Tick Eggs Research Articles

Genetic parameters of infectious bovine keratoconjunctivitis and its relationship with weight and parasite infestations in Australian tropical Bos taurus cattle

BackgroundInfectious bovine keratoconjunctivitis (IBK) or ‘pinkeye’ is an economically important ocular disease that significantly impacts animal performance. Genetic parameters for IBK infection and its genetic and phenotypic correlations with cattle tick counts, number of helminth (unspecified species) eggs per gram of faeces and growth traits in Australian tropically adapted Bos taurus cattle were estimated.MethodsAnimals were clinically examined for the presence of IBK infection before and after weaning when the calves were 3 to 6 months and 15 to 18 months old, respectively and were also recorded for tick counts, helminth eggs counts as an indicator of intestinal parasites and live weights at several ages including 18 months.ResultsNegative genetic correlations were estimated between IBK incidence and weight traits for animals in pre-weaning and post-weaning datasets. Genetic correlations among weight measurements were positive, with moderate to high values. Genetic correlations of IBK incidence with tick counts were positive for the pre-weaning and negative for the post-weaning datasets but negative with helminth eggs counts for the pre-weaning dataset and slightly positive for the post-weaning dataset. Genetic correlations between tick and helminth eggs counts were moderate and positive for both datasets. Phenotypic correlations of IBK incidence with helminth eggs per gram of faeces were moderate and positive for both datasets, but were close to zero for both datasets with tick counts.ConclusionsOur results suggest that genetic selection against IBK incidence in tropical cattle is feasible and that calves genetically prone to acquire IBK infection could also be genetically prone to have a slower growth. The positive genetic correlations among weight traits and between tick and helminth eggs counts suggest that they are controlled by common genes (with pleiotropic effects). Genetic correlations between IBK incidence and tick and helminth egg counts were moderate and opposite between pre-weaning and post-weaning datasets, suggesting that the environmental and (or) maternal effects differ between these two growth phases. This preliminary study provides estimated genetic parameters for IBK incidence, which could be used to design selection and breeding programs for tropical adaptation in beef cattle.

Pathogenicity of Metarhizium anisopliae towards Rhipicephalus (Boophilus) microplus under laboratory and field conditions

Metarhizium anisopliae is one of the most studied agents of biological control of several arthropod plagues, including the cattle tick Rhipicephalus (Boophilus) microplus. Studies have been conducted to assess the fungal complex infection process towards its hosts. To accomplish that, mutant strains overexpressing or lacking assumed determinant genes for the process were constructed over the years. A fundamental experiment to demonstrate a particular gene or set of genes participation is the bioassay. The comparison of bioassays using wild and engineered strains is an essential tool to affirm a given gene is crucial in the process. Therefore, the in vitro bioassays should mimic the results obtained in tests under field conditions. In this study, tests under laboratory and filed conditions were done and a correlation analysis was performed in order to statistically validate in vitro bioassays. Tick egg laying, larvae hatching and host mortality were recorded in each experiment through 21 days, both under laboratory and field conditions. In all cases, M. anisopliae treatments were statistically different from the control treatments. A linear regression analysis was performed between the cases. Laboratory results showed a statistically significant correlation with the field conditions using the Pearson’s Correlation Test (P < 0.01 host mortality – 0.969, tick egg laying – 0.977 and larvae hatching – 0.956). These results legitimize the in vitro bioassays and, therefore, constitute them as a valid tool for studying this fungus behavior, so they can be used to infer M. anisopliae response towards R. (Boophilus) microplus. Key words: Metarhizium anisopliae, Rhipicephalus (Boophilus) microplus, bioassay, cattle tick

Syringe test (modified larval immersion test): A new bioassay for testing acaricidal activity of plant extracts against Rhipicephalus microplus

We report a new bioassay “syringe test” (modified larval immersion test) for in vitro evaluation of acaricidal activity of crude plant extracts. Prepared syringes, containing eggs of tick, were incubated until 14d after hatching of eggs, when the bioassay was performed on the larvae. Lethal concentrations for 50% of larvae (LC50), LC90 and LC99 values were calculated for each tested product. 95% confidence intervals for LC50 were very narrow, indicating a high degree of repeatability for the new bioassay on larvae of R. microplus. Bioassays were applied to six crude aqueous-methanol extracts from five plants (Acacia nilotica, Buxus papillosa, Fumaria parviflora, Juniperus excelsa, and Operculina turpethum), of which three showed discernible effects. Twenty-four hours post exposure, LC99 values were 11.9% (w/v) for F. parviflora, 20.8% (w/v) and 29.2% (w/v) for B. papillosa and A. nilotica, respectively. After six days of exposure these values were; 9.1% (w/v), 9.2% (w/v) and 15.5 (w/v) for F. parviflora, A. nilotica and B. papillosa, respectively.

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.

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (Ki 0.65nM), neutrophil elastase (Ki 21nM) and bovine thrombin (Ki 57pM), while D1 inhibited trypsin and neutrophil elastase (Ki of 2.0 and 129nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.

Efficiency of the latex from Euphorbia splendens var. hislopii (N.E. Br.) Ursch &amp; Leandri, Euphorbiaceae, in the control of Rhipicephalus (Boophilus) sanguineus (Latreille, 1806) (Acari: Ixodidae)

The aim of this study was to evaluate the effect of the latex from Euphorbia splendens var. hislopii (N.E. Br.) Ursch & Leandri, Euphorbiaceae, on the eggs and engorged larvae of Rhipicephalus (B.) sanguineus (Latreille, 1806) (Acari: Ixodidae). Six aqueous concentrations: 25, 50, 100, 125, 250 and 500 μL/L of the latex of this plant were tested. The control group was tested only with distilled water. The latex of E. splendens var. hislopii presented an LD50 of 18.031 μL/L and LD90 of 84.610 μL/L against the eggs of R. (B.) sanguineus. The larvae of all the groups treated with the latex presented a low survival rate of 0% at 25 μL/L, 1% at 50 μL/L, 2% at 100 μL/L, 3% at 125 μL/L, 9% at 250 μL/L, 5% at 500 μL/L when compared with the control group (91%). On day 7 and 14 after the application the latex killed more efficiently the treated groups (25, 50, 100, 125, 250 and 500 μL/L). As from day 21 the latex became less effective for all treatments. Our results clearly show that the aqueous concentration of the latex have a strong effect on tick eggs and larvae suggesting that it could become an important acaricide.

Identification of the heat shock protein 70 (HLHsp70) in Haemaphysalis longicornis

A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal “KDEL” that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4–37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks.

Seasonal dynamics and hosts of Amblyomma triste (Acari: Ixodidae) in Argentina

The seasonal dynamics and host usage of Amblyomma triste in Argentina were analyzed. Adults of A. triste were present from early winter to mid-summer, with the peak of abundance from late winter to mid-spring (August to October). Larvae and nymphs were found from December to June, with the peak of abundance in summer. There were no differences among the biological parameters (pre-moult period of larvae and nymphs, pre-oviposition period of females, and minimum incubation period of eggs) of engorged ticks exposed to different photoperiod regimens at the laboratory, but the periods for each biological parameter obtained from ticks exposed in the field were significantly longer than those from the laboratory. Field results fit better with the data of seasonal distribution of each stage. Morphogenetic diapause was not detected, but complementary studies should test the presence of behavioral diapause. Rodents of the subfamily Sigmodontinae (Akodon azarae, Oligoryzomys flavescens, Oligoryzomys nigripes, Oxymycterus rufus and Scapteromys aquaticus) are the principal hosts for immature stages of A. triste, the caviid Cavia aperea could be another potential host for these stages, and birds are exceptional hosts for larvae and nymphs. Regarding hosts of adults in Argentina, domestic and wild large-sized mammals belonging to different orders (cattle, dog, horse, Blastocerus dichotomus and Hydrochoerus hydrochaeris) serve as hosts for adults of this tick species. In conclusion, A. triste has a life cycle of 1 year with adults feeding on large endemic and introduced mammals and immature stages using sigmodontine and caviid rodents as hosts.

Amblyomma imitatorTicks as Vectors ofRickettsia rickettsii, Mexico

Real-time PCR of Amblyomma imitator tick egg masses obtained in Nuevo Leon State, Mexico, identified a Rickettsia species. Sequence analyses of 17-kD common antigen and outer membrane protein A and B gene fragments showed to it to be R. rickettsii, which suggested a potential new vector for this bacterium.

Effect of GSK-3 activity, enzymatic inhibition and gene silencing by RNAi on tick oviposition and egg hatching

Glycogen synthase kinase-3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. It has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. This enzyme has already been described in Rhipicephalus (Boophilus) microplus and the ovaries of females appeared to be the major site of GSK-3 transcription. The treatment with GSK-3 specific inhibitor (alsterpaullone, bromo-indirubin-oxime 6 and indirubin-3-oxime) caused a reduction in oviposition and egg hatching in completely engorged female ticks. The effect was more pronounced in partially engorged females when alsterpaullone was administrated by artificial capillary feeding. Moreover, GSK-3 gene silencing by RNAi in partially engorged females reduced significantly both oviposition and hatching. The study of tick embryogenesis and proteins that participate in this process has been suggested as an important means for the development of novel strategies for parasite control. GSK-3 is an essential protein involved in embryonic processes and for this reason it has already been suggested as a possible antigen candidate for tick control.

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick ( Rhipicephalus ( Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus ( Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC 50, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. ( B.) annulatus in vitro.

Potential role of ticks as vectors of bluetongue virus

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus.

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

BackgroundTicks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation.ResultsThe genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs.ConclusionsOur study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100 %) and moderate temperature (25 °C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30 °C or reducing humidity to 55–75 % induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25 °C and 100 % RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 μm long, 2.5 ± 0.2 μm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Neem-tree ( Azadirachta indica Juss.) extract as a feed additive against the American dog tick ( Dermacentor variabilis) in sheep ( Ovis aries)

Acaricides can be conveyed to ticks via the blood of their hosts. As fruit and kernel extracts from the Meliaceae family, and, in particular the tetranortriterpenoid azadirachtin (AZA) inhibits tick egg production and embryogenesis in the Ixodidae ticks, we investigated the effects of Neem Azal ®, an extract containing 43% AZA, given as a feed additive to lambs artificially infested with engorging adult Dermacentor vairiabilis ticks. After tick attachment, the lambs were allotted to three dietary treatments: AZA0 (control, n = 10), AZA0.3 ( n = 5), and AZA0.6 ( n = 5), with feed containing 0%, 0.3%, and 0.6% AZA on DM basis, respectively. In half of the AZA0 lambs, ticks were sprayed on day 4 after attachment with an ethanol:water:soap emulsion containing 0.6% AZA (AZA0S). In spite of its very pungent odor, the neem extract was well accepted by all but one lamb. No differences were found between treatment groups in liver enzymes in blood, and there was no indication of toxicity. The plasma AZA concentrations after 7 and 14 days of feeding AZA were (4.81 and 4.35 μg/mL) for the AZA0.6 and (3.32 and 1.88 μg/mL) for the AZA0.3 treatments, respectively ( P < 0.0001). Treatments were not lethal to ticks, but tick weights at detachment were 0.64, 0.56, 0.48, and 0.37 g for ticks from the AZA0, AZA0.3, AZA0S, and AZA0.6 treatments ( P < 0.04), respectively, suggesting that blood AZA impaired blood-feeding. The highest mortality rate after detachment was for AZA0.6 ( P < 0.09). As AZA affects embryo development and ticks at the molting stages, we expect that following treatments of hosts for longer periods, one-host ticks will be more affected than the three-host tick D. variabilis.

Pathogenicity ofMetarhizium anisopliae(Hypocreales: Clavicipitaceae) to Tick Eggs and the Effect of Egg Cuticular Lipids on Conidia Development

The ovicidal efficacy of two entomopathogenic hyphomycetes fungi--Metarhizium anisopliae variety acridum (M. an. ac.) Driver and Milner (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae variety anisopliae (M. an. an.) (Metschn.) Sorokin (Hypocreales: Clavicipitaceae)--was evaluated against eggs of three tick species (Acari: Ixodidae)--Hyalomma excavatum (Koch), Rhipicephalus (Boophilus) annulatus (Say), and Rhipicephalus sanguineus (Latereille)--by placing eggs, laid by surface-sterilized females, on conidia-impregnated filter paper. Although M. an. an. strains differed in their virulence to the tested ticks, they reduced the hatching percentages of eggs of all three tick species to 0-32% compared with 80-90% in the control eggs. The M. an. ac. strains were found highly virulent to H. excavatum and R. sanguineus eggs, reducing the hatching percentages to 2-6% but had no influence on hatching of R. annulatus eggs. Older tick eggs were more susceptible to fungal infection than newly laid ones. The effects of polar and nonpolar lipid fractions, extracted from the surface of tick eggs, on the development of conidia were tested. Both germination of M. an. an. conidia and formation of appressoria were stimulated by extracts from egg cuticles of all three tested tick species. However, the stimulating effect was lower when the conidia were exposed to lipids from relatively less susceptible R. annulatus eggs than when exposed to lipids from H. excavatum or R. sanguineus eggs. Unlike those of M. an. an., conidia of M. an. ac. exposed to such lipid extracts did not germinate and did not form appressoria.

Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations.

BackgroundThe cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia pastoris.ResultsRecombinant Ba86 (Israel strain) was used to immunize cattle to test its efficacy for the control of B. annulatus (Mercedes, Texas, USA strain) and B. microplus (Susceptible, Mexico strain) infestations. Bm86 (Gavac and Mozambique strain) and adjuvant/saline were used as positive and negative controls, respectively. Vaccination with Ba86 reduced tick infestations (71% and 40%), weight (8% and 15%), oviposition (22% and 5%) and egg fertility (25% and 50%) for B. annulatus and B. microplus, respectively. The efficacy of both Ba86 and Bm86 was higher for B. annulatus than for B. microplus. The efficacy of Ba86 was higher for B. annulatus (83.0%) than for B. microplus (71.5%). The efficacy of Bm86 (Gavac; 85.2%) but not Bm86 (Mozambique strain; 70.4%) was higher than that of Ba86 (71.5%) on B. microplus. However, the efficacy of Bm86 (both Gavac and Mozambique strain; 99.6%) was higher than that of Ba86 (83.0%) on B. annulatus.ConclusionThese experiments showed the efficacy of recombinant Ba86 for the control of B. annulatus and B. microplus infestations in cattle and suggested that physiological differences between B. microplus and B. annulatus and those encoded in the sequence of Bm86 orthologs may be responsible for the differences in susceptibility of these tick species to Bm86 vaccines.

Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission ofBabesiaparasite

A cDNA encoding the vitellogenin receptor of the ixodid tick, Haemaphysalis longicornis Neumann (HlVgR) was cloned and characterized. The full-length cDNA is 5631 bp, including an intact ORF encoding an expected protein with 1782 amino acids. The deduced amino acid sequence of the HlVgR cDNA revealed two ligand-binding domains with four class A cysteine-rich repeats in the first domain and eight in the second domain similar to those of insect VgRs. The immunoblot analysis detected approximately 197 kDa protein in both tick ovary and egg. The developmental expression profile demonstrated that HlVgR mRNA exists throughout the ovarian development, and the transcriptional level is especially high in the previtellogenic period. Immuno electron microscopy analysis demonstrated that the localization of HlVgR is detected on the external surface of oocyte plasma membrane. RNAi showed that eggs of HlVgR dsRNA-injected adult ticks had not developed into fully mature oocytes and laid abnormal eggs. The Babesia parasite DNA was not detected in the eggs of HlVgR dsRNA-injected tick that fed on Babesia gibsoni infected dog, whereas it was detected in the eggs of PBS-injected ticks and noninjected ticks. Expression of HlVgR was increased by the vitellogenic hormone 20-hydroxyecdysone. These results indicate that HlVgR, which is produced by the developing oocytes, is essential for Vg uptake, egg development in the H. longicornis tick, and transovarial transmission of Babesia parasites.

Vaccine potential of a tick vitellin-degrading enzyme (VTDCE)

VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus ( Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs ( Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.