Experimental immunisation of crossbred cattle with glycoproteins isolated from the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus

In India, control of tick infestations is a major problem facing the owners of highly productive crossbred cattle. To develop suitable immunoprotective measures against the multitick infestations, specific problem encountered by the Indian farmers, an attempt has been made to identify candidate protein molecules in the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus. Employing two steps affinity chromatography method, glycoproteins of 34 and 29 kDa were isolated from H. a. anatolicum and B. microplus, respectively. The additive effect of the isolated glycoproteins was tested by the immunisation of crossbred male calves at a dose of 200 microg/animal of each glycoprotein, three divided dosages. Following challenge infestations by the stages of two species of ticks, a significant rejection of (73.6%; P < 0.01) of larvae and adults (75.0%; P < 0.01) of H. a. anatolicum and adults (89.8%; P < 0.01) of B. microplus were noted. The direct effect of immunisation on the stages of the challenged ticks (%DT) was calculated as 69%, 52% against larvae and adults of H. a. anatolicum and 60% against adults of B. microplus. A direct corelation between protection and anti-glycoprotein antibody titre was detected. The effect of the isolated glycoproteins on limiting the tick infestation is discussed in detail.     

Hyalomma anatolicum anatolicum: the role of humoral factors in the acquisition of host resistance

A significant degree of resistance to Hyalomma anatolicum anatolicum can be adoptively transferred to naive recipients with immune serum from rabbits repeatedly infected with adult H. a. anatolicum. Ticks fed on recipients of immune serum took longer to become engorged and showed a significant decrease (P less than 0.01) in engorgement weight and oviposition compared with ticks that fed on recipients of normal serum. A direct correlation between resistance and anti-saliva IgG antibodies was indicated by a progressive increase in the degree of resistance and IgG antibody titres following successive tick infestations. Challenge feeding sites on actively sensitised hosts and recipients of immune serum revealed significantly greater infiltration of basophils and eosinophils compared with feeding sites on recipients of normal serum. However, both the degree of resistance and the accompanying cutaneous basophil and eosinophil responses in recipients of immune serum were considerably weaker than those induced by active tick feeding, thus suggesting that nonhumoral (cell-mediated) mechanisms might also be involved in acquired host resistance to H. a. anatolicum.     

Immunization of cattle against Hyalomma anatolicum anatolicum using larval antigens

Development of immunity in cross-bred (Bos taurus x Bos indicus) calves against Hyalomma anatolicum anatolicum, vector of bovine tropical theileriosis, was studied using larval antigen (LS) in Freund's complete adjuvant (FCA). Calves immunized with LS + FCA showed significant rejection of larvae (57.25 +/- 6.8) and nymphs (45.75 +/- 5.16). Abnormally fed larvae (11.4 +/- 0.8) and nymphs (8.25 +/- 1.2) were also recovered from immunized calves. This abnormal feeding may possibly be attributed to their inability to gain access to the blood vessels owing to the host immunological reactions. Consequently, feeding of extravascular fluid leads to white colour of fed ticks. Sera from all immunized calves after a week of immunization were positive for anti-LS antibodies in ELISA. The investigation indicates that LS in FCA enhanced anti-tick immunity.     

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.     

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae)

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10⁸ conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25 ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3–5 days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.     

Comparative immunopotentiating properties of saponin and incomplete Freund's adjuvant coupled to affinity purified larval antigen of Hyalomma anatolicum anatolicum

Comparative immunostimulatory properties of saponin and incomplete Freund's adjuvant (IFA) were studied using affinity purified 39 kDa larval antigen of Hyalomma anatolicum anatolicum. Significant antibody response to 39 kDa antigen was detected in the sera of rabbits immunized with both 39 kDa plus saponin and 39 kDa plus IFA in comparison to their corresponding control animals. Insignificant differences were noted in the antibody response between the animals of two immunized groups. Upon challenge, the animals immunized with 39 kDa plus IFA rejected 76.2 +/- 9.7% of larvae and 45.8 +/- 4.1% of adults while in group of animals injected with 39 kDa plus saponin rejected 80.9 +/- 11.2% of larvae and 47.2 +/- 5.7% of adults.     

Immunization of cross-bred cattle against Hyalomma anatolicum anatolicum by purified antigens

Extracts prepared from unfed larvae of Hyalomma anatolicum anatolicum were purified by immunoaffinity chromatography using anti-gut IgG as ligand. Affinity purified antigen (Aff-GHLAg) was used to immunize cross-bred (Bos taurus×B. indicus) calves of 6–7 months of age. Immunized calves rejected 70.6% larvae, 54.5% nymphs and 61.9% adults. No significant changes in the engorged weight of females was observed; however, significant decrease in the engorgement weight of larvae and nymphs was recorded. There was a significant decrease in the emerging nymphs (p<0.05) and adults (p<0.01) of the tick stages fed on immunized animals. SDS-PAGE analysis revealed three antigenic proteins of 100, 59.4 and 37kDa responsible for induction of resistance in the host.     

Effects of 60Co irradiation of the early developmental stages of an ixodid tick, Hyalomma anatolicum.

Various developmental stages of Hyalomma anatolicum were subjected to a range of doses of 60Co radiation from 0-10 000 R, and their subsequent development was observed. Some of these ticks were infected with Theileria annulata. Doses of 2000 R and over prevented all but a very limited development of ticks. After 1000 R a proportion of exposed individuals continued development in a near-normal manner. Lower doses had proportionately less effect. 1000 R to ticks infected with T. annulata failed to prevent transmission of typical theileriasis to calves.     

Effects of 60Co irradiation on unfed adults and engorged females of the tick Hyalomma anatolicum.

Unfed and engorged adults of the cattle tick Hyalomma anatolicum, known to be biological vector of Theileria anmulata in India, were exposed to various levels of gamma-radiation from 0-60 000 R, and the effect on engorgement and redproduction was observed. The radiation tolerance limit for unfed adults is 1000 R for engorgement and reproduction. The sterilizing dose for most of the males is 2000 R but the females can tolerate a slightly higher dose. Engorged females have a tolerance limit of 10 000 R for oviposition and 1000 R for larval development. Higher dosages (20 000-60 000 R) have adverse effects on development, completely inhibiting egg-laying at 60 000 R, but these females (60 000 R) live longer (10 weeks) than those irradiated at lower levels.     

A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum

The pathogenicity and morphology of a large Babesia species, Babesia sp. Xinjiang, are described here. The parasite has very low virulence for sheep, and caused no detectable clinical symptoms. Splenectomized sheep infected with the parasite showed mild fever and low parasitemia and would recover gradually. If splenectomized sheep were immuno-suppressed with dexamethasone, the parasitemia could reach 8.5%, and death occurred. A splenectomized calf could not be infected with the Babesia species. Paired parasites were the typical form of the Babesia species in erythrocytes and the average size of a pair of parasites was 2.42 (±0.35) μm × 1.06 (±0.22) μm. Merozoites were found in the gut, salivary gland, haemolymph, ovary and eggs of female Hyalomma anatolicum anatolicum engorged on sheep infected with the parasites. The results of experimental transmission showed that the larval, nymph and adult stages of H. a. anatolicum could transmit the Babesia species to sheep.     

Molecular characterization of HAO3, the homologue of the Bm86 tick vaccine antigen,from the Iranian isolate of Hyalomma anatolicum anatolicum

Hyalomma anatolicum anatolicum tick is widely distributed in many parts of Iran and while the commercial vaccines based on the application of midgut-derived recombinant Bm86 antigen are used for its control, limited information about the efficiency of this vaccination in Iran is available. Herein, with the final aim of evaluation of Bm86-based heterologous vaccination, as the primary step the Bm86 homologue of the H. a. anatolicum (Hao3) from an Iranian isolate was characterized and compared with the commercialized Bm86 and other Bm86 homologoue sequences available in GenBank.     

The salivary glands of Hyalomma anatolicum anatolicum: structural changes during attachment and feeding

The histology and ultrastructure of the salivary glands of male and female H.a. anatolicum ticks have been examined m unfed and feeding ticks with special emphasis on aspects related to the feeding process. The salivary glands of H.a. anatolicum consisted of three types of acinus (acinus I, II and III) in females and an additional type IV acinus in males. The type I acinus was agranular and showed slight morphologic changes during feeding. The presence of cells with ultrastructural features characteristic of epithelia involved in the secretion of hyperosmotic fluids supports the hypothesis that these acini secrete hygroscopic saliva during questing stages to absorb water from an unsaturated atmosphere. There were five granular cell types (a, b, c₁–c₃) in type II acinus, three granular cell types (d, e, and f) in type III acinus, and one granular cell type (g) in type IV acinus. The cells a, d and e secreted most of their granules early in feeding and are considered to be cement precursors. The b and c cells appeared to synthesise and secrete their products throughout feeding and so are likely to secrete anticoagulants, enzymes and other pharmacologically active agents required during feeding. The interstitial cells, which were insignificant in acinar types II, III and IV of unfed ticks, became more distinct during feeding. The type III acinus in females showed remarkable cell transformations, during the course of feeding. The ablumenal interstitial cells of type III acinus, in females formed a basal labyrinth of extraordinary complexity by interdigitating with the basolateral membranes of transformed f cells to form a network of extracellular channels to excrete fluid during feeding. There was an enormous increase in the secretory granules of g cells as the feeding advanced. The secretory granules were released by a process of exocytosis, by direct fusion with the apical membranes and through channels connecting several granules.     

Expression of recombinant HAO3 from an Iranian isolate of Hyalomma anatolicum anatolicum in Pichia pastoris and evaluation of its antigenicity

Hyalomma anatolicum anatolicum tick is considered as one of the main problem of ruminants’ productivity in endemic countries such as parts of Africa, the Middle East and India. The disease is economically important and hence, its control and eradication is a priority. This problem reinforces the need for alternative approach like vaccine to control tick infestations instead of continuous application of acaricide which led to the natural selection of the acaricide-resistant ticks. Therefore, the present study provided evidence for the construction of transformant containing the chromosomally integrated multi-copy expression cassettes of HAO3, its successful and efficient expression in Pichia pastoris yeast and purification of the secreted protein by ultrafiltration (UF) system in a high level yield and purity.The result of antigenicity assay for the rHAO3 protein pointed well toward its capability for the elicitation of antibody response in immunized rabbits. Interestingly, the results indicated that the expressed HAO3 protein reacted well with mid gut antigen (MGAg) and rBm86 (Gavac) antisera in ELISA and western blot assays making it evident that the epitopes present in expressed protein are well recognized by the antibodies against MGAg and rBm86 proteins. Moreover, the presence of cross-reactive epitopes between rHAO3 protein with its native antigen from mid gut cells was also determined.     

Theileria annulata and Babesia ovis: ultracytochemical lactic dehydrogenase activity of sporozoites in salivary glands of female ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa

The occurrence of a marker enzyme of glycolysis, lactic dehydrogenase (LDH) (EC 1.1.1.27.), was studied ultracytochemically in sporozoites of Babesia ovis in the tick Rhipicephalus bursa and in sporozoites of Theileria annulata in the tick Hyalomma anatolicum excavatum. Female ticks infected transstadially were fed on rabbits and dissected 3–5 days post infestationem. The salivary glands were removed and incubated in the cytochemical medium unfixed or after fixation in buffered paraformaldehyde solution. A modified ferricyanide medium adjusted to pH 6.5 was used for incubation. Controls were performed by preincubating specimens in 10^?3 M iodine or by omitting NAD⁺ or lactate in the incubation medium. Following incubation, the specimens were fixed in buffered solutions of paraformaldehyde or glutaraldehyde, postfixed in osmium tetroxide, and embedded in Durcupan ACM. Mature “schizonts” consisting of an abundant number of sporozoites were examined in both piroplasmean species. In sporozoites of B. ovis the final enzymic product was deposited within the nucleus. No cytoplasmic reaction was observed. However, the membrane delimiting the schizont from the adjacent glandular cells was distinctly reactive. In T. annulata reactivity was usually confined to the cytoplasm. Sometimes, a reaction within mitochondria could be observed. The reaction product had formed aggregations which often appeared to be attached to micronemes. There was no nuclear reactivity in this species.The results suggest the existence of a glycolytic metabolic pathway with different subcellular localizations in sporozoites of the two piroplasmean species.     

Transstadial transmission of Theileria annulata through common ixodid ticks infesting Indian cattle.

Transstadial transmission of Theileria annulata with Hyalomma anatolicum anatolicum, H. dromedarii and H. marginatum isaaci, and Haemaphysalis bispinosa, Rhipicephalus haemaphysaloides haemaphysaloides and Boophilus microplus was determined. It was found that the infection was successfully transmitted by H. a. anatolicum from larva to nymph and nymph to adult in all attempts. When larvae were fed on an infected calf the succeeding adults transmitted the infection when the intervening nymphs fed on a rabbit (non-susceptible host) but not when fed on a calf (susceptible host). Infective adult ticks transmitted the parasite during the first 24 h of feeding on a calf. When the feeding was interrupted after 24 h, and the tick transferred to another calf, the infection was transmitted to the latter as well. H. dromedarii successfully transmitted the infection from larva to nymph and from nymph to adult. Larvae of H. marginatum isaaci did not feed on calves but the infection was successfully transmitted from nymph to adult. Haemaphysalis bispinosa, Rhipicephalus h. haemaphysaloides and Boophilus microplus did not transmit Th. annulata from larva to nymph to adult.     

Muscari turcicum (Liliaceae/Hyacinthaceae), a new species from south Anatolia, Turkey

Muscari turcicum Uysal, Ertugrul & Dural (Liliaceae/Hyacinthaceae) is described and illustrated from south Anatolia, Turkey. This species grows on alpine steppe in the Middle Taurus (C4 Konya Province). Muscari turcicum , an endemic confined to the Middle Taurus, is closely related to M. discolor Boiss. & Hausskn. and M. anatolicum Cowley & Özhatay. Diagnostic morphological characters are discussed and compared with those of closely related taxa.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 233–236.     

The distribution of Hyalomma spp. ticks from domestic ruminants in Iran

The distribution of Hyalomma species on domestic animals was studied in four zoogeoghraphical zones. Nine hundred and ninety-two Hyalomma ticks were collected from sheep, goats, cattle and camels. A total of seven tick species consisting of: Hyalomma anatolicum Koch, 1844 (28.93%), H. excavatum Koch, 1844 (12.5%), H. asiaticum Schulze & Schlottke, 1930 (13.5%), H. marginatum Koch, 1844 (27.01%), H. detritum Schulze, 1919 (9.67%), H. schulzei Olenev, 1931 (4.03%) and H. dromedary Koch, 1844 (4.33%) were recorded. The results indicated that H. anatolicum, H. asiaticum, H. marginatum and H. detritum were present in all zones whereas H. excavatum was absent in Zone I. The result also showed that H. dromedari was presented in Zones III and IV whereas H. schulzei was present in Zones II and III. During this study, the tick species collected are candidates for investigation as vectors of pathogenic organisms in Iran.     

Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.     

Ultrastructural study on the development of Babesia equi (Coccidia: Piroplasmia) in the salivary glands of its vector ticks

The formation of Babesia equi sporozoites in the salivary glands of three tick species (Hyalomma anatolicum anatolicum, H. a. excavatum, Rhipicephalus turanicus) was studied by electron microscopy. The development was identical in all three vectors. On the 8th day post repletionem kinetes of B. equi had invaded alveoli of the nymphal salivary glands and were transformed to sporonts bounded by a single membrane. The sporonts were polymorphous bodies each with a highly lobed nucleus and numerous mitochondria. These stages persisted during ecdysis of the tick nymph to the adult stage. After attachment of these newly molted adults to a new host the formation of sporozoites was completed within five days. The sporonts occupied most of the infected alveolus and were extensively divided into cytoplasmic portions of various size. On the 4th day after attachment of the tick, sporozoite-anlagen, into each of which a nucleus and a mitochondrion were incorporated, appeared at the periphery of the sporonts. An apical complex with a polar ring, rhoptries, and micronemes was formed at the tip of each protruding anlage. Finally thousands of pyriform sporozoites (3.0 X 1.2 microns) filled the hypertrophied alveolus. This development is similar to sporogony in the genus Theileria.