The effects of salivary gland extracts from Boophilus microplus ticks on mitogen-stimulated bovine lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.

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.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3+, CD4+, CD8+ and [gamma][delta] T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for [gamma][delta] T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

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.

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin).

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.

Comparison of bioassay responses to the potential fungal biopesticide Metarhizium anisopliae in Rhipicephalus(Boophilus) microplus and Lucilia cuprina.

Quantal response bioassays were conducted with cattle ticks and sheep blowflies with three different isolates of Metarhizium anisopliae and different methods of inoculation. Ticks were either topically dosed with 2 mu l or immersed in the conidial preparations. Blowflies were either topically dosed with 2 mu l of the conidial preparation or fed on conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95% of ticks after 2 days and 88 (+/- 2)% of blowflies after 4 days. Strikingly different mortality patterns indicated that virulence is dependent on different mechanisms in ticks and blowflies. The pattern of mortality seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose.

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.

Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations. (C) 2012 Elsevier Ltd. All rights reserved.

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.

Dinámica de la garrapata (Boophilus microplus) en el municipio de Siuna, Región Autónoma del Atlántico Norte (RAAN)

No existe información sobre el tiempo que requiere la garrapata B. microplus para su desarrollo no parasítico en áreas ganaderas del municipio de Siuna, R.A.A.N. El estudio se realizo en 10 fincas localizadas en tres comunidades pertenecientes al lugar antes mencionado, con temperatura media anual de 26º C; 80% ±5% y 2000 mm de precipitación, se estudiaron las fases no parasíticas de preovopo sición, ovoposición, incubación y supervivencia larval de B. microplus a partir de huevos depositados por teleoginas. Se utilizó un diseño completamente aleatorio (DCA), para lo cual se tomaron 108 animales que se dividieron en tres categorías, 36 vacas en producción Láctea, 36 terneros (as) menores de un año y 36 vacas vacías, a los que se les realizaba recuento de garrapatas cada dos semanas para determinar la carga parasitaria existente en cada visita a la finca. La observación de los cultivos “ in vitro ” se realizó tres veces al día para determinar el desarrollo de las fases de: Preovoposición 2 a 5 días; ovoposición, de 13 a 22 días; incubación de 23 a 33 días; y supervivencia larval de 53 a 96 días. El análisis que se realizó fue descriptivo (AD) el cual determinó que existían diferencias significativas en las fechas de recuento. La máxima capacidad vital larvaria (MCV) observada aisladamente fue de 100 días. Los resultados están correlacionados con los factores en que fue realizado el estudio, esto puede variar al contraponerlo a los factores naturales. En relación a la aplicabilidad del presente estudio para programas de control bioecológico de la garrapata Boophilus microplus , se recomendaría un período de descanso de potreros superior a la MCV como estrategia de manejo sanitario, y la introducción de bovinos resistentes naturalmente a la garrapata, como medida de control racional.

Efeito acaricida in vitro de extratos de plantas do Pantanal no carrapato de bovinos, Rhipicephalus (Boophilus) microplus.

Estudou-se a ação acaricida dos extratos brutos etanólicos de 14 plantas do Pantanal sobre fêmeas ingurgitadas do carrapato-do-boi. Os bioensaios foram realizados em triplicatas pelo método de imersão das teleóginas e estimadas a produção de ovos, a eclodibilidade e a eficácia dos extratos. Extratos do lenho da raiz e da casca da raiz de Annona dioica, da raiz e da casca do caule de Simarouba versicolor, da raiz de Annona cornifolia e de Duguetia furfuracea tiveram atividade acaricida entre 50% e 100% e resposta dose-dependente. Extratos de Dimorphandra mollis, Magonia pubescens, Protium heptaphyllum, Hyptis crenata, Sebastiana hispida, Aspidosperma australe, Senna occidentalis e de Elyonurus muticus mostraram atividade acaricida baixa ou ausente (0% a 10%). Extratos da parte aérea de A. cornifolia, da planta inteira de Croton glandulosus e da casca do caule de Stryphnodendron obovatum mostraram atividade acaricida intermediária (10% a 20%). A alta atividade acaricida observada nos extratos de A. dioica, A. cornifolia e D. furfuracea indica ação de acetogeninas, moléculas naturais com várias atividades farmacológicas já descritas e presentes, principalmente, nas plantas da família Annonaceae.

Situação atual da resistência do carrapato-do-boi Rhipicephalus (Boophilus) microplus aos acaricidas no Brasil.

Rhipicephalus microplus é o principal ectoparasita hematófago dos bovinos, presente nas diversas regiões do país. É causador de enormes perdas econômicas pela espoliação que causa ao hospedeiro, além de ser transmissor da tristeza parasitária bovina. O principal método de controle utilizado atualmente é o controle químico, mas é crescente o número de relatos que apontam um aumento das populações resistentes de carrapatos a diversos princípios químicos presentes nos acaricidas. As bases moleculares da resistência em R. microplus ainda não são totalmente conhecidas, mas muitos estudos indicam alguns mecanismos associados. O estudo dos mecanismos de resistência no carrapato aos acaricidas é de fundamental importância para a eficiência do seu controle no rebanho bovino. O objetivo deste trabalho é apresentar uma visão geral da resistência dos carrapatos aos acaricidas e a situação atual deste problema no Brasil.