Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Molecular characterization of HAO3, the homologue of the Bm86 tick vaccine antigen,from the Iranian isolate of Hyalomma anatolicum anatolicum

Hyalomma anatolicum anatolicum tick is widely distributed in many parts of Iran and while the commercial vaccines based on the application of midgut-derived recombinant Bm86 antigen are used for its control, limited information about the efficiency of this vaccination in Iran is available. Herein, with the final aim of evaluation of Bm86-based heterologous vaccination, as the primary step the Bm86 homologue of the H. a. anatolicum (Hao3) from an Iranian isolate was characterized and compared with the commercialized Bm86 and other Bm86 homologoue sequences available in GenBank.     

Laboratory rearing of <Emphasis Type="Italic">Theileria annulata</Emphasis>-free <Emphasis Type="Italic">Hyalomma anatolicum anatolicum</Emphasis> ticks

Hyalomma anatolicum anatolicum is a three-host tick which transmits Theileria annulata infection in Indian cattle. Laboratory rearing of ixodid ticks is an essential requirement of any laboratory engaged with research on ticks and tick borne diseases. The Entomology laboratory of Indian Veterinary Research Institute is fully equipped with all the facilities and skilled manpower to maintain a homogenous H. a. anatolicum population throughout the year. The continuous supply of eggs, larvae and adults of H. a. anatolicum is maintained to meet out the demand of different experiments viz., preparation of tick antigens for immunization of animals, experimental challenge, isolation of genomic DNA and RNA. Maintenance of a H. a. anatolicum colony free of T. annulata infection is imperative for the experimental challenge infestation on cross-bred (Bos indicus × B. taurus) calves, in order to prevent the transmission of T. annulata infection to the experimental animals. A system has been developed in the laboratory in which the larvae of H. a. anatolicum were fed on New Zealand white rabbits and the dropped fed nymphs molted to adults are fed on cross-bred calves free of T. annulata infection. This synthetic cycle prevents the transstadial transmission of T. annulata as the rabbits are unsusceptible to T. annulata infection and only the adults were fed on cross-bred animals. Moreover, absence of transovarial transmission of T. annulata prevents the chance of carry over infection to experimental animals in the next cycle. Declaration: The experiments have been conducted in accordance with the approved guidelines of Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA). Besides, the institute animal ethics committee continuously monitored the animal experimentation.     

Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

Background  

For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.     

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac TM

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac^TM has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac^TM in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995–2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.     

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.     

Shifts in the distributional ranges of <Emphasis Type="Italic">Boophilus</Emphasis> ticks in Tanzania: evidence that a parapatric boundary between <Emphasis Type="Italic">Boophilus microplus</Emphasis> and <Emphasis Type="Italic">B. decoloratus</Emphasis> follows climate gradients

Based on a national tick survey conducted in Tanzania between 1998 and 2001, predictive maps of tick distribution for B. microplus and B. decoloratus were computed and compared with historical maps that originated about 40 years ago. The new data collected showed that except for extremely cold and dry areas B. microplus has extended its distribution range and is now present in all the northern regions of Tanzania, and that high suitability is currently recorded for most of the previously non-occupied areas. In contrast, B. decoloratus, once abundant in Tanzania, has largely retreated to highlands in north and central Tanzania and several other high-altitude refuges. Geostatistical analysis revealed a dissimilar character of distribution for the two species at a local and regional scale, as well as an antagonistic relationship between them in a spatial context. The Tanzania data suggest that an equilibrium phase with a stable parapatric boundary that follows a climate gradient has been achieved. The zone of overlap in the distribution ranges of B. microplus and B. decoloratus is characterized by a decreased infestation rate as well as mutual exclusion on infested cattle. This finding matches studies on parapatry with hybrid zones, where the latter zones that separate species tend to rest in density troughs. Analysis of the Tanzania data shows that the critical factor for the advance of B. microplus and the retreat of B. decoloratus is associated with the 58 mm isohyet and the 22–23°C isotherm and indicates a well developed higher-temperature tolerance for B. microplus. It can be anticipated that climate changes may enhance the spread of B. microplus and consequently Babesia bovis into new areas of the African continent.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.     

Electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections

Background

Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.

Results

In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35–42%) and purity (80–85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.

Conclusion

These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.     

In vitro Detection of Acaricide Resistance in Boophilus microplus (Acari: Ixodidae)

The cattle tick Boophilus microplus causes significant economic losses in cattle in the tropical areas of Central and South America. Acaricides are widely used to control tick infestations, with the consequent selection of tolerant and resistant subpopulations. Detoxifying enzymes, and esterases in particular, have been associated with organophosphate resistance in Mexican isolates of B. microplus ticks. Here, we present results of the biochemical detection of esterases and oxidases in 20 isolates of larvae and adult ticks and the effect of esterase and oxidase inhibitors on larvae and adult stages of the tick. Our results indicate the presence of significant differences both in vivo and in vitro between esterase and oxidase activities of OP-susceptible and OP-resistant isolates, even when samples were collected in the same area. The importance of such differences in regionalized integrated pest management and in the US Tick Eradication Program is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of enzyme linked immunosorbent assays to measure Bm86 antigen of Boophilus microplus (cattle tick) and to detect anti-Bm86 antibodies in serum samples

The immunization of cattle with the Boophilus microplus Bm86 antigen has been successful for the control of cattle tick infestations. To monitor the Bm86 production process and to measure the anti-Bm86 antibody titers in vaccinated cattle, mAb-based ELISA were developed and validated. The development of both immunological methods is essential to obtain a product with high quality and immunogenic properties and to monitor the immunological protection induced in vaccinated cattle against B. microplus.     

The Known Distribution and Ecological Preferences of the Tick Subgenus Boophilus (Acari: Ixodidae) in Africa and Latin America

A compilation of the known distribution of Boophilus ticks in Africa and Latin America is presented, together with details on climate preferences. B. annulatus is recorded mainly in the western part of a strip from the equator to parallel 20° N. It associates with woodlands and forests (lowland rain forest and secondary grassland). This species is also present in the Mediterranean region, associated to woodland and open areas. B. decoloratus extends southern to parallel 20° N, in woodland with montane vegetation and Zambezian miombo; some records have been collected in the highveld grassland. B. geigyi is mainly collected in the western range of a stripe extending between parallels 5° N and 18° N, associated with Sudanian woodland, lowland rain forest with secondary grassland and woodland. Confirmed records of microplus in Africa are restricted to Malagasy region and south and eastern Africa, being predominant in the Zambezian miombo, deciduous forest with secondary grassland, and woodland. In Latin America, microplus is abundant in the Mesoamerican corridor to Venezuela and Colombia, and southern in Brazil and Argentina. The tick is mainly associated to the biomes of Chaco and Pampas in Argentina, the North-central moist Andes, the Atlantic forest (southern range) and the moist Meso-American vegetation (northern range). Most collections of B. annulatus and B. geigyi came from areas where winter minimum temperature is above 15 °C, maximum temperatures remain between 33 and 36 °C and maximum rainfall is recorded between June and September. B. decoloratus and African B. microplus are recorded in sites with low temperatures in May–September. Minimum temperature requirements are similar for both B. decoloratus and African B. microplus, and both are around 4 °C less than the value recorded for collections of Latin-American B. microplus. The rainfall pattern observed for decoloratus shows a minimum in May and June. The requirements of total rainfall are highest for B. microplus in Latin America, while records of African B. microplus are concentrated in areas of low rainfall between May and October, and high rainfall between November and March (low rainfall in the same period for B. decoloratus). Statistical analysis revealed the existence of populations (demes) with ecologically different requirements within each tick species. Both B. annulatus and B. decoloratus showed many different demes clearly associated to defined areas. The collections of Latin American B. microplus are very homogeneous according climate preferences and well separated from the African counterpart.     

The effect of orally administered Aloe marlothii leaves on Boophilus decoloratus tick burdens on cattle

The effect of a decoction of pulverized aloe leaves (Aloe marlothii) mixed with tap water and administered orally through a gastric tube was determined against ticks on cattle. The decoction was administered to six randomly selected calves seven times at 3-day intervals while the remaining calves received an equivalent amount of tap water only. Five hours after first treatment the calves were infested with Boophilus decoloratus larvae hatched from one gram of eggs. Resultant engorged female ticks were collected, counted and weighed daily. Fertility estimates were determined for 30 engorged female ticks collected from each calf in both the treated and control groups of calves. The treatment had no significant effect on total or daily numbers of engorged females collected per group, nor on their biomasses. Fertility estimates showed the treatment group to yield a marginally higher, yet insignificant, egg laying response (ELR) and reproductive estimate (RE).     

A note on <Emphasis Type="Italic">Hyalomma nitidum</Emphasis>, its distribution and its hosts

The paper summarises the morphological characteristics, the known geographical distribution and hosts of Hyalomma nitidum, a tick which has never been properly characterised. H. nitidum was first described in Cameroon in 1919, long considered as a synonym of Hyalomma truncatum and recognised as a separate and valid species only since the early 1970s. H. nitidum occurs from Senegal to the Central African Republic in more humid conditions than H. truncatum, roughly to the south of the isohyet of 900 mm. Adults are parasitic on various wild and domestic ungulates, on which immatures have not been found. Nymphs, identified after moulting to adults, have been found on a hare, and tentatively identified larvae and nymphs of this species have also been collected on hare as well as on a zebra mouse. The tick has been found infected with the virus of Crimean-Congo haemorrhagic fever, so far no other disease associations are known.Gerrit Uilenberg - A Surgente     

Partial strategic tick control within a herd of European breed cattle in the state of Rio Grande do Sul,southern Brazil

A trial is described, in the state of Rio Grande do Sul, southern Brazil, as one of a series suggested to investigate the effects of strategic but selective acaricide treatments of cattle within herds against Boophilus microplus. They are aimed at considering the repercussions of farmer attempts at immediate reductions in acaricide costs and the potential for creation of ‘refugia’ of untreated ticks. Half (Group 1) of a small experimental herd of European breed heifers were treated strategically against ticks, three times during the late spring–early summer and twice during autumn (southern hemisphere), with an injectable avermectin endectocide, designed to act directly against the first and third generations of parasitic B. microplus per ‘cattle tick year’ at this site, respectively. The consequent levels of infestations on all of the member cattle in their common pasture were monitored. Group 1 showed low to zero tick counts during the 28-day treatment interval periods and up to ca. 14 days after the last of such a series. Treated cattle, however, became re-infested outside of these periods and to levels that would be considered as unacceptable by farmers in the state. The untreated cattle (Group 2) showed infestations at generally higher levels, than their contemporaries, within and outside of the treatment periods. There were thus ample sources of larvae in the pasture, derived principally from falling, untreated engorged female ticks, re-infesting both the treated and untreated cattle. Advantages of maintaining chemically untreated cattle ticks within a herd, compared to their disadvantages as contaminants to classical strategic control procedures, merit re-evaluation, especially in relation to the recent, world-wide resurgence of acaricide resistance in B. microplus. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

Prevalence of ixodid ticks on cattle in Mazandaran province, Iran

A survey was carried out to investigate the prevalence of hard tick species (Acari: Ixodidae) on cattle in Mazandaran province, Iran. A total of 953 ticks were collected from 86 infested cattle during activating seasons of ticks during 2004-2005. Nine species were identified: Boophilus annulatus (51.3%), Rhipicephalus bursa (16.8%), Haemaphysalis punctata (6.3%), Ixodes ricinus (6.8%), Hyalomma marginatum (12.5%), Hyalomma anatolicum excavatum (5.2%), Hyalomma asiaticum (0.6%), Hyalomma detritum (0.2 %), and Dermacentor spp. (0.1%). The results show that Boophilus annulatus, Rhipicephalus bursa, and Hyalomma species are dominant tick species in the surveyed area.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

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

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae)

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10⁸ conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25 ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3–5 days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.     

Prevalence of Theileria in the tick Hyalomma detritum detritum in the Doukkala region,Morocco

Abstract. The overall prevalence of Theileria species, mainly, if not exclusively, T.annulata, in 901 Hyalomma detritum detritum collected from cattle in the Doukkala region of Morocco over a period of 2 years was 21.5%. The quantity of infection (number of sporoblasts per infected tick) followed the negative binomial distribution with between one and 250 sporoblasts per infected tick. Infected ticks were found in eight of fourteen areas examined whilst T.annulata was present in all fourteen. There were significant differences in both the prevalence and the quantity of infection between ticks collected from different farms, and between nymphs collected in the autumn from these farms, and moulted in the laboratory, and adults collected in the following summer. The prevalence, but not the quantity, of infection was higher in female than in male ticks. No correlations were established between infection of engorged nymphs and the breed, sex and Theileria piroplasm parasitaemia of the host animal. However, calves infected a greater proportion of nymphs than adult cattle and the heavier the infestation of nymphs on an animal, up to a plateau, the higher the prevalence of infection in those nymphs. There were no differences in infection between ticks moulted at 24^oC and 37^oC, after the engorged nymphs had been stored at 18^oC to simulate over-wintering.