Lack of Immunological Cross-reactivity of 36-kDa Secretory Salivary Gland Antigen of Hyalomma anatolicum anatolicum with Hyalomma dromedarii and Boophilus microplus Ticks

During feeding of ticks of the species Hyalomma anatolicum anatolicum, most of the proteins in salivary gland extracts (SGEs) remained unchanged from the unfed to the fully fed state (from day 1 to day 7 of the experiment), as revealed by SDS-PAGE. However, a 45-kDa protein band disappeared and 26-, 32- and 33-kDa bands appeared when feeding commenced. Some of the protein bands changed their intensity. When probed with anti-H. anatolicum anatolicum hyperimmune sera, transblotted SGE proteins of unfed H. anatolicum anatolicum and Hyalomma dromedarii revealed two common bands of 105 and 80 kDa. A 36-kDa protein band present in H. anatolicum anatolicum SGE could not be detected in H. dromedarii. None of these proteins were detected in partly fed Boophilus microplus when probed with anti-H. anatolicum anatolicum hyperimmune serum. This H. anatolicum anatolicum specific 36-kDa protein was strongly recognized throughout feeding, and thus may be an immunogen of importance for the development of an H. anatolicum anatolicum specific serodiagnostic assay.     

Relationship between the resistance of crossbred cattle to ticks, Boophilus microplus (Canestrini, 1887) and Hyalomma anatolicum anatolicum (Koch, 1844)

Studies were undertaken to determine the effect of repeated pure infestations with Boophilus microplus on susceptibility to subsequent pure infestations with Hyalomma anatolicum anatolicum, and the effects of pure infestations with both species of tick on susceptibility to a series of mixed infestations. Crossbred (Bos indicus X Bos taurus) calves were infested with Boophilus microplus (17 times), H. a. anatolicum (four times), followed by five mixed infestations of B. microplus and H. a. anatolicum. The decline in B. microplus engorgement from a mean yield of 274.4 +/- 60.3 ticks per host after the first exposure, to a mean yield of 9 +/- 4.6 per animal after the seventeenth exposure, was observed in animals exposed to only B. microplus. This might be due to acquired resistance. However, these animals were found to be as susceptible to H. a. anatolicum as animals which had never been exposed to ticks of either species. A decline in the yield of H. a. anatolicum from a mean yield of 92.1 +/- 10.7 after the first exposure to 54.7 +/- 11.3 after the fourth exposure, indicated that the cattle could also acquire resistance to repeated pure infestations with this species. After repeated pure infestations with both tick species, cattle reacted to five mixed infestations showing a high degree of resistance to B. microplus and low resistance to H. a. anatolicum (mean yield for B. microplus was only 10 +/- 8.1 ticks per host after the first mixed exposure and declined to 1.3 +/- 1.7 after the fifth, whereas the mean yield for H. a. anatolicum was 71.4 +/- 11.3 ticks per host following the first exposure and declined to 37.3 +/- 7.8 after the fifth). Host responses elicited to one species do not provide cross-resistance to the second species used in this study.     

Relative role of male and female Hyalomma anatolicum anatolicum ticks in Theileria transmission

The relative role of male and female Hyalomma anatolicum anatolicum ticks in the epidemiology of Theileria transmission was studied by detecting Theileria sporozoites in the dissected salivary glands of 568 ticks by the methyl green pyronin staining method. Detailed frequency distribution of Theileria-positive acini in the salivary glands of the 264 (46.48%) positive ticks from a field collection in Haryana indicated that the number of infected salivary acini per positive tick was greater in females than in males. This suggests that female ticks have a more important role in Theileria transmission than male ticks. This finding assumes greater significance in the light of the observation that the natural male:female ratio is also in favour of female ticks.     

Effect of temperature on transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum ticks.

The effect of temperature on the transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum was studied. Variation in temperature (4-40 degrees C) had a significant effect on moulting rate of the ticks and transmission of theilerial parasites from nymphs to resultant adults. The temperatures above 40 degrees C and below 12 degrees C prevented moulting. Maximum infection levels were obtained in salivary glands of adult ticks when the infected engorged nymphs were incubated at 24-28 degrees C. The infection rate in salivary glands was assessed using a methyl green pyronin technique.     

Lack of infectivity of a Brazilian Anaplasma marginale isolate for Boophilus microplus ticks

Previous studies have shown that one Brazilian Anaplasma marginale isolate presents an inclusion appendage (tail), while other isolates do not present such inclusion. Studies on tick transmission have been carried out with tailless isolates but little is known about transmission of tailed isolates by Boophilus microplus. Two splenectomized calves were experimentally inoculated with the tailed A. marginale isolate. During ascending rickettsemia, B. microplus larvae, free from hemoparasites, were fed on the calves and the resulting nymphs, adult males and engorged females were examined by optic and electronic microscopy. No A. marginale colonies were observed in the gut cells of engorged females and the larvae originated from them did not transmit A. marginale to susceptible calves. In addition, no colonies of A. marginale were seen in the gut cells or in salivary glands of adult males and nymphs. These results suggest that B. microplus is not the biological vector for this tailed isolate.     

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher (p<0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.     

Natural infection rates and transmission of Theileria annulata by Hyalomma anatolicum anatolicum ticks in the Sudan

Hyalomma anatolicum anatolicum nymphs were collected from two localities in the Sudan: Eddamer in Northern Sudan and Wad-Medani in Central Sudan. They were allowed to moult to adult ticks, which were assessed for Theileria infection in their salivary glands using Feulgen stain. At Eddamer, 49.6% of 123 ticks examined were infected with Theileria and the mean intensity of infection was 1.3 (i.e. the number of infected acini/number of infected ticks). At Wad-Medani, 8.6% of 162 ticks were infected and the mean intensity of infection was 7.9. The prevalence of infection was higher in female than in male ticks at both localities. When adult H. a. anatolicum were applied onto two susceptible calves, both animals developed the severe form of theileriosis.     

Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies.     

Potential of immunoprophylaxis using cobalt-60 irradiated Theileria annulata in salivary gland suspensions of the tick Hyalomma anatolicum

The possibility of the attenuation of the infective stage (sporozoite) of Theileria annulata within salivary gland suspension of the tick, Hyalomma anatolicum, was investigated using ⁶⁰Co irradiation at levels of 5 and 10 kiloröntgens (kR). Inoculation of the irradiated ineffective material in susceptible calves induced a relatively less severe disease and longer incubation period than those in control animals inoculated with non-irradiated organisms. Milder reactions and longer incubation periods were observed in animals inoculated with sporozoites irradiated with 10 kR than those inoculated with sporozoites irradiated with 5 kR. Irradiation at these levels did not alter the morphology of the parasites. Because only few animals were used in these trials, further investigations are recommended to test the suitability of the technique for immunoprophylaxis.     

The production of nymphs of Hyalomma anatolicum anatolicum for experimental infection with Theileria annulata.

Methods are described for the production of nymphs of Hyalomma anatolicum anatolicum for later use in transmission experiments with Theileria annulata where the timing of the application of nymphs to infected cattle needs to be accurately controlled. Larvae are fed on the torsos of rabbits where approximately 98% undergo a two-host feeding cycle. This cycle is interrupted 2 days after the first larvae moult into nymphs by killing the rabbits and the nymphs are then collected with a suction pump. Nymphs produced by the interrupted larval feeding method feed well on cattle, in regard to timing of detachment and weight, compared with nymphs produced by interrupted feeding on rabbits.     

小亚璃眼蜱对牛巴贝斯虫未定种和环形泰勒虫传播的试验研究

利用蜱传播试验确定小亚璃眼蜱对中国新分离的牛的巴贝斯虫未定种和环形泰勒虫的传播能力与传播方式，进而明确其在中国牛梨形虫病传播中的流行病学意义。试验结果表明：小亚璃眼蜱可在雌虫阶段受到巴贝斯虫未定种的感染，并可在次代若虫（2／2）和成虫（3／3）阶段将病原传播给试验牛；小亚璃眼蜱幼虫和若虫吸入环形泰勒虫，饱血脱落的幼虫和若虫所蜕化发育的饥饿若虫（2／2）和成虫（2／2）均可将病原传递给敏感动物；感染巴贝斯虫未定种的小亚璃眼蜱饱血雌虫所孵育出的次代幼虫和若虫仍可被环形泰勒虫感染，所发育出的若虫（2／2）和成虫（2／2）可在一次传播试验中将环形泰勒虫和巴贝斯虫未定种同时传播给试验动物。     

Experimental transmission of an unnamed bovine Babesia by Hyalomma spp., Haemaphysalis longicornis and Boophilus microplus

Experiments were undertaken to determine the mode of transmission to cattle of an unnamed Babesia sp. by Hyalomma anatolicum anatolicum, Hyalomma detritum, Hy. rufipes koch, Haemaphysalis longicornis and Boophilus microplus. The unnamed Babesia species designated Babesia U sp. was isolated by infesting cattle with nymphs from female Hy. a. anatolicum ticks collected from Xinjiang province. Adults of laboratory reared Hy. a. anatolicum, Hy. detritum and Hy. rufipes koch were infected with Babesia U sp. by feeding on infected cattle, isolated with nymphal ticks of Hy. a. anatolicum derived from females collected from field. The experiments revealed that Hy. a. anatolicum was capable of transmitting Babesia U sp. transovarially in larval (2 of 4 calves), nymphal (6 of 6 calves) and adult (3 of 8 calves) stages, with prepatent periods of 16, 12, and 8 days, respectively, and that this Babesia was also transovarially transmitted by both the nymphal and adult stages of Hy. detritum and Hy. rufipes. Attempts to transmit this Babesia U sp. transovarially with Hae. longicornis and B. microplus, and transstadially with Hyalomma spp., were carried out, and the results proved to be negative.     

Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24-27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats.     

小亚璃眼蜱源性牛环形泰勒虫裂殖子表面抗原(Tams1)基因检测及同源性分析

针对牛环形泰勒虫裂殖子表面抗原(Tams1)基因设计特异性引物,对提取的奶牛全血DNA和小亚璃眼蜱组织DNA进行环形泰勒虫病原的PCR扩增,并对PCR产物进行克隆、测序和同源性分析。结果显示,血源性和小亚璃眼蜱蜱源性环形泰勒虫Tams1基因序列大小分别为534bp和546 bp,经NCBI BLAST后发现,血液源性环形泰勒虫与环形泰勒虫印度株同源性为99%,而蜱源性环形泰勒虫与环形泰勒虫意大利株有99%的同源性,表明新疆地区存在环形泰勒虫不同株型的流行。     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.     

Identification of Theileria infections in the salivary glands of Hyalomma anatolicum anatoli cum and Rhipicephalus appendiculatus using isoenzyme electrophoresis

Lysates prepared from the salivary glands of uninfected Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus and from ticks of these species infected with 2 strains of Theileria annulata and 1 strain of T. parva respectively have been examined for enzyme polymorphism using thin layer starch gel electrophoresis. Representative enzymes from 3 of the major metabolic pathways, the tricarboxylic acid cycle, the glycolytic pathway and the pentose phosphate pathway were investigated. Only 1 enzyme, glucose phosphate isomerase, showed differences between the host cell activity and the parasite activity and also inter- and intraspecific differences between the parasites. This technique represents a useful complement to the present histochemical methods for identification of Theileria infections in the salivary glands of ticks.     

Analysis of BoLA class II microsatellites in cattle infested with Boophilus microplus ticks: class II is probably associated with susceptibility

The aim of this study was to determine the role of certain bovine lymphocyte antigens (BoLA) regions in the resistance or susceptibility to Boophilus microplus tick infestation in two different breeds of cattle. The breeds were maintained, one in natural conditions and the second one in an experimental setting at the research station in Martinez de la Torre, Veracruz, Mexico. The study took place from June to August 2001 (natural infestation) using 33 crossbreed steers (crossbreed is here defined as 3/4 European = 1/2 Simmenthal x 1/4 Holstein x 1/4 Zebu, a cross resulting from F1 x Simmenthal), ranging from 15 to 20 months old. Fifty-nine F1 cows (1/2 Holstein x 1/2 Zebu) were included in the experimental setting, infested and followed during 25 days in November 2001 and 2002. Experiment A included thirty-one 2-7-year-old F1 cows, and experiment B included twenty-eight 18-24-month-old F1 heifers. Both groups were analysed separately and were not comparable because of the different infestation methods and genetic background. All ticks > or =4mm long were counted on the total body of F1 animals and on one side of the 3/4 European steers. In this case, susceptible animals were defined when having ticks = X + 1S.D. (29 +/- 16). In the experimental setting susceptibility was defined when the number of ticks was over the 75 percentile (> or =79). DNA was extracted from peripheral blood samples of all animals. The BoLA DRB3, DRBP1, RM185 and BM1815 microsatellite loci were amplified using a PCR method. Genescan software was used for analysis in an ABI sequencer. The SPSS statistical program was used and the comparisons were assessed using the Fisher's exact test. In the naturally infested animals, DRB3-184 was found positively associated with tick infestation (P = 0.018; Pc = NS; OR = 5; EF = 28%). DRBP1-128 was also found to be increased (P = 0.03; Pc = NS; OR = 6; EF = 42%). In the experimentally infested animals, two more loci were found to be associated, BM1815-152 (P = 0.01; Pc = NS; OR = 15; EF = 74%) and DRBP1-130 (P = 0.05; Pc = NS; OR = 4; EF = 77%). None of them remained significant after correction, indicating that a larger sample size is needed to confirm the results. This is the first study showing MHC genes associated with tick infestation based on class II microsatellite polymorphisms. Further studies are needed to confirm the susceptibility traits and to determine haplotype segregation in families.