Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

e carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.     

Feeding performance ofAmblyomma variegatum (Acarina: Ixodidae) fed repeatedly on rabbits

Rabbits infested with different stages ofAmblyomma variegatum Fabricius, 1794 became resistant to subsequent infestations by the same tick. Resistance was manifested by a reduction of 73.6% in the mean engorged weight of female ticks by the 3rd infestation. By the 5th infestation, only 70% of the nymphs engorged and their mean engorged weight was 57.7% of that of nymphs fed on tick-naive control rabbits. In the case of larvae, there was a 22.8% reduction in their mean engorged weight by the 6th infestation. Based on increases in body length while feeding on tick-naive rabbits, the nymphs could be divided into four feeding categories, i.e. Nl (unfed), N2, N3 and N4 (increasing states of engorgement). While 92–96% of ticks which dropped from susceptible rabbits fitted into feeding category N4, only 28% of those that dropped from tick-resistant rabbits fitted into that category. The remainder of the ticks fitted into categories N3 (60%) and N2 (12%). The majority of those ticks with reduced weight developed into adults which had an atypical scutal ornamentation pattern, in that an additional pair of lateral spots was regularly observed on the males. The females of such ticks deposited small numbers of eggs from which no larvae hatched.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

Haemaphysalis concinna (Acari: Ixodida): Persistent efficacy of doramectin in rabbits under laboratory conditions

In the present study, the persistent efficacy of doramectin (DOR) in rabbits infested by Haemaphysalis concinna was observed. DOR (200 µg/kg) was administered once to infested rabbits by subcutaneous injection. A total of 15 rabbits were allocated to three groups, which were observed for efficacy. On days 1–7, 8–14, 15–21 and 22–28 post-inoculation, the percent reduction of tick larvae in DOR treated rabbits was 100.0, 100.0, 86.7 and 61.7%, respectively; the percent reduction of nymphs was 100.0, 90.0, 75.0 and 65.0%, respectively; and the percent reduction of adults was 85.0, 65.0, 55.0 and 55.0%, respectively. In comparison, in untreated rabbits at the same time points (control group), the percent reduction of larvae was 5.0, 3.3, 5.0 and 5.0%, respectively, and there was no reduction of nymphs and adults in the untreated rabbits.     

Vector competence ofRhipicephalus sanguineus andDermacentor variabilis for American isolates ofBabesia gibsoni

To determine the identity of the tick vector of enzooticBabesia gibsoni in California, two common ixodid ticks were allowed to engorge uponB. gibsoni infected dogs. Sporozoites were observed in the salivary glands of prefed nymphalRhipicephalus sanguineus ticks that fed as larvae onB. gibsoni-infected dogs. A higher proportion (31%) of nymphal ticks that prefed on an uninfected dog for 48 hours contained sporozoites in their salivary glands than did ticks which had fed for 24 hours (13%). Sporozoites were not observed in the salivary glands of prefedR. sanguineus nymphs which were derived from the eggs of adult females that fed on an infected dog, in adults that were fed as nymph on an infected dog, or in the nymphal and adult uninfected controls.Dermacentor variabilis ticks appeared not to become infected. Although attempts to transmitB. gibsoni to susceptible, splenectomized dogs were unsuccessful,R. sanguineus would appear to be the most likely tick vector to maintain this piroplasm in California. This study was supported by grants from the Companion Animal Disease Laboratory, School of Veterinary Medicine, University of California, Davis.     

Observations on Antricola ticks: small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccessful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gut.     

Antigens of Amblyomma americanum ticks recognized by repeatedly infested sheep

Sera were taken from 3 sheep that had been infested 5 times with Amblyomma americanum and that exhibited manifestations of humoral depression to homologous antigens and anti-tick resistance. Proteins extracted from the intestine or salivary glands of unfed ticks or salivary glands from partially (3-day) fed ticks were analyzed by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Antigens recognized by the sheep in the same materials before and after each infestation were analyzed by western blots. The sheep responded to 44 antigens. Nine to 23 antigens were recognized by the preinfestation sera and the sera of 2 gnotobiotic sheep. Four antigens (34,000, 36,500, 38,000, and 115,000 MW) were revealed conspicuously by the serum of the first infestation but very weakly or not at all by the sera of the third infestation onward. Two antigens (35,500 and 29,000 MW) from fed salivary glands were revealed only by sera taken after manifestations of resistance had appeared. These antigens may be responsible for anti-tick protection. The 29,900 MW antigen was present also in salivary extracts of Boophilus microplus.     

Histological examination ofBorrelia burgdorferi infections in unfedIxodes ricinus nymphs

Borrelia burgdorferi, the agent of Lyme borreliosis, is vectored in Europe byIxodes ricinus. In unfed ticks, the spirochaete resides primarily in the midgut, but a low percentage (5.5%; 4/73) of naturally infected ticks may present a systemic infection involving organs such as the salivary glands and central ganglion (Burgdorferet al., 1983). In this study, we examined 79 unfed nymphs collected in two sites in Switzerland (Neuchatel and Aarberg), and 35 unfed nymphs which were fed as uninfected larvae on 3 infectedApodemus sylvaticus mice. Dieterle silver staining was used to visualize the spirochaetes in the ticks. Of the unfed field-collected nymphs, 21/79 (27%) were infected of which 2/21 (10%) had systemic infections. Taking account of the site of collection, we observed that 0/12 ticks from Neuchatel were systemically infected whereas 2/9 (22%) from Aarberg had a disseminated infection. Out of the 35 unfed nymphs examined after an infectious blood meal on rodents, 14 (40%) were infected and 2 (14%) had a disseminated infection. A total of 4/35 (11%) unfed infected nymphs presented a systemic infection which represents a higher percentage than previously described. The presence of spirochaetes in salivary glands of systemically infected ticks before the initiation of feeding may reduce the time delay generally recorded for the tick-borne transmission ofB. burgdorferi.     

Secretory process of salivary glands of female Amblyomma cajennense (Acari: Ixodidae) ticks fed on resistant rabbits

Ticks have great economic and health importance since infested animals have reduced milk and meat production, and, besides that, they are expensive ectoparasites to control. While feeding, ticks can transmit to their hosts a large amount of pathogens, including Rickettsia rickettsii responsible for the ??spotted fever?? or ??fever of the mountains.?? It is known that animals infested with ticks or artificially immunized with their salivary gland extracts develop resistance, which is related to a decrease in engorged female weight, in egg-laying by adults, in egg viability and, in some cases, in the capacity of pathogens transmission. The present study aimed to examine morpho-histochemically the female salivary glands of semi and engorged Amblyomma cajennense fed on resistant rabbits. The results revealed that acinus I had no changes when compared to that of females fed on naive rabbits. The c cells of acinus II showed signs of early degeneration, which may result in feeding efficiency decrease. In acinus III d cells, activity time was longer. Such occurrence was associated with the time of female fixation, which increased in females fed on resistant hosts.     

Changes in immunity to Ixodes ricinus by rabbits infested at different levels

Abstract. Two groups of rabbits were infested twice with different numbers of Ixodes ricinus adults: one group (high infestation) with twenty-five females and twenty-five males and the other group (low infestation) with five pairs. A third infestation was performed in both groups with fifteen adult pairs. Tick biology was monitored for resistance effects. At the second infestation, the feeding and the egg production were more perturbed in ticks fed on high infestation rabbits. The embryogenesis was only affected in ticks from high infestation rabbits. At the third infestation, resistance was increased only in low infestation rabbits, which became more resistant than high infestation rabbits. The blastogenic response of peripheral blood lymphocytes and antibody production against ticks were assessed. A salivary gland extract and an integumental antigen from Lricinus adult females were able to initiate lymphocyte proliferation. The response was significantly higher in high infestation rabbits, especially at the end of the second infestation, and higher in low infestation rabbits during the third infestation. Non-specific proliferation with concanavalin A was temporarily decreased in both rabbits groups during the first and the second infestations. Specific antibody response to salivary and integument antigens were always the highest in high infestation rabbits. The involvement of tick-induced immunosuppression is discussed.     

Prevalence and abundance of Ixodes pacificus immatures (Acari: Ixodidae) infesting western fence lizards (Sceloporus occidentalis) in northern California: temporal trends and environmental correlates.

The prevalence and abundance of immature Ixodes pacificus ticks on western fence lizards (Sceloporus occidentalis) were examined in relation to time of year, host attributes (i.e., age, gender, and presence or absence of blood parasites), and 5 environmental characteristics, including topographic exposure and ground cover substrate, over a 2-year period in northern California. Lizards were infested with subadult ticks from early March until late July or early August, with peak median numbers of larvae and nymphs recorded in late April and early May of both years. Peak larval and nymphal abundances differed between years. The overall ratio of larvae to nymphs on adult male lizards was low, ranging from 0.80 in 1999 to 2.41 in 2000. Such intensive feeding of nymphs versus larvae on these lizards, which are reservoir-incompetent for Borrelia burgdorferi spirochetes, may explain previous observations of decreasing spirochetal infection prevalence from the nymphal to adult stage in northwestern California. Adult male lizards were more likely to be infested with nymphs and harbored greater abundances of larvae and nymphs than adult females. Lizards uninfected with blood parasites had more nymphs than infected lizards. The measured environmental characteristics could explain only a small percentage of the total variation observed in larval prevalence (22%) and in larval and nymphal abundance (12 and 3%, respectively).     

Tick infestation of dogs in Thessaloniki,northern Greece

Ticks were collected from 249 dogs, admitted over a one-year period (1996-1997) to a Veterinary Teaching Hospital and four private veterinary practices located in the county of Thessaloniki, Greece. A total of 2812 ticks (Acari: Ixodidae) were collected, of which 2511 (89.3%) were Rhipicephalus sanguineus (Latreille) adults (1070 males, 1441 females), 156 (5.5%) R. turanicus Pomerantsev adults (35 males, 121 females), 100 (3.6%) Rhipicephalus spp. nymphs and 45 (1.6%) Rhipicephalus spp. larvae. Dogs living outdoors, in rural areas and in close proximity to farm animals, were infested with higher numbers of ticks than dogs living indoors. Tick attachment sites were recorded on 237 dogs, harbouring a total of 1711 ticks. The most heavily infested sites were, in decreasing frequency, the ear pinnae, neck, interdigital skin folds, trunk, head, ventrum, extremities and the tail. Cutaneous lesions at the attachment sites were noticed in 129 dogs and then mainly in the more severely infested animals.     

The effect of feeding immature Karoo paralysis ticksIxodes rubicundus (Acari: Ixodidae) on the metabolic rate of the rock elephant shrew

The effect of feeding immature Karoo paralysis ticks (Ixodes rubicundus) on the resting metabolic rate (RMR) of their principal natural host, the rock elephant shrew (Elephantulus myurus), was investigated under laboratory conditions. The elephant shrews were artificially infested with numbers of ticks simulating natural burdens. The RMR of the elephant shrews was quantified by measuring the oxygen consumption in an open through-flow system. The RMR of hosts infested only with larvae did not differ from that of the control group. Those infested with nymphs, or nymphs and larvae, had significantly lower RMR's compared to the control animals. There were no signs of paralysis in any of the infested hosts.     

Experimental evaluation of birds as disseminators of the cosmopolitan tick Rhipicephalus sanguineus (Acari: Ixodidae)

Rhipicephalus sanguineus is believed to be the most widespread tick species of the world and its dissemination seems to rely on the diffusion of its main host, the dog. Empirical observations indicate that several bird species in urban areas regularly steal dog food. Such circumstances create a chance for R. sanguineus ticks to climb on birds and carry ticks to another site. In this work we evaluated experimentally the likelihood of birds (chicks) to either feed and/or carry R. sanguineus ticks from an infested site to another and to infest a host (rabbit) in the new location. Chicks were not suitable hosts for R. sanguineus ticks. Not a single adult tick engorged on chicks, yield as well as weight of engorged larvae and nymphs were very low and feeding period of these ticks was very long. However, a few larvae and, chiefly, nymphs were delivered to a new location either mechanically or after attachment and engorging total or partially on chicks. A few of these ticks fed successfully on rabbits. Further evidence on the capacity of birds to introduce R. sanguineus into non-infested dog settings should be provided by systematic examination of birds from urban areas, close to tick infested households.     

Developmental changes in salivary glands of nymphs and adults of the spinose ear tick Otobius megnini

Otobius megnini has an autogenous 1 host life cycle, where larvae and nymphs stay attached inside the ear canal for long periods, but the adult tick is free living and can lay several egg batches without feeding. In order to obtain information about anatomical structures involved in this particular life cycle, nymphs and adults of O. megnini were dissected and salivary gland images were obtained in situ with the use of scanning electronic microscopy. Measurements of salivary alveoli were obtained with the use of ImageJ 1.40 g software. In the nymphs, the Type I alveoli are relatively small (mean diameter: 19.6 μm) compared with those of the adults (mean: 43.4 μm) and other soft ticks in the literature. Type II alveoli in nymphs are similar (mean: 82.6 μm) to previously described alveoli in adult soft ticks. In contrast, the adults of O. megnini Type II alveoli are smaller (mean: 36.8 μm) and have a wrinkled surface. These findings provide more evidence that Type I alveoli take part in absorption of moisture during the free-living tick stages.     

The feeding behaviour of larvae, nymphs and adults of Rhipicephalus appendiculatus.

The pre-feeding and feeding periods of larvae, nymphs and adults of Rhipicephalus appendiculatus on rabbits (Oryctolagus cuniculus) were investigated. Larvae and nymphs required at least 8-9 days after hatching and moulting respectively before they could attach and start feeding, while adults required at least 6-9 days. But longer periods of starvation improved the proportion of ticks that successfully fed. After attachment, there was always an initial period of slow feeding, which was followed by a phase of very rapid feeding before the engorged ticks detached from the host. Larvae detached 4-5 days after attachment, nymphs detached after 5-6 days and adults detached 7-9 days after attachment.     

C3 levels in the sera of rabbits infested and reinfested withIxodes ricinus L. and in midguts of fed ticks

Serum C3 levels of rabbits infested 3 times withIxodes ricinus L. females and C3 in midgut extracts of fed ticks have been measured by a single radial immunodiffusion test.From the first tick attachment, the mean serum C3 level of hosts increased. A peak of C3 occurred 6 days after the beginning of each infestation and was highest during the third infestation (about 8 times more pronounced than the level measured before the first). After the end of all infestations of the rabbits, the serum C3 level decreased and approached normal levels.Modifications of serum C3 levels during reinfestations influenced C3 contents in the blood meal of fed ticks. Midguts of ticks fed during the third infestation contained more C3 than midguts of ticks of the same engorged weights fed during the first or second infestations.     

Risk factors in habitats of the tick Ixodes ricinus influencing human exposure to Ehrlichia phagocytophila bacteria

Ixodes ricinus L. (Acari: Ixodida) were sampled during 1996-99 in southern Scotland, on vegetation using cloth drags, on humans by removal from clothing and on roe deer (Capreolus capreolus L.) by searching legs of culled deer. Developmental microclimate was recorded by automatic recorders and questing microclimate by portable instruments during tick collections. Ticks and deer were examined for infection with Ehrlichia phagocytophila bacteria (Rickettsiales) using microscopy and polymerase chain reaction. This pathogen causes tick-borne fever of sheep in Europe and human granulocytic ehrlichiosis in North America, but in Europe human clinical ehrlichiosis due to E. phagocytophila has not been recorded despite serological evidence of exposure. Among three types of habitat, coniferous woodland was most infested with questing ticks (560 ticks/km of drag; mean numbers collected on long trousers: 24.3 larvae, 13.5 nymphs and 0.8 adult ticks/km walked), deciduous woodland had slightly lower infestation (426 ticks/km drag) and upland sheep pasture had much lower infestation (220 ticks/km drag). Of the three main vegetation types, bracken was least infested (360 ticks/km drag), ericas most (430 ticks/km drag) and grassland had intermediate infestation density (413 ticks/km drag). Questing and developmental microclimates were poor predictors of exposure within these habitats, except lower infestation of pastures was attributed to greater illumination there. Collectors who walked a total of 300 km through all habitats (taking 360 h in all seasons), wearing cotton trousers hanging outside rubber boots, were bitten by only four nymphs and 11 larvae of I. ricinus (but no adult ticks). There was a negative correlation between densities of deer and ticks collected, although presence of deer remains a major indicator of exposure. The proportion of infected ticks was fairly uniform at four sites studied. Overall prevalence of E. phagocytophila in I. ricinus was 3.3% in nymphs (40/1203) but only approximately 1.5% in adults of both sexes (although males do not bite). It was estimated that nymphs of I. ricinus gave 4.4% probability of one infected bite/person/year (for occupational exposure during this research) due to presence in all seasons and habitats, their human biting rate of 0.011 nymphs/h or 0.013 nymphs/km and widespread infection with E. phagocytophila. The frequency distribution of intensity of infection in ticks was approximately normal (mean 98 morulae/nymph infected), thus there is a high risk of receiving a high dose from any one infected tick bite.     

Life cycle and host specificity of Amblyomma triste (Acari: Ixodidae) under laboratory conditions

We report biological data of two generations of Amblyomma triste in laboratory and compared the suitability of different host species. Infestations by larval and nymphal stages were performed on guinea pigs (Cavia porcellus), chickens (Gallus gallus), rats (Rattus norvegicus), rabbits (Oryctolagus cuniculus), wild mice (Calomys callosus), dogs (Canis familiaris) and capybaras (Hydrochaeris hydrochaeris). Infestations by adult ticks were performed on dogs, capybaras and rabbits. Tick developmental periods were observed in an incubator at 27 degrees C and RH 90%. Guinea pigs were the most suitable hosts for larvae and nymphs, followed by chickens. The remaining host species were less suitable for immature ticks as fewer engorged ticks were recovered from them. Mean larval feeding periods varied from 3.8 to 4.7 d between different host species. Mean larval premolt periods ranged from 8.9 to 10.4 d. Nymphal mean feeding periods varied from 4.2 to 6.2 d for ticks fed on different host species. Premolt period of male nymphs (mean: 15.4 d) was significantly longer than that of female nymphs (14.7 d). Female nymphs were significantly heavier than male nymphs. The overall sex ratio of the adult ticks emerged from nymphs was 0.9:1 (M:F). Capybaras were the most suitable host for the tick adult stage as significantly more engorged females were recovered from them and these females were significantly heavier than those recovered from dogs or rabbits. The life cycle of A. triste in laboratory could be completed in an average period of 155 d. The potential role of guinea pigs, birds and capybaras, as hosts for A. triste in nature, is discussed.     

A cement protein of the tick Rhipicephalus appendiculatus,located in the secretory e cell granules of the type III salivary gland acini,induces strong antibody responses in cattle

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.