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.     

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.     

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.     

Non-specific immunization against bovine tropical theileriosis (Theileria annulata) using killed Corynebacterium parvum

Killed Corynebacterium parvum was used as an adjuvant for the production of non-specific resistance against Theileria annulata in cattle. Groups of cross-bred (Bos indicus X Bos taurus) calves were administered C. parvum adjuvant subcutaneously and were then challenged with T. annulata-infected ticks on 45, 60 or 90 days later. The challenge caused mild reactions in the protected calves. None of the 10 immunized calves died due to theileriosis, whereas all three paris of susceptible control calves died due to theileriosis. It appears from this pilot study that cattle can be protected non-specifically with C parvum adjuvant against T. annulata.     

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.     

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。     

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.     

Reduction of <Emphasis Type="Italic">Theileria annulata</Emphasis> Infection in Ticks Fed on Calves Immunized with Purified Larval Antigens of <Emphasis Type="Italic">Hyalomma anatolicum anatolicum</Emphasis>

Larval antigen of Hyalomma anatolicum anatolicum, the vector of Theileria annulata, was purified by two-step affinity chromatography using anti-tick gut-specific rabbit IgG and IgG from immunized cattle. The purified antigen showed the presence of a single polypeptide of 37 kDa (GHLAgP) on SDS-PAGE. Two groups (I and II) of naive crossbred calves (Bos taurus × B. indicus) were immunized with 1 mg of GHLAgP in three divided doses. Immunized calves of group I were also infected with a sublethal dose of T. annulata along with a group of non-immunized calves (group III). Animals in groups I, II, III as well a control group (group IV) were challenged with live nymphs of H. a. anatolicum on the 10th day of immunization. There was a significant reduction in the number of emerging adults of 56.9% ± 1.67% in calves of group I (p < 0.01) and 63.09% ± 1.26% in calves of group II (p < 0.001) compared to the controls. The calves of groups I and II showed antibody responses to tick antigen up to day 70 post immunization. Infection with T. annulata was determined in the salivary glands of adult ticks that developed from the nymphs used for challenge infection. In ticks taken from group I calves, there was a 75.0% ± 0.00% infection compared with only 85.0% ± 2.88% infection in ticks taken from calves of group III. Using PCR, a lower infection (83.33% ± 3.33%) was detected in ticks that developed from calves of group I compared with calves from group III (90.00% ± 2.88%). The ground-up tick supernatants (GUTS) of the ticks taken from calves of group III yielded higher infection rate and exhibited higher infectivity titre in in vitro infection assay of bovine mononuclear cells than the GUTS of the ticks taken from calves of group I. The results suggest a partial reduction in growth rate of T. annulata in ticks feeding on calves immunized with GHLAgP.     

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.     

Cross-bred Cattle Protected against Hyalomma anatolicum anatolicum by Larval Antigens Purified by Immunoaffinity Chromatography

Antigens from larvae of Hyalomma anatolicum anatolicum were extracted and purified by immunoaffinity chromatography using immunoglobulin ligands from cross-bred animals immunized with soluble larval antigen. Affinity-purified antigen (Aff-TLE) and a total larval extract (TLE) were used to immunize cross-bred (Bos indicus×Bos taurus) cattle. The group immunized with Aff-TLE rejected 71.6% of larvae and 77.3% of nymphs. However, the rejection percentages were lower in the TLE-immunized group. No significant changes in the feeding period, moulting percentages or moulting period of engorged larvae and nymphs were recorded. There was, however, a significant decrease in the number of resultant nymphs p<0.01) and adults (p<0.01) in the ticks fed on the Aff-TLE-immunized group. The Aff-TLE antigen was 93.3% purified. SDS-PAGE analysis identified a 39 kDa protein, reported for the first time, as the antigen responsible for the induction of resistance in the host.     

Immunization of Crossbred Cattle (Bos indicus×Bos taurus) with Fractionated Midgut Antigens against Hyalomma anatolicum anatolicum

Crossbred calves (Bos indicus×Bos taurus) were immunized with a fractionated midgut supernate antigen (GS-F Ag from Hyalomma anatolicum anatolicum). The first inoculation on day 0 was given intramuscularly after emulsification with Freund's complete adjuvant; the second was given subcutaneously on day 14 in incomplete Freund's adjuvant; and the third on day 35 was given subcutaneously without adjuvant. Each injection comprised 1 mg of antigen protein. Ten days after the last inoculation, the immunized calves were challenged simultaneously with 1000 larvae and 20 pairs (20 males and 20 females) of adult H. a. anatolicum on one ear and a similar number of Hyalomma dromedarii ticks on the other ear. There was a significant decrease in the percentage larval engorgement and larval rejection of up to 34% on the immunized calves. A significant increase in the engorgement and preoviposition periods and a significant decrease in the engorged weight, egg mass weight and reproductive index were observed for adult female ticks when fed on the immunized calves. The GS-F Ag also induced a considerable degree of cross-protection in calves against H. dromedarii larval ticks.     

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.     

Evaluation of purified Theileria annulata sporozoite antigen from the tick Hyalomma anatolicum anatolicum

Rabbits immunized with purified sporozoites from Hyalomma anatolicum anatolicum produced antibody to Theileria annulata specific antigens as measured by the indirect fluorescent antibody test. Nonspecific fluorescence which compounded the interpretation of the test was observed. Incorporation of Eriochrome black and Evans blue as a counterstain in the diluent buffer for the conjugate significantly reduced the background fluorescence.     

Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.     

Immunoprotective Efficacy of a Purified 39 kDa Nymphal Antigen of Hyalomma anatolicum anatolicum

Soluble nymphal antigens (HNAg) were purified by immunoaffinity chromatography using CNBr-activated Sepharose 4B coupled with immunoglobulin ligands from animals immunized with HNAg and 69–71% protected against challenge infestations, and 8% recovery of the purified protein (Aff-HNAg) was obtained. Following immunization of crossbred calves (Bos indicus×Bos taurus) with 1600 g of Aff-HNAg in three divided doses, significant rejections of larvae (p<0.001, 84.2%), nymphs (p<0.05, 61.4%) and adults (p<0.05, 58.7%) were recorded. No significant changes were recorded in the engorgement weights of the larvae and nymphs, but there was a significant (p<0.05) reduction in the weight of the engorged adults. Immunization conferred a significant decrease in the numbers of resultant nymphs (p<0.001) and adults (p<0.001) that had fed on the immunized animals. SDS-PAGE analysis identified a 39 kDa protein, previously isolated from larvae of Hyalomma anatolicum anatolicum, as the antigen responsible for the induction of resistance against all the stages of the tick.     

Prophylactic efficacy of buparvaquone in experimentally induced Theileria annulata infection in calves

The antitheilerial activity of buparvaquone (BW 720C) was evaluated in experimentally induced Theileria annulata infections in cross-bred male calves. T. annulata infections were induced by injecting a suspension of infected ground tick tissue suspension (GUTTS) equivalent to two ticks subcutaneously into each calf. Buparvaquone at a dose of 2.5 mg kg-1 body weight was given as a single injection (intramuscularly) on Day 0 (Group 1), Day 8 (Group 2) and Day 12 (Group 3) post-infection. The animals in Groups 4 and 5 were untreated and challenged controls, respectively. All of the recovered animals from Groups 1-4 were challenged with a lethal dose of T. annulata at 6 weeks post-infection. The immunized animals were resistant to the homologous challenge, which killed three of four control animals (Group 5); the controls showed typical antemortem and post-mortem lesions of theileriosis.     

Determination of duration of immunity of calves vaccinated with the Theileria annulata schizont cell culture vaccine

Bovine tropical theileriosis caused by Theileria annulata is a serious haemoprotozoan disease of cattle affecting exotic cattle, their crossbreeds and young indigenous calves. Cell culture vaccines have been developed and used effectively in various countries for the control of this disease. However, the duration of immunity provided by these vaccines is poorly understood. The present experiments were planned to study the duration of immunity in animals after vaccination with the T. annulata (Hisar) schizont cell culture vaccine. Two groups of calves were vaccinated and challenged after a period of 3 and 6 months, respectively. There was no fever in any of the vaccinated calves after challenge. However, the vaccinated animals exhibited mild to moderate enlargement of lymph nodes and parasitological reactions. The parasitological reactions were very mild in calves challenged after 3 months and moderate in calves challenged after 6 months. There was a mild but significant decrease in the haematological values of calves after challenge. A significant rise in the anti-theilerial antibody titres was observed in all calves after vaccination, which increased further, by many folds after challenge. On the other hand, all the challenge control calves showed symptoms of acute theileriosis and died. The observations suggested that the T. annulata (Hisar) schizont cell culture vaccine provided immunity in vaccinated animals for at least 6 months in the absence of field tick challenge. However, there was some decline in immunity after 6 months, if the animals are not exposed to ticks during this period.     

Immunization of neonatal bovines against Theileria annulata by an infection and treatment method

Sixty three cross-bred (Bos taurus X Bos indicus) 4-5-day-old calves were divided into 16 groups (A-P). Each calf in Groups A and B was given ground-up-tick supernate prepared from Theileria annulata-infected or non-infected Hyalomma anatolicum anatolicum (GUTS) equivalent to 5 ticks (50 infected acini). Groups D and E received infected GUTS equivalent to 2 ticks (20 infected acini) and Groups G and H were given infected GUTS equivalent to 1 tick (10 infected acini). Each calf in Groups J, K and L received infected GUTS equivalent to 5 infected acini (0.035 tick), and Group O was inoculated with non-infected GUTS equivalent to 5 ticks. Each calf in Groups A, D, G, J, K and O was also given a single intramuscular injection of long acting oxytetracycline, 20 mg kg-1 body weight just after inoculation of GUTS. Severe reactions resulted in the death of five of eight, three of eight, five of six, one of five and one of five calves in Groups A, D, G, J and K respectively and all of the calves in Groups B, E, H, and L. The surviving calves of Groups A, D, G, J, K and O were challenged on Day 45 post-immunization along with freshly introduced susceptible control calves of Groups C, F, I, M, N and P. All the calves of Groups A, G, J and K withstood the challenge dose; in Group D four of five and in Groups C, F, I, M, N, O, and P all the calves died of theileriosis. It is concluded that though the infection and treatment method of immunization may be used for neonatal bovines, the dose of immunogen should be based on actual counts of infected salivary acini of ticks instead of the number of ticks.     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: I. In vivo responses.

In a series of experiments, sporozoite stabilates of a Theileria lestoquardi (Lahr) and a T. annulata (Ankara) stock prepared from Hyalomma anatolicum anatolicum ticks, were used to examine the infectivity of both parasite species for sheep and cattle and to study the development of cross-immunity between these parasite species. In the first experiment sheep and cattle were inoculated with T. lestoquardi sporozoites. Surviving animals and naive sheep and cattle were, in the second experiment, inoculated with T. annulata. In the third experiment, naive sheep and sheep previously infected with T. annulata, were inoculated with T. lestoquardi. The following responses to inoculations were monitored: clinical and haematological signs of infection, appearance of parasitic stages of the parasites in lymph node biopsies and in peripheral blood and serological response to T. lestoquardi and T. annulata schizont antigens. While T. lestoquardi readily infected sheep and caused severe disease, it did not infect cattle. On the other hand, T. annulata infected both cattle and sheep. However, whereas cattle became severely affected, infected sheep showed mild clinical symptoms only and piroplasms did not develop. Despite their different behaviour in the host species examined, cross-immunity studies suggested that the parasite species are very closely related. Experiments in sheep indicated that T. lestoquardi infection protected against subsequent T. annulata infection. On the other hand, recovery from T. annulata infection did not prevent infection by sporozoites of T. lestoquardi, resulting in the establishment of schizonts and their subsequent development into piroplasms, although it protected against the major clinical effects of T. lestoquardi infection.     

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.