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.     

Factors influencing the toxicity of salivary gland extracts of Ixodes holocyclus Neumann

A bioassay using mice was developed to compare the toxin content of extracts of salivary glands of I. holocyclus at various stages of feeding. The quantity of toxin increased rapidly from the third day of feeding. Toxin production continued and increased in ticks removed after 3–5 days on mice and held at 30°C at 92% RH for 24 h, whereas no toxin was detected in the salivary glands of ticks fed for 3 days and treated similarly. It is suggested that major physiological changes occur in the salivary glands of I. holocyclus on the third day, which once stimulated continue independently of feeding. Toxin production in ticks was not suppressed by passively immunizing mice with anti-tick toxin but was in ticks fed upon hosts with a previous experience of tick feeding.Thus, to obtain salivary glands containing high concentrations of toxin for chemical analysis, it is necessary for salivary glands to develop 5 days from the initial attachment of the tick to a host with no previous experience of tick feeding. This can be achieved by passively immunizing mice against toxin, thus enabling the tick to feed 5 days without killing the mouse or by keeping the tick for 24 h at 30°C at 92% RH following the death of the mouse on the fourth day.     

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.     

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.     

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.     

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.     

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.     

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.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

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.     

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).     

Resistance in cattle to Haemaphysalis (Kaiseriana) longicornis

Samples of each instar of the 3-host tick, Haemaphysalis (Kaiseriana) longicornis were collected from grazing Bos taurus cattle to investigate their survival on hosts with previous exposure to the tick and on previously unexposed ‘naive’ hosts. The ticks were allocated to categories based on their size to observe at what stage of development mortality occurred. Analysis of the age structure of the ticks showed that small but variable proportions of larvae, nymphs and females matured on previously exposed hosts and larger proportions on the naive hosts. This suggests that cattle acquire and express different levels of resistance against each instar of the tick. The technique used has potential for estimating levels of resistance of hosts to 3-host ticks under field conditions.     

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.     

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.     

Immunological responses of the rabbit host to infestation by the brown ear-tickRhipicephalus appendiculatus (Acarina: Ixodidae)

Rabbits developed resistance toRhipicephalus appendiculatus instars following repetitive infestations. Rejection was accompanied by elevated IgM and IgG titres. Extracts of salivary gland, mouthpart cement and whole ticks induced a dose-related lymphocyte transformation.Skin-provocation tests with tick extracts elicited an immediate type-I hypersensitivity reaction with a delayed time-course which was influenced by antihistamines. Passive-transfer studies indicated that resistance was partially transferred with serum.A comparative histological study of the attachment sites of larvae on resistant and naive hosts demonstrated the role of eosinophils and macrophages during the initial phase of infestation. Possible rejection mechanisms are discussed in the light of these and other findings.     

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.     

Amblyomma americanum (L): Tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.     

Microarray analysis of gene expression changes in feeding female and male lone star ticks,Amblyomma americanum (L)

A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport‐associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less. © 2009 Wiley Periodicals, Inc.