Resistance in cattle againstRhipicephalus appendiculatus with an assessment of cross-resistance toR. pulchellus (Acari: Ixodidae)

A comparison of the yield of engorged nymphs ofRhipicephalus appendiculatus andR. pulchellus was carried out between cattle highly resistant toR. appendiculatus but susceptible toR. pulchellus, and cattle and rabbits susceptible to both species.Resistant cattle yielded fewer and smallerR. appendiculatus nymphs than either susceptible cattle or rabbits. More nymphs of both tick species dropped from susceptible cattle but no differences were detected in the mean weight of engorgedR. pulchellus nymphs fed onR. appendiculatus-resistant and susceptible cattle, although resistant animals yielded more nymphs ofR. pulchellus than ofR. appendiculatus.An acute inflammatory reaction, accompanied by intense pruritus and grooming, was observed in the ears of the resistant cattle infested withR. appendiculatus, but no reaction was detected in the opposite ears whereR. pulchellus was feeding.The low cross-resistance between these two closely related tick species suggests that different antigens may be involved in the development of resistance in cattle to different tick species.     

Monitoring of naturally acquired and artificially induced immunity toAmblyomma variegatum andRhipicephalus appendiculatus ticks under field and laboratory conditions

The ability of rabbits, goats and cattle to acquire immunity to the ixodid ticksAmblyomma variegatum andRhipicephalus appendiculatus was studied under laboratory and field conditions. Rabbits were successfully immunized with crude salivary gland extract (SGE) and midgut extract (ME) obtained from flat or partly fed femaleR. appendiculatus ticks. The lowest numbers of larvae were produced by females fed on rabbits immunized with unfed midgut extract. Similar reductions in larval production could be induced after three infestations of rabbits with adultR. appendiculatus. Also, successive feedings of nymphs ofR. appendiculatus on rabbits resulted in significantly reduced engorgement weights. Skin testing with SGE induced delayed-type hypersensitivity reactions, which could be correlated with immunity toR. appendiculatus in rabbits. Moreover, circulating antibodies were detected in rabbits with an ELISA using SGE ofR. appendiculatus.Immunity toA. variegatum nymphs could be induced in rabbits by repeated infestations, but this failed in goats. Immunization of goats with midgut extract from adultA. variegatum did not protect against subsequent nymphal challenge, but strong skin reactions were noticed when adults ticks fed on immunized goats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of SGE and ME fromA. variegatum revealed the presence of 48 protein bands in SGE and 29 bands in midgut extract. Western blotting employing serum from a rabbit immune toR. appendiculatus recognized a number of bands in SGE fromR. appendiculatus, but also in SGE ofA. variegatum.Immunity acquired by cattle to ixodid tick infestations under field conditions was monitored by skin testing with SGE and western blot analysis. In general, cattle with the lowest tick numbers manifested the strongest delayed-type hypersensitivity responses. Finally, western blot analysis employing sera from tick-infested and tick-naive cattle could not be related to actual immune status.     

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).     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

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.     

<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.     

Sequential Histopathology at the Rhipicephalus Sanguineus Tick Feeding Site on Dogs and Guinea Pigs

The tick Rhipicephalus sanguineus is a very common parasite of dogs worldwide. Dogs seem unable to acquire resistance against this tick species, whereas guinea pigs demonstrate a very strong resistance following primary infestation. We studied the inflammatory reaction at the R. sanguineus tick feeding site on dogs and guinea pigs during primary and tertiary infestations at different time intervals after attachment. Biopsies were collected after 4, 24, 48 and 96 hours. Changes that were found in all experimental groups included a cone of cement around the mouthparts of the tick, epidermal hyperplasia, edema and inflammatory cell infiltration in the dermis directly underneath the tick attachment site. Dogs reacted to ticks mainly with neutrophils, particularly after repeated exposure. Mast cells and mononuclear leukocytes were also present. Guinea pigs reacted to R. sanguineus mainly with mononuclear cells, eosinophils and basophils. These cells were particularly numerous after repeated exposure to R. sanguineus. Our results suggest that basophils and eosinophils are involved in resistance of guinea pigs to R. sanguineus and that neutrophils in dogs have little effect against this tick species.     

Immunology of interactions between ticks and laboratory animals

There is good reason to believe that the resistance to ixodid ticks acquired by guinea pigs, rabbits and mice is immunologically mediated. One proposed mechanism for this resistance, which may well be common to all these laboratory animals, involves cutaneous hypersensitivity reactions. Basophils accumulate at tick attachment sites in the skin of resistant animals and degranulate in response to tick salivary antigens, releasing histamine and other mediators. The mediators may directly cause ticks to cease salivating and feeding and then to detach, or they may induce reflex grooming reactions by the host, leading to the removal of ticks from the itching skin.There are gaps in the evidence supporting this hypothesis, and it is likely that other modes of tick resistance remain to be described. However, it should be recognized that, although there have been a few details added to the story in the last fifty years, William Trager's original classic observations and conclusions still stand as the core of the current dogma.     

Ecology and phenology of cattle ticks in Zambia: Development and survival of free-living stages

A study on development and survival of free-living stages of three important cattle ticks in Zambia,Amblyomma variegatum Fabricus,Boophilus decoloratus Koch, andRhipicephalus appendiculatus Neumann, was carried out to complement studies on seasonal dynamics of parasitic stages.Different instars of engorged ticks were exposed under quasi-natural conditions according to the season in which they occur naturally. Generally, development rates of all stages of the three species were related to temperature, whilst the duration of survival was influenced mainly by rainfall and consequent relative humidity.Observations on the effect of age and climate on the behaviour of ticks on pastures were also made. BothA. variegatum andR. appendiculatus completed only one generation per year. InA. variegatum, engorged females detaching early in the adult season (August to October) undergo morphogenetic diapause. Adults ofR. appendiculatus emerging between August and October enter a period of behavioural diapause before becoming active in December. These mechanisms effectively synchronize the life-cycles of these two univoltine species. The one-host tick,B. decoloratus, is able to complete three to five generations each year with no indication of seasonal synchronization.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Immunisation of rabbits against the brown ear tick,Rhipicephalus appendiculatus using tick haemolymph

Tick naive rabbits were immunised with haemolymph components from partially fed Rhipicephalus appendiculatus adult ticks and subsequently challenged with all the developmental instars of the tick. The results obtained showed that the rabbits were rendered resistant to all the instars of the tick. However, the resistance was more pronounced in adult ticks than in the immature stages. The resistance was manifested as a reduction in the number of ticks that fed successfully to engorgement, reduced engorgement weights and reduced fecundities. Re challenging the resistant experimental rabbits with all the developmental instar stages of Rhipicephalus appendiculatus showed that resistance was maintained in subsequent infestations.     

Tick antigens recognized by serum from a guinea pig resistant to infestation with the tick Rhipicephalus appendiculatus

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the cattle disease East Coast Fever, was induced in a guinea pig by repeated tick infestation. This resistance is expressed as the ability of the host to interfere with tick feeding. Resistance to ixodid tick feeding is an acquired response mediated by host antibody. We report the use of antibodies from a resistant host animal, in immunoblotting, to characterize the tick antigens recognized. The major tick antigens identified had molecular weights of 120,000, 94,000, 88,000, 77,000, 58,000, 46,000, 35,000, 31,000, 28,000, 25,000, 20,000 and 16,000. Most of these antigens were found in tick salivary glands. The presence and concentration of many tick salivary antigens appeared to vary with relation to the tick feeding cycle. Many of the antigens present in salivary glands were also detected in tick cement. Tick gut extract, although a poorer source of antigens, contained more of the 31,000 dalton antigen than salivary glands. Larval and nymphal tick extract lacked many of the antigens present in adult ticks. The data suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens.     

Ecology and phenology of ticks in Zambia: Seasonal dynamics on cattle

A study of the seasonality and infestation rates of ticks was carried out in 11 cattle herds in different ecological habitats in Zambia between 1980 and 1982. Wherever possible supplementary data were obtained from opportunistic collections from cattle and other hosts.Analysis of over 1000 tick collections from cattle indicated that infestation rates of the most important species,Amblyomma variegatum andRhipicephalus appendiculatus vary in different ecological habitats: (i) In Western Province, infestations are much lower than elsewhere; (ii) in Central and Southern Provinces, moderate to high infestations occur; and (iii) in Eastern Province,R. appendiculatus numbers are generally low andA. variegatum numbers are moderate.These two species, however, have similar life cycles throughout their range with one generation per year. Larvae occur mainly from March to May, nymphae from May to September, and adults ofA. variegatum from October to December and ofR. appendiculatus from December to April. Boophilus decoloratus appears to have two to four generations per year but is uncommon during the rainy season. In some areas in central ZambiaRhipicephalus compositus adults are seasonally common in September–October whereasRhipicephalus evertsi is more or less ubiquitous. Low to moderate infestations ofHyalomma truncatum andHyalomma rufipes occur in most areas.At least 14 other less common or rare species ofRhipicephalus, Amblyomma, Haemaphysalis andIxodes were taken infrequently from cattle. These and other host-specific species were also collected from dogs, sheep, various wildlife hosts and the environment.Infestation rates, seasonality and host-relationship of tick species are discussed in relation to their ecology. Relevant biosystematic and disease relationships are reviewed briefly. The baseline data derived from this study are adequate for integrated analysis with those from other ecological and economic investigations to formulate tick control strategies.     

Rhipicephalus appendiculatus: Larval feeding sites in guinea pigs actively sensitized and receiving immune serum

Histological analyses of larval Rhipicephalus appendiculatus feeding sites in naive and actively sensitized guinea pigs were made at 6, 24, 48, 72, and 96 hr post-tick attachment. As primary feedings progressed the cavity at the entrance of the ticks mouthparts into the uppermost dermis, and the surrounding cellular infiltrate (lesion) both increased. Early (6 hr) lesions were dominated by eosinophils (46% of the infiltrate), neutrophils predominated at 24 (55%) and 48 hr (68%), eosinophils again predominated at 72 hr (44%), and finally basophils were dominant at 96 hr (78%). At sites of secondary feedings in animals expressing acquired resistance, lesion size increased as tick feeding progressed and at each observation time was at least twice that observed in primary feedings. Dermal cavities at the site of entrance of the ticks mouthparts were occasional in occurrence and were reduced in size indicating altered tick feeding. Basophils were dominant at all observation times ranging from 61 to 91% of the infiltrate. The second cell type of significance was the eosinophil, ranging in abundance from 7 to 21%. Recipients of immune serum had a smaller cellular infiltrate around feeding ticks, but basophils were also dominant. Basophils appear to be the principal host cell involved in acquired resistance to tick feeding as indicated by the profound cutaneous basophil reaction that characterized the immune response to larval ticks both in actively and passively sensitized hosts. The finding of significant eosinophil accumulations at tick feeding sites of both hosts indicates that these cells may also contribute to acquired resistance.     

The effects of nutritional status of rabbits and sheep on their resistance to the ticksRhipicephalus evertsi evertsi andR. appendiculatus

Rabbits and sheep were exposed to low-and high-protein diets and subsequently infested three times with adults ofRhipicephalus appendiculatus andRhipicephalus evertsi evertsi. The mean weight ofR.e. evertsi females which dropped from rabbits maintained on a high-protein diet decreased from 515.0±24.9 mg (naive) to 381.5±25.0 (second infestation) to 340.3±23.3 mg (third infestation) while the weight of ticks fed on animals which were exposed to a low-protein diet did not change significantly (2.7%). The mean weight of engorged females ofR. appendiculatus which completed their blood meal on rabbits (high protein) decreased from 520.9±31.8 (naive) to 369.3±39 mg (3rd infestation), a significant decrease of 29.1% compared to a 12.3% decrease in weight between the 1 st and 3rd infestation of females fed on animals on a low-protein diet.Rhipicephalus e. evertsi fed on sheep exhibited the same phenomenon. The mean decrease in weight of 4rd-infestation ticks which dropped from sheep fed lucerne was 26.2% compared to 16.6% for ticks from sheep which were fed on grass.Hosts maintained on a low-protein diet failed to acquire resistance to ticks, lost weight and developed anaemia while those on a high-protein diet developed resistance, maintained weight and did not develop anaemia.The nutritional stress of the hosts and its application in South Africa are discussed.     

Experimental infection of the rabbit tick,<Emphasis Type="Italic"> Haemaphysalis leporispalustris</Emphasis>, with the bacterium<Emphasis Type="Italic"> Rickettsia rickettsii</Emphasis>, and comparative biology of infected and uninfected tick lineages

The present study consisted of two experiments that evaluated experimental infections of Haemaphysalis leporispalustris ticks by a Brazilian strain of Rickettsia rickettsii, and their effect on tick biology. In experiment I, ticks were exposed to R. rickettsii during the larval, nymphal or adult stages by feeding on rabbits (Oryctolagus cuniculus) needle-inoculated with R. rickettsii, and thereafter reared on uninfected rabbits for the entire next tick generation. Regardless of the tick stage that acquired the infection, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 1.3 to 41.7%), and were able to transmit R. rickettsii to uninfected rabbits, as demonstrated by rabbit seroconversion, guinea pig inoculation with rabbit blood, and PCR on rabbit blood. In Experiment II, ticks were exposed to R. rickettsii during the larval stage by feeding on rabbits co-infested with R. rickettsii-infected adult ticks, and thereafter reared on uninfected rabbits until the next generation of larvae. Again, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 3.0 to 40.0%), and were able to transmit R. rickettsii to uninfected rabbits. Thus, it was demonstrated that larvae, nymphs, and adults of H. leporispalustris were able to acquire and maintain the R. rickettsii infection by transstadial and transovarial transmissions within the tick population, with active transmission of the bacterium to susceptible rabbits by all parasitic stages. Analyses of biological parameters of uninfected and R. rickettsii-infected tick lineages were performed in order to evaluate possible deleterious effects of R. rickettsii to the infected tick lineages. Surprisingly, all but one of the four R. rickettsii-experimental groups of the present study showed overall better biological performance than their sibling uninfected control ticks. Results of the present study showed that H. leporispalustris could support infection by a high virulent strain of R. rickettsii for at least two generations, in which infected tick lineages tended to have better performance than uninfected ticks. Our results support a possible role of H. leporispalustris in the enzootic maintenance of R. rickettsii in Latin America, as previously suggested by earlier works.     

Histopathological changes in the skin of small mammals in the area of feeding of Ixodes trianguliceps, I. Persulcatus i I. Ricinus (Ixodidae) ticks

Histopathological changes in the places of attachment and feeding of the Ixodes trianguliceps, I. persulcatus, and I. ricinus on natural hosts are similar. They pass through the stages of skin inflammation: perforation, exudation and proliferation. The mouthparts of tick contact with the host tissues. The fibrin deposit and callogen capsule surround the mouthparts. These species of ticks do not form the cement. Formation of the feeding cavity is caused by the salivation of ticks and neutrophil infiltration into surrounding tissues. Unnatural hosts (mice, rabbits) do not form the callogen capsule in the places of feeding, the callogen fibres are disposed irregular. Mast cells accumulate in the tick attachment site in the skin of natural host and degranulate in response to the tick salivary antigens releasing the histamine. Mediators involve cutaneous hypersensitivity reactions.     

In-vivo effects of ivermection onRhipicephalus appendiculatus: the influence of tick feeding patterns and drug pharmacokinetics

Sublethal effects seen amongstRhipicephalus appendiculatus feeding on ivermectin-treated rabbits were diverse and dependent both on drug dose, pharmacokinetics and tick feeding patterns: changes in drug formulation, the time of infestation relative to treatment, and the tick instar used, profoundly influenced acaricidal activity. Death was a sequel to paralysis only if tick feeding was interrupted for sufficient time to produce irreversible dehydration. Concurrent pharmacokinetic investigations revealed that, for the larvae ofR. appendiculatus, the mean critical lethal dose of ivermectin imbibed over a 5-day engorgement period was 3500 g/kg. This quantity of ivermectin was achieved in the blood-meals of larvae feeding on rabbits treated subcutaneously with a single dose of Ivomec injection (MSD)^*800 g/kg, provided infestation took place within 24 h of treatment. At lower drug doses, or if larval infestations were delayed for>24 h post-treatment, the quantity of circulating ivermectin (and thus imbibed by the tick larvae) fell below 3500 g/kg and an increasing percentage of larvae successfully engorged and detached. More than 90% of such larvae moulted to the nymphal stage. Nymphae and larvae exhibited similar susceptibility to ivermectin on treated rabbits which could be explained by similar feeding patterns. However, adult female and male ticks were markedly less susceptible and interpretation of ivermectin-induced effects was more complex.     

Artificial immunization of rabbits with Hyalomma dromedarii tick-derived midgut antigen

New Zealand white rabbits were immunized with partly fed Hyalomma dromedarii tick-derived midgut concealed antigens (supernate and pellet fractions) and Freund's complete adjuvant (FCA). The rabbits received three inoculations subcutaneously on days 0, 14 day 21 at a dose rate of 1 mg antigen per animal. The effects of the immunity induced was determined by infesting the rabbits with adult H. dromedarii ticks. In immunized rabbits a significant reduction in tick yield, engorgement weight, oviposition period, egg mass weight and percentage of egg hatchability was found. The gut supernatant antigen fraction induced the best protection in terms of reduced feeding and reproductive performance of the ticks.     

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.