Acquired resistance to ixodid ticks induced by tick cement antigen

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa,Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera fromR. appendiculatus-resistant guinea pigs also recognized some salivarygland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum andA. gemma). Antibodies against the most strongly recognizedR. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified fromR. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight inR. pulchellus andR. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding ofR. appendiculatus ticks but slight enhanced feeding ofR. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.     

Mating,male Ixodes scapularis express several genes including those with sequence similarity to immunoglobulin-binding proteins and metalloproteases

We created a cDNA library from mating, male Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate genes common to feeding female and mating male I. scapularis ticks. A total of seven unique cDNAs were identified in this screen. One cDNA had sequence similarity to the IGBP-MC gene from Rhipicephalus appendiculatus, and another cDNA potentially encodes a protein with similarity to metalloproteases. RT-PCR, using RNA isolated from male and female I. scapularis ticks, confirmed that these genes are expressed in male, but not female ticks. The remaining five cDNAs did not match any sequences in the GenBank database. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> splenocyte proliferation responses of BALB/c mice to salivary gland extracts of three ixodid tick species (Acari: Ixodidae)

In vitro proliferation and cytokine production were investigated in BALB/c mice splenic cell cultures that were stimulated with concanavalin A (ConA) and lipopolysaccharide (LPS) and simultaneously exposed to salivary gland extracts (SGE) of unfed and partially fed adult ixodid ticks (Ixodes ricinus, Rhipicephalus appendiculatus, Amblyomma variegatum). Generally, tick SGE enhanced proliferation of unstimulated splenocytes and SGE of unfed ticks suppressed mitogen induced proliferation. Partially fed R. appendiculatus and A. variegatum suppressed ConA responses, while partially fed I. ricinus stimulated both ConA and LPS induced proliferation. A. variegatum and R. appendiculatus females slightly enhanced LPS responses 2 days after attachment but suppressed them at the end of the slow feeding phase. In 72 h ConA induced cell cultures, interferon gamma (IFN-γ) production was suppressed by SGE of all ticks, interleukin (IL)-10 production was enhanced by unfed I. ricinus and partially fed A. variegatum males and IL-5 production was enhanced by feeding R. appendiculatus females and A. variegatum males. The study revealed variability in the responsiveness of murine splenocytes to SGE of different ixodid tick species, whereby patterns of host immunomodulation within one tick species differed between sexes and changed during feeding.     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Further evidence for geographic differentiation in R. appendiculatus (Acari: Ixodidae) from Eastern and Southern provinces of Zambia

Studies in the biology, ecology and behaviour of R. appendiculatus in Zambia have shown considerable variation within and between populations often associated with their geographical origin. We studied variation in the mitochondrial COI (mtCOI) gene of adult R. appendiculatus ticks originating from the Eastern and Southern provinces of Zambia. Rhipicephalus appendiculatus ticks from the two provinces were placed into two groups on the mtCOI sequence data tree. One group comprised all haplotypes of specimens from the Eastern province plateau districts of Chipata and Petauke. The second group consisted of a single haplotype of specimens from the Southern province districts and Nyimba, an Eastern province district on the fringes of the valley. This variation provides additional evidence to the earlier observations in the 12S rDNA and ITS2 data for the geographic subdivision of R. appendiculatus from Southern province and Eastern province plateau. The geographic subdivision further corresponds with differences in body size and diapause between R. appendiculatus from these geographic areas. The possible implications of these findings on the epidemiology of East Coast fever (ECF) the disease for which R. appendiculatus is one of the vectors are discussed. Nucleotide sequence data reported in this paper are available in the GenBank^TM under the Accession Numbers DQ859259–DQ859266     

Artificial-membrane feeding of the ixodid tick,Rhipicephalus appendiculatus,to repletion

An artificial feeding device was constructed with a Baudruche, membrane bearing olfactory and tactile stimuli, to induce voluntary attachment and feeding of the brown ear tick,Rhipicephalus appendiculatus. Preparation of the membrane and experimental conditions used are described. Unfed adults ofR. appendiculatus, 2–3 weeks after moulting, were confined on the membrane, which was treated with cattle ear-wash and other stimuli. They attached and commenced sucking blood within 12–48 h. Fully engorged ticks started detaching from the membrane on the 6th day. After a 5–6-day incubation period, they started laying eggs which hatched into normal larvae.     

Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log₁₀ plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.     

The role ofRhipicephalus appendiculatus andR. evertsi evertsi males in inducing resistance in laboratory animals: preliminary studies

Guinea-pigs infested with male ticks of the speciesRhipicephalus appendiculatus, and rabbits infested withR. evertsi evertsi, acquired immunity to conspecific female ticks. The hosts were first infested with male ticks and thereafter were challenged with males and females of the same species. The mean weight of the engorged females ofR. appendiculatus fed on guinea pigs previously infested with male ticks was 509.0 (±41.4) mg compared with that of females fed on control guinea pigs (651.2±31.8 mg). Similar weight differences were observed forR.e. evertsi females which fed on rabbits previously infested three times with male ticks. The mean weight of the female ticks which fed on these rabbits was 520.1 (±29.8) mg compared with 640.7 (±30.2) mg ofR.e. evertsi females which fed on control hosts. The concentration of gammaglobulins in the sera of rabbits was monitored at various intervals after the first infestation. It was found, for the first time, that infestation of laboratory animals with male ticks conferred immunity, but to a lesser degree than infestation with both sexes. It was also shown that the level of gammaglobulins increased from 3.4±0.28 g l^–1 to 7.3±0.24 g l^–1 in sera of rabbits hosts as a result of the feeding activity of males, but to a lesser extent than in sera of rabbits on which both sexes had fed (10.8±2.4 g l^–1).     

Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.     

Cloning and Characterization of a Rice Field Eel vasa-Like Gene cDNA and Its Expression in Gonads During Natural Sex Transformation

The vasa (vas)-related gene encodes an RNA helicase protein member of the DEAD-box family and plays key roles in germ-cell formation in higher metazoans. Using degenerate PCR and RACE, we cloned the vasa gene of the rice field eel (Monopterus albus), which is homologous to the Drosophila vasa gene. We named it ma-vas (Monopterus albus vas). Ma-vas encodes a protein of 618 amino acids, which contains all of the known characteristics of vasa homologs. RT-PCR analysis revealed that ma-vas was exclusively expressed in the gonads of the female, intersex, and male. During gonadal natural sex reversal, ma-vas is expressed in oocytes at all stages of oogenesis, in degenerating oocytes of ovotestis, and in spermatogonia and spermatocytes at early stages. The vasa positive signal was also observed in the peripheral layer of late ovary. It was not found, however, in that layer of the testis. Alkaline phosphatase (AKP) staining on the ovary and testis also indicated that some cells had differentiational potential in the peripheral layer of the ovary, suggesting that spermatogonia might arise from cells with AKP and vasa-positive staining in the peripheral layer of the female gonad.     

Cattle ticks of the genera <Emphasis Type="Italic">Rhipicephalus</Emphasis> and <Emphasis Type="Italic">Amblyomma</Emphasis> of economic importance in Tanzania: distribution assessed with GIS based on an extensive field survey

In order to implement a robust integrated tick and tick-borne disease control programme in Tanzania, based on ecological and epidemiological knowledge of ticks and their associated diseases, a national tick and sero-surveillance study was carried out in all 21 regions of the mainland, as well as on Mafia Island, between 1998 and 2001. The current distributions of Rhipicephalus appendiculatus, R. pravus, Amblyomma variegatum, A. gemma, and A. lepidum are illustrated and discussed. Tick distribution maps were assessed using the Weights-of-Evidence method (WofE), and employing temperature, humidity, NDVI, rainfall, and land-cover predictive data. Ground-truthing was done to check correspondence both of the data employed in prediction with land-cover characteristics discerned in the field as well as of the surveyed and predicted tick distributions. Statistical methods were used to analyse associations of the tick species with their environment, cattle density, and other ticks. Except for R. appendiculatus, no appreciable changes were demonstrated in the predicted and observed tick distributions compared to the existing maps that originated in the 1950–1960s. Cattle density influenced the distribution of A. variegatum and, to a certain extent, of A. lepidum, but had no appreciable influence on the distribution of any of the other ticks discussed in this paper, neither did livestock movement. Distinct differences for environmental requirements where observed between different tick species within the same genus. The predictive maps of R. appendiculatus and R. pravus suggest their mutually exclusive distribution in Tanzania, and simultaneous statistical analysis showed R. pravus as a greater specialist. Of the three Amblyomma species, A. variegatum is the most catholic tick species in Tanzania, while both A. gemma and A. lepidum belong to the more specialized species. Despite dissimilar habitat preferences, all three Amblyomma spp. co-exist in central Tanzania, where very heterogeneous habitats may simultaneously satisfy the environmental requirements of all three species. The current study, conducted about 4 decades after the last major survey activities, has shown that changing livestock policies, unrestricted livestock movement and a continuous change in climatic/environmental conditions in Tanzania have brought about only limited changes in the distribution patterns of R. appendiculatus, R. pravus and the three Amblyomma species investigated. Whether this observation indicates a relative indifference of these ticks to environmental and/or climate changes allows room for speculation.     

Ixodid ticks feeding on humans in South Africa: with notes on preferred hosts,geographic distribution,seasonal occurrence and transmission of pathogens

This paper records the identities of 558 ixodid ticks feeding on 194 humans in South Africa. These ticks belonged to 20 species in six genera and those most frequently encountered were Amblyomma hebraeum, Haemaphysalis leachi, Hyalomma marginatum rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus gertrudae and Rhipicephalus simus. With the exception of the larvae of R. appendiculatus, the incidents of these ticks feeding on humans correlated well with their seasonal occurrences on preferred hosts. Ticks were also collected at monthly intervals, for 14 consecutive months, from the clothing of a game-guard providing protection for field-workers engaged in the collection, by means of flannel strips, of free-living ticks from the vegetation of four localities in the southern region of the Kruger National Park. In addition, with the exception of 3 months when the particular worker was absent, ticks that fed on one of the field-workers were collected over the same 14-month period. A total of 54,429 free-living ticks belonging to 14 species and six genera were collected from the vegetation at the four localities during this time and 3751 ticks belonging to 11 species and six genera from the clothing of the game-guard. The larvae of A. hebraeum and Boophilus decoloratus were the most numerous of the immature ticks, and H. leachi and R. simus of the adults on both the vegetation and the guard’s clothing. Ticks fed on the field-worker on six occasions and 14 were collected, all of which were A. hebraeum larvae. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Immunization of cattle with a semi-purified fraction of solubilized membrane-bound antigens extracted from the midgut of the tickRhipicephalus appendiculatus

Boran cattle were immunized with a semi-purified fraction (F₂) of solubilized membrane-bound antigens extracted from the midgut of the tickRhipicephalus appendiculatus. A subsequent challenge with all stages ofR. appendiculatus showed protection of the cattle to a significant degree. There was a decrease in engorgement weight of ticks, the number which fed above critical engorgement weight was considerably reduced, and about 38% of the larvae which fed to repletion did not moult. The major cause of death of adult female ticks was the increased grooming by cattle as a result of increased dermal irritation.     

Dynamics of Borrelia burgdorferi infection in nymphal Ixodes ricinus ticks during feeding

We report the sequential developmental events of Borrelia burgdorferi in histological sections of Ixodes ricinus nymphs before, during and after feeding. During the blood meal a decrease of approximately 50% in the number of infected ticks was recorded (eight out of 76, 11%) in comparison with the infection rate of unfed ticks (12 out of 56, 21%). Spirochetes were detected in tick salivary glands only after 2 days of attachment. From day 3 until drop-off, the number of infected ticks increased to 31% (15 out of 49). A quadratic logistic regression analysis showed that the variation in the number of infected ticks was significant, but only during the blood meal. The drop in the percentage of infected ticks during the first hours following attachment to the host is explained by our observation of spirochetes in the faeces of the ticks. The increase in the infection rate of replete ticks may be due to an uptake of spirochetes from the host skin at the feeding site.     

Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth

In the gypsy moth, Lymantria dispar, release of sperm bundles from the testis into the upper vas deferens (UVD) and subsequent transfer of sperm bundles into the seminal vesicles (SV) occurs in a daily rhythm. The UVD undergoes different types of contractions despite the fact that its musculature appears to receive no innervation. Patterns of the UVD movements were recorded throughout the daily sperm release and transfer cycle. In males kept in light-dark cycles, transfer of sperm from the UVD to the SV was accompanied by a characteristic pattern of UVD contractions of high frequency and amplitude. In males kept in constant light, which fail to transfer sperm, this contraction pattern was absent. It is concluded that the vas deferens muscles undergo daily changes in contraction pattern in phase with the light-dark cycle. The increased muscular contractions appear to be a causal factor in the gated sperm transfer from the UVD to the SV.Abbreviations LD light-dark - LL constant light - SV seminal vesicle - UVD upper vas deferens     

The effect of rainfall on tick challenge at Kyle Recreational Park,Zimbabwe

The effect of rainfall pattern on tick challenge was investigated at Kyle Recreational Park, Zimbabwe, from 1991 to 1992 using drag and removal plot methods to sample environmental tick density. The abundance of adults and nymphs of the brown ear-tick Rhipicephalus appendiculatus and larvae of the bont tick Amblyomma hebraeum was positively correlated with monthly rainfall, whereas no relationship with rainfall was revealed for larval R. appendiculatus, adults of the red-legged tick R. evertsi, or larvae of the blue tick Boophilus decoloratus. A comparison between 1991 (490 mm rainfall) and the drought year of 1992 (161 mm) revealed significant differences in the abundance of R. appendiculatus, A. hebraeum, and B. decoloratus. During the wet season, R. appendiculatus adults were 2–3 time more numerous in the environment during the higher rainfall year of 1991. A. hebraeum larval abundance exhibited a similar pattern to that of R. appendiculatus adults, but B. decoloratus larvae were more abundant in the drought year of 1992 during both the wet and dry seasons. Comparable tick abundance data collected at Kyle during the above-average rainfall years of 1975–1977 (mean = 1029 mm) were compared with tick challenge during the below-average rainfall years of 1991–1992 (mean = 326 mm). In grassland sand habitat and all habitats combined R. appendiculatus adults, nymphs, and larvae were much more abundant during the high rainfall years. In contrast, larvae of B. decoloratus were more numerous during the drier years. A. hebraeum larvae were also more abundant during the drier years. The strong positive correlation of adult R. appendiculatus abundance with rainfall and the coincidence of increased adult tick challenge with increased rainfall indicates that adult R. appendiculatus tick burden on hosts would be heaviest during the wet season and high rainfall years.