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.     

Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation

Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.     

Cholesteryl esters on the body surfaces of the camel tick,Hyalomma dromedarii (Koch, 1844) and the brown dog tick,Rhipicephalus sanguineus (Latreille, 1806)

Cholesteryl esters were found to constitute a major component of the lipids coating the body cuticle of females of the camel tick, Hyalomma dromedarii and the brown dog tick, Rhipicephalus sanguineus. One or more cholesteryl esters, alone or in combination, have been shown to serve as the mounting sex pheromone of several species of ixodid ticks. Consequently, knowledge of these compounds is important for an understanding of the mating behavior of these ticks. Based on thin layer chromatography, cholesterol and cholesteryl esters were the most abundant neutral lipids found on the body surfaces of fed females of these two species. Analysis using HPLC demonstrated significant quantities of the following compounds, tentatively identified as cholesteryl esters (expressed in micrograms per female equivalent), in H. dromedarii: Cholesteryl acetate 18.2; cholesteryl laurate, 6.8; cholesteryl linoleate, 24.8; cholesteryl oleate, 12.9; cholesteryl palmitate, 0.3; and cholesteryl stearate 1.7.In contrast, the same method revealed only 3 cholesteryl esters in extracts of females of R. sanguineus: Cholesteryl acetate, 2.0; cholesteryl linoleate, 8.5; and cholesteryl oleate, 3.0. In both species, two unidentified peaks, with the spectral characteristics of cholesteryl esters, were also observed. Identification of the cholesteryl esters was confirmed: by (1) positive bioassay results with conspecific (H. dromedarii) males and heterospecific (Dermacentor variabilis) males; (2) similarity of ultraviolet spectra between identified sample peaks and authentic standards; and (3) demonstration of cholesterol and the corresponding free fatty acid following enzymatic digestion of each of the HPLC-separated fractions containing the different cholesteryl esters. Comparisons with the cholesteryl ester composition of the mounting sex pheromone of other metastriate Ixodidae are discussed. These findings, along with studies reported previously, suggest that differences in the mounting sex pheromones of ixodid ticks are an important factor in minimizing heterospecific matings in nature.Supported by Cooperative Agreement No. 263-0152-A-00-2207 with the U. S. Agency for International Development to the Department of Infectious Diseases. College of Veterinary Medicine, University of Florida, Gainesville, FL and a subcontract, ODURF No. 536521, to the Department of Biological Sciences, Old Dominion University, Norfolk, VA     

Biological aspects of <Emphasis Type="Italic">Amblyomma brasiliense</Emphasis> (Acari: Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20°C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.     

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.     

The impact of RNA interference of the subolesin and voraxin genes in male <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) on female engorgement and oviposition

Reducing or replacing the use of chemical pesticides for tick control is a desirable goal. The most promising approach would be to develop vaccines that protect hosts against tick infestation. Antigens suitable for the development of anti-tick vaccines will likely be those essential for vital physiological processes, and in particular those directly involved in feeding and reproduction. In this study genes from Amblyomma hebraeum Koch that encode for subolesin and voraxin were studied in male ticks by RNA interference (RNAi). Males (unfed or fed) were injected with dsRNA of (1) subolesin, (2) voraxin, (3) subolesin plus voraxin or (4) injection buffer, after which they were held off-host overnight and then allowed to feed on rabbits together with normal female A. hebraeum. Females that fed together with male ticks injected with subolesin or subolesin + voraxin dsRNA had a higher rate of mortality, weighed substantially less and produced a smaller egg mass than the controls. However, females feeding with males injected with voraxin dsRNA alone were not significantly different from the controls with respect to mortality, engorged weight or fecundity. However, as assessed by semi-quantitative RT-PCR, voraxin was not silenced in this study, the reasons for which remain unknown. The results of this study suggest that A. hebraeum subolesin is worthy of further testing as a candidate tick vaccine antigen.     

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.     

Characterization of protective antigens from the midgut of Amblyomma variegatum ticks

Separation of midgut membrane proteins from the tick,Ambylomma variegatum, using a non-ionic detergent (TritonX-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (M_r ∼27,000, 67,000, 86,000 and 95,000,)and 2 proteins (M_r ∼54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins M_r∼27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, there productive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

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.     

Effects of immunization of rabbits on establishment, survival, and reproductive biology of the tick Hyalomma anatolicum anatolicum.

Four antigenic preparations, viz. salivary gland antigen (SG Ag), whole tick extract antigen (WTE Ag) and 30,000-g supernatant fraction, and pellet of WTE Ag (TES Ag and TEP Ag, respectively), were made from partially fed adult female Hyalomma anatolicum anatolicum. Four groups of 5 rabbits each were immunized with the antigens, and a fifth group was kept as control. Following challenge with adult H. a. anatolicum, a significant decrease in engorgement weight and egg mass weight and an increase in engorgement period and preoviposition period were observed in WTE Ag-immunized rabbits. Similar results were observed with TES Ag and SG Ag, except that change in the engorgement period was insignificant. However, none of the tick parameter measurements showed significant changes with TEP Ag. None of the antigens produced significant changes in the percentage of engorgement or oviposition period of the challenged ticks. Thus, WTE Ag was the most effective in altering tick performances.     

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.     

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.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

Molecular characterization of ticks and tick-borne piroplasms from cattle and camel in Hofuf,eastern Saudi Arabia

The aims of the present study were to characterize ticks infesting the dromedary camel and cattle in Hofuf, Eastern Saudi Arabia and to determine the piroplasms that they may harbor. DNA was extracted from ticks, collected from camels and cattle, using commercial kits and subjected to polymerase chain reaction using specific primers for the amplification of ticks and piroplasms DNA. The cytochrome oxidase subunit I mitochondrial gene (COI) was used for characterization of ticks whereas partial 18S rRNA gene (18S rRNA) was used for piroplasms characterization. Ticks were genetically identified as Hyalomma dromedarii and Hyalomma anatolicum. Both cattle and camel in Hofuf, were found to be infested with both species. Both ticks identified as H. dromedarii and H. anatolicum from camels and cows showed 100% identity to COI sequences from the same species available in GenBank. Only Theileria annulata DNA was amplified from both H. anatolicum and H. dromedarii infesting cattle. None of the ticks collected from camels revealed DNA of piroplasms. T. annulata DNA was reported for the first time from Hofuf and the role of both H. anatolicum and H. dromedarii as potential vectors for this parasite in cattle in Saudi Arabia has been documented for the first time.     

The Effect of Male Ticks on the Feeding Performance of Immature Stages of Rhipicephalus Sanguineus and Amblyomma Americanum (Acari: Ixodidae)

A unique group of immunoglobulin-binding proteins (IGBPs), produced by ixodid male ticks during the latter half of their prolonged feeding period, improves the feeding performance of co-feeding females. As a follow-up to this observation, we investigated whether male tick feeding also affects the feeding of other developmental stages. Immature stages of Rhipicephalus sanguineus (Latreeille) and Amblyomma americanum (L.) were fed on rabbits in the presence or absence of conspecific males. The mean weight of larvae and nymphs of both species that fed around males and detached from the host on the first day of dropping was significantly higher than when the immature ticks fed on rabbits in the absence of males. However, larvae of both species and nymphs of R. sanguineus that fed slower and detached on the second day of dropping did not show significant differences in weight. A similar pattern was observed for A. americanum nymphs although, unlike R. sanguineus, the presence of males also influenced the feeding performance of the nymphs that fed slowly and detached on the second day of drop-off. The improved feeding performance demonstrated by immature ticks in the presence of males may be due to immunomodulatory saliva proteins, such as immunoglobulin-binding proteins (IGBPs) that are introduced into the co-feeding site. The results are considered in relation to the distribution of ixodid tick species on their natural hosts.     

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.     

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).     

Biology and life cycle of <Emphasis Type="Italic">Amblyomma incisum</Emphasis> (Acari: Ixodidae)

Amblyomma incisum Neumann is a major tick species in the Atlantic Forest of Brazil. Tapir is the main host for adult ticks and a high aggressiveness of nymphs to humans has been reported. In this work data on the biology and life cycle of this tick species is presented for the first time. It was shown that horse is a suitable host for A. incisum adults and rabbit for larvae and nymphs. It was also shown that A. incisum is a big tick species (mean engorged female weight of 1.96 g) with a long life cycle which lasts 262.3 days when maintained at 27°C and 85% RH. These laboratory conditions were, however, inappropriate and egg hatching rate (1.2%) was very low. Nevertheless egg hatching of ticks in a forest patch increased considerably (72.2%) indicating that this A. incisum population is highly dependent on a forest-like environment.     

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction

Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.