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.     

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.     

Natural infection rates of Hyalomma anatolicum anatolicum with theileria in Sudan

Hyalomma anatolicum anatolicum ticks were collected around cattle pens at two locations close to Khartoum. They were assessed for theileria infection by four methods. Salivary glands were stained whole with methyl green pyronin and examined for parasite masses. Adult ticks were partially fed on rabbits, ground up in medium, and the suspensions were examined in Giemsa stained smears and by inoculation into bovine lymphocyte cultures. Ticks were fully fed on calves which were monitored for developing theileriosis. H a anatolicum were found infected with parasite masses similar to those seen in experimental infections with T annulata. At one site 38 per cent of 102 ticks were infected and the mean number of parasite masses per tick for the whole sample was 37. At the other site 86 per cent of 156 ticks were similarly infected and the mean parasite masses per tick was 19.5. Suspensions of sporozoites contained sporozoites typical of those found in experimental preparations of T annulata. Sporozoites harvested from ticks from both locations infected and transformed normal bovine lymphocyte cultures. H a anatolicum ticks from both locations produced fatal theileria infection in susceptible calves.     

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.     

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.     

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.     

The effects of bovine tick resistance on the susceptibility of Hyalomma anatolicum anatolicum to infection with Theileria annulata (Ankara)

Two Bos taurus calves were made resistant to tick infestation by exposing them to approximately 500 rabbit-reared nymphs of Hyalomma anatolicum anatolicum twice at a 2-week interval. These two calves, together with a tick-susceptible control calf, were inoculated with a stabilate of Theileria annulata (Ankara). Patent infection resulted in all three calves. Seven-hundred and fifty gerbil-reared nymphs were then applied on each of these calves as well as another tick-susceptible calf that was Theileria free. This infestation was carried out on Day 8 post-inoculation. Ticks that dropped on Day 13 post-inoculation were examined to note the development of T. annulata in them and the histological changes that occurred in the gut and salivary glands. During the second phase of feeding, the gut epithelia of the ticks from the tick-resistant calves were less active. There were no notable differences in the characteristics of the developmental stages of T. annulata between the ticks from the tick-resistant calves and those from the susceptible calf. However, ticks from one calf that acquired a higher level of tick resistance were significantly less susceptible to infection by T. annulata. Bovine tick resistance therefore compromises the vector capacity of H. a. anatolicum and this may be of epidemiological significance in the endemic areas of tropical theileriosis.     

Immunization of cattle with nymphal Hyalomma anatolicum anatolicum extracts: effects on tick biology

Antigens derived from partially engorged nymphs of Hyalomma anatolicum anatolicum were used in immunizing crossbred (Bos indicus×Bos taurus) cattle against larval, nymphal and adult H. a. anatolicum and H. dromedarii. The cattle were either infected with Theileria annulata at low parasitaemia or were uninfected. Whole nymphal extract (WNE), nymphal membrane antigens (NMA) and nymphal soluble antigens (NSA) were used for immunization. The group immunized with WNE showed significant and better rejection of H. a. anatolicum ticks as compared to calves immunized with either NMA or NSA. The moulting rates of both engorged larvae and nymphs remained unaffected. Nymphs which engorged on the immunized calves were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of Theileria infections in the resulting adult ticks from immunized and unimmunized Theileria infected cattle. These ticks also transmitted fatal theileriosis to susceptible calves.     

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.     

Infectivity of ground-up tick supernates prepared from Theilerai annulata infected Hyalomma anatolicum anatolicum.

Groups of Theileria annulata infected Hyalomma anatolicum anatolicum adults prefed on a calf for 1 to 6 days were separately ground in tissue culture medium-199, supplemented with bovine albumin powder, Fraction-V. Supernatant fluid was collected, made up with additional medium, so that each millilitre represented material from 25 ticks, and was injected subcutaneously into groups of cross-bred male calves. The results indicated that ground-up tick supernate (GUTS) prepared from unfed ticks was not infective, whereas that prepared from 1 to 6 days prefed ticks was infective. GUTS prepared from 3 days prefed ticks appeared to contain highest infectivity and 1 ml of it induced fatal theileriosis.     

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.     

High infection rates of the tick Hyalomma anatolicum anatolicum with Trypanosoma theileri

A crossbred calf (3 months old) obtained from a farm where regular control of ticks was practised and found to be free of blood parasites was inoculated with 20 ml pooled blood collected from four field cattle which had very low Trypanosoma theileri parasitaemias (one parasite per 70 microl blood as determined by the haematocrit centrifugation technique). Trypanosoma theileri was present in the blood 6 days after injection and a peak parasitaemia of 42 parasites per 70 microl blood was recorded by day 12. Hyalomma anatolicum anatolicum nymphs were applied on the ears of the calf on day 8 and they dropped engorged by days 13 and 14. The resulting adult ticks were examined for the presence of T. theileri by severing a leg and making a smear of the clear haemolymph which exuded from the wound. The smear was fixed in methanol and stained with Giemsa stain. The infection rate with T. theileri in the ticks was 43.3% (26 out of 60). The intensity of infection was very high and various developmental stages of the flagellates were observed (epimastigotes, sphaeromastigotes, trypomastigotes and other intermediate stages). The haemolymph from 12 ticks was also collected in tissue culture medium and the trypanosomes survived for 25 weeks before eventually dying. The results demonstrated unequivocally the high vectorial capacity of the tick H. a. anatolicum for T. theileri.     

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。     

Rapid quantitative assessment ofTheileria infection in ticks

A simplified method for methyl green pyronin staining is described for Theileria parva and T. annulata in whole salivary glands of Rhipicephalus appendiculatus and Hyalomma anatolicum subspecies respectively. The stain gives results comparable with Feulgen staining and can be used after the ticks have been in cold storage for 3 days. There is considerable variability in the rate and intensity of infection of these ticks with theilerial parasites and it is concluded that the method permits large samples (60 ticks per person per day) to be examined to overcome this variability when assessing infection quantitatively.     

Cattle infestation by Hyalomma ticks and prevalence of Theileria in Hyalomma species in the east of Turkey

A total of 2388 cattle and 442 shelters, from two provinces (Elazig and Malatya) endemic for tropical theileriosis in the east of Turkey, were studied for Hyalomma tick populations from July 1993 to July 1995 in Elazig and from May 1998 to January 1999 in Malatya. Four thousand five hundred and eighty one of 7455 Hyalomma ticks were collected from cattle, the other ticks (2874) were collected from shelters. All of the ticks collected from shelters were Hyalomma anatolicum anatolicum. Two thousand eight hundred and ninety five (63.1%) of 4581 Hyalomma ticks collected from cattle were H.a. anatolicum. 23.8% (1047/4581), 11.7% (536/4581) and 0.6% (3/4581) of Hyalomma ticks were Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum, respectively. A total of 5909 Hyalomma adult ticks collected from cattle (3362/5909) and shelters (2447/5909) were dissected and salivary glands were stained with Methylgreen/Pyronin method. Thousand one hundred and fifty (46.9%) of 2447 H.a. anatolicum collected from shelters and 412 (19.1%) of 2147 H.a. anatolicum collected from cattle were positive for Theileria infection. Twenty (2.4%) of 820 H.a. excavatum and 23 (4.6%) of 495 H. detritum collected from cattle were positive. The mean number of infected acini per infected male and female ticks collected from cattle were 11.3 and 22.4 in H.a. anatolicum, 4 and 6.8 in H.a. excavatum, 17.9 and 18.3 in H. detritum, respectively. In H.a. anatolicum collected from shelters, the above rates were 11.8 and 17.6 in male and female ticks, respectively. The prevalence and intensity of Theileria infection was greater in female ticks than in males.     

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.     

A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata

In this comparative study unfed nymphs of four Hyalomma tick species (Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum) were allowed to engorge on calves experimentally infected with Theileria annulata. The infection prevalence in the salivary glands of the adult female and male ticks of each Hyalomma species used in the study were assessed. The infection prevalence with T. annulata was high and did not vary markedly in the four Hyalomma tick species. The mean number of infected acini per tick in female and male ticks was different with female ticks having higher numbers of infected acini than the male ticks. The sex difference was more significant between H.a. anatolicum and H.a. excavatum than between H. detritum and H.m. marginatum. This study clarifies the roles of four Hyalomma tick species, and their sex, in the development of T. annulata.     

湖北省水牛巴贝斯虫病调查研究 Ⅴ.镰形扇头蜱经卵传递牛巴贝斯虫的证实

1986年从病牛身上摘下饱血雌蜱,经实验室饲养,孵化为第二代成蜱,人工越冬。于1987年将上述蜱100只接种于已去脾23天的一岁龄健康水牛,以后每天从该牛身上取蜱查其唾液腺。结果在吸血期间,始终查到梨形虫体,其平均大小为1.3×0.68微米。接种后第4天,牛血中开始出现巴贝斯虫,至第19天,红细胞感染率高达9.6％,单梨形虫体大小为0.84～2.64×0.42～1.22微米,双梨形虫体大小为0.64～1.64×0.37～0.97微米,以后逐渐下阵。该牛出现体温升高)41.6℃)、严重贫血(血红蛋白量2克,红细胞压积14％,红细胞总数177万/毫米~3)、黄疸及精神萎顿等症状。上述结果表明,该牛感染了巴贝斯虫病,由此证实镰形扇头蜱是经卵传递巴贝斯虫,其感染阶段是成蜱。     

Maturation of Theileria sergenti in salivary glands of Haemaphysalis longicornis induced by temperature stimulation

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.     

Infection rates of Theileria sergenti in Haemaphysalis longicornis ticks collected from the field in Japan

For a period of one year Haemaphysalis longicornis ticks were collected in a pasture from vegetation by dragging flannel cloth and from soil samples at monthly intervals. Nymphal ticks were assessed for Theileria infection. Salivary glands were stained with methylgreen-pyronin and examined for parasite masses. Nymphal H. longicornis infected with Theileria sergenti were found in all samples during 12 months including the winter. After shortening the prefed period on rabbits to 24 hours, the parasite masses could be detected in the salivary glands of nymphs collected in the grazing season, from May to October, while no parasite masses were detected in other season. It was suggested that the environmental factors in the grazing season might influence on the maturation of parasites in the salivary glands of ticks.