance has become the method of choice for non-chemical control of cattle tick. Previous studies have suggested that larval stages are most susceptible to host resistance mechanisms. To gain insights into the molecular basis of host resistance that occurs during R. microplus attachment, we assessed the abundance of proteins (by isobaric tag for relative and absolute quantitation (iTRAQ) and Western blot analyses) and mRNAs (by quantitative reverse transcription PCR (qRT-PCR)) in skin adjacent to tick bite sites from high tick-resistant (HR) and low tick-resistant (LR) Belmont Red cattle following challenge with cattle tick. We showed substantially higher expression of the basal epidermal keratins KRT5 and KRT14, the lipid processing protein, lipocalin 9 (LCN9), the epidermal barrier catalysing enzyme transglutaminase 1 (TGM1), and the transcriptional regulator B lymphocyte-induced maturation protein 1 (Blimp1) in HR skin. Our data reveals the essential role of the epidermal permeability barrier in conferring greater resistance of cattle to tick infestation, and suggest that the physical structure of the epidermal layers of the skin may represent the first line of defence against ectoparasite invasion. Crown Copyright. © Australian Society for Parasitology Inc.

Fungal Biopesticide for cattle tick and buffalo fly control

Cattle ticks and buffalo flies impose significant economic burdens on the Northern Australian cattle and dairy industries. With the increased temperatures expected under climate change the range of parasites such as these is likely to extend. Current control options for these ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides offer a sustainable and promising alternative method of control. Laboratory and animal studies have established the potential for the fungus Metarhizium in tick control and provided data that suggests a secondary effect of buffalo fly control is possible. Small field trials are required to obtain a proof of concept for the control of ticks and buffalo flies on animals.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Repellent effects of Melaleuca alternifolia (tea tree) oil against cattle tick larvae (Rhipicephalus australis) when formulated as emulsions and in β-cyclodextrin inclusion complexes

Rhipicephalus australis (formerly Boophilus microplus) is a one host tick responsible for major economic loss in tropical and subtropical cattle production enterprises. Control is largely dependent on the application of acaricides but resistance has developed to most currently registered chemical groups. Repellent compounds that prevent initial attachment of tick larvae offer a potential alternative to control with chemical toxicants. The repellent effects of Melaleuca alternifolia oil (TTO) emulsions and two β-cyclodextrin complex formulations, a slow release form (SR) and a modified faster release form (FR), were examined in a series of laboratory studies. Emulsions containing 4% and 5% TTO applied to cattle hair in laboratory studies completely repelled ascending tick larvae for 24 h whereas 2% and 3% formulations provided 80% protection. At 48 h, 5% TTO provided 78% repellency but lower concentrations repelled less than 60% of larvae. In a study conducted over 15 days, 3% TTO emulsion applied to cattle hair provided close to 100% repellency for 2 days, but then protection fell to 23% by day 15. The FR formulation gave significantly greater repellency than the emulsion and the SR formulation from day 3 until the end of the study (P < 0.05), providing almost complete repellency at day 3 (99.5%), then decreasing over the period of the study to 49% repellency at day 15. Proof of concept is established for the use of appropriately designed controlled-release formulations to extend the period of repellency provided by TTO against R. australis larvae.

Expression of intracellular calcium signalling genes in cattle skin during tick infestation

It is widely acknowledged that changes in intracellular calcium ion (Ca2+) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca2+ signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca2+ signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca2+ and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.

Identification of genes involved with tick infestation in Bos taurus and Bos indicus

Tick resistant cattle could provide a potentially sustainable and environmentally sound method of controlling cattle ticks. Advances in genomics and the availability of the bovine genome sequence open up opportunities to identify useful and selectable genes controlling cattle tick resistance. Using quantitative real-time PCR and the Affymetrix bovine array platform, differences in gene expression of skin biopsies from tick resistant Bos indicus (Brahman) and tick susceptible Bos taurus (Holstein-Friesian) cattle following tick challenge were examined. We identified 138 significant differentially-expressed genes, including several immunological/host defence genes, extracellular matrix proteins, and transcription factors as well as genes involved in lipid metabolism. Three key pathways, represented by genes differentially expressed in resistant Brahmans, were identified; the development of the cell-mediated immune response, structural integrity of the dermis and intracellular Ca 2+ levels. Ca2+, which is implicated in host responses to microbial stimuli, may be required for the enhancement or fine-tuning of transcriptional activation of Ca2+- dependant host defence signalling pathways. Animal Genomics for Animal Health International Symposium, Paris, October 2007: (Proceedings)

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Commercial products from bio-active extractives in cypress milling residues.

Extractive components obtained from milling residues of white cypress were studied for chemical identity and bioactivity with a view to developing a commercial use for these components, thus increasing the value of the residues and improving the economics of cypress sawn wood production. Extracts obtained by solvent or steam extraction techniques from cypress sawdust were each fractionated by a range of techniques into groups of similar compounds. Crude extracts and fractions were screened against a range of agricultural pests and diseases, including two fungi, subterranean termites, fruit spotting bugs, two-spotted mites, thrips, heliothis, banana scab moths, silverleaf whiteflies, cattle tick adults and larvae, and ruminant gastrointestinal nematodes. Additional screening was undertaken where encouraging results were achieved, for two-spotted mites, thrips, silverleaf whiteflies, cattle tick adults and ruminant gastrointestinal nematodes. After considering degrees of efficacy against, and economic importance of, the agricultural pests, and likely production costs of extracts and fractions, the crude extract (oil) produced by steam distillation was chosen for further study against silverleaf whitefly. A useful degree of control was achievable when this oil was applied to tomato or eggplant at 0.1%, with much less harmful effects on a beneficial insect. Activity of the oil against silverleaf whitefly was undiminished 3.5 years after it was generated. There was little benefit from supplementing the extract with co-formulated paraffinic oil. From the steam distilled oil, fifty-five compounds were characterised, thirty-five compounds representing 92.478 % of the oil, with guaiol (20.8%) and citronellic acid (15.9%) most abundant. These two compounds, and a group of oxygenated compounds containing bulnesol and a range of eudesmols, were found to account for most of the activity against silverleaf whitefly. This application was recommended for first progression to commercialisation.

CattleTickBase: An integrated Internet-based bioinformatics resource for Rhipicephalus (Boophilus) microplus

The Rhipicephalus micro plus genome is large and complex in structure, making it difficult to assemble a genome sequence and costly to resource the required bioinformatics. In light of this, a consortium of international collaborators was formed to pool resources to begin sequencing this genome. We have acquired and assembled genomic DNA into contigs that represent over 1.8 Gigabase pairs of DNA from gene-enriched regions of the R. micro plus genome. We also have several datasets containing transcript sequences from a number of gene expression experiments conducted by the consortium. A web-based resource was developed to enable the scientific community to access our datasets and conduct analysis through a web-based bioinformatics environment called YABI. The collective bioinformatics resource is termed CattleTickBase. Our consortium has acquired genomic and transcriptomic sequence data at approximately 0.9X coverage of the gene-coding regions of the R. microplus genome. The YABI tool will facilitate access and manipulation of cattle tick genome sequence data as the genome sequencing of R. microplus proceeds. During this process the CattleTickBase resource will continue to be updated. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.