Immunological relationship between strains of Theileria annulata Dschunkowsky and Luhs 1904

The immunological relationship between the Ludhiana and Hissar strains, and between the Uruli-Kanchan, Bangalore and Jaipur strains of Theileria annulata was studied. Calves were immunised against the Ludhiana strain by the infection-treatment method, against the Uruli-Kanchan strain by the infection-treatment method or by untreated low-grade infection and against the Jaipur strain by untreated low-grade infection. After 45 days, groups of immune calves and susceptible controls were challenged with 10-tick equivalent stabilates. The ensuing host responses, body temperature, swelling of the regional lymph node, appearance of schizonts in the regional lymph node and piroplasms in blood and mortality, were studied. All five strains proved very virulent and produced severe disease in susceptible calves. Immunisation with the Ludhiana, Uruli-Kanchan and Jaipur strains conferred absolute protection against severe homologous challenges. Immunisation with the Ludhiana and Uruli-Kanchan strains conferred substantial protection against heterologous challenges with the Hissar strain, and the Bangalore and Jaipur strains, respectively. Immunisation with the Uruli-Kanchan strain gave less protection against heterologous challenge with the Jaipur strain and vice versa as some of the challenged calves showed slight swelling of the regional lymph node, two had fever, all exhibited a low-grade parasitaemia and four developed anaemia.     

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.     

The epidemiology of tropical theileriosis (Theileria annulata infection in cattle) in an endemic area of Morocco.

A longitudinal study of tropical theileriosis was performed on 12 farms in the Doukkala region of Morocco during 1990. Adult Hyalomma detritum detritum were collected between March and early October and a peak in numbers was observed at the end of June. Nineteen percent (24/127) were infected with Theileria species and, amongst these, over 50% had five or more sporoblasts in their salivary glands (range 1-151). Hyalomma d detritum larvae and nymphs were found on cattle between September and early December with the highest numbers in late October. The prevalence of T. annulata piroplasm carriers at the beginning of the year was 48.5% (47 positive out of 97) and there were 14 new infections during the disease season (March to September) of which five developed into clinical cases. The incidence rates of new infection and clinical disease were 0.156 and 0.056 per animal-season, respectively. Differences were observed between age categories of cattle in both tick and parasite infections. A significantly lower number of adult H.d. detritum were collected from calves than from adult cattle. The prevalences of piroplasm carriers before the disease season were 0%, 36% and 76%, respectively, in (a) calves which had been born since the previous disease season, (b) calves born before then and (c) adults. However, the incidence rates of infection and disease for uninfected animals in the two categories of calves were approximately the same: 0.299 and 0.378 new infections, and 0.085 and 0.126 clinical cases per animal-season for (a) and (b), respectively. The date predicted for the appearance of adult H.d. detritum, based on published tick development times and local temperature records, was within 2 weeks of the study visit when the highest number of adults were collected from cattle. However, the date predicted for the appearance of larvae was 6 weeks earlier than the observed peak populations and may indicate that H.d. detritum delays either egg laying in the summer or larval host searching in the autumn.     

Relative infectivity of Theileria annulata (Dchunkovsky and Luhs, 1904) stabilates derived from female and male Hyalomma ecavatum (Koch, 1844) ticks

Partially engorged female and male Hyalomma excavatum ticks infected with Theileria annulata were triturated separately in Eagle's minimum essential medium complemented with fetal calf serum. Glycerol was added to the supernatant obtained after centrifugation and the material was frozen at—79°C. Infectivity of the stabilate was titrated by inoculating susceptible calves with dilutions of the material.Stabilate prepared from female ticks was considerably more infective on a per tick basis than stabilate from males. All calves inoculated with an estimated equivalent of 0.1 or more female ticks, or 3.3 or more male ticks, became infected.Time to fever, prepatent period, and mortality did not appear to be dose-dependent in the range of tick equivalents employed.     

Immunization of cattle against East Coast fever using Theileria parva (Marikebuni) and relaxation of tick control in North Rift, Kenya.

A total of 90 animals was immunized against East Coast fever (ECF) using Theileria parva (Marikebuni) stock on three large-scale farms in Kiminini Division, Trans-Nzoia District, North Rift, Kenya. Another 90 cattle served as non-immunized controls. Following immunization the number of cattle with significant indirect fluorescent antibody (IFA) titres increased from 43.9% to 84.4% and 6.7% of the cattle developed clinical ECF reactions. Two months after immunization, the immunized and non-immunized cattle were divided into two groups one of which was dipped every 3 weeks and the other dipped when total full body tick counts reached 100. All the animals were monitored for 51 weeks for incidences of ECF and other tick-borne diseases. Twenty-four cases of ECF were diagnosed among the non-immunized cattle compared to four cases among the immunized cattle; a difference that was significant (P > 0.05). There was no significant difference in the incidences of babesiosis and anaplasmosis between the immunized and non-immunized cattle.     

Invasion and intracellular development of Theileria annulata sporozoites in lymphoblastoid cell lines already transformed by T. annulata (Hissar), T. annulata (Ankara) and T. parva (Muguga)

Interactions between Theileria annulata sporozoites and lymphoblastoid cell lines already transformed by the Hissar and Ankara strains of T. annulata [T. a. (H) and T.A. (A), respectively] and the Muguga strain of T. parva [T.P. (M)] were studied in vitro. Although sporozoites of the Hissar strain of T. annulata attached to and entered peripheral blood lymphocytes (PBL) and lymphoblastoid cell lines transformed by T. a. (H) and T. a. (A), they neither attached to nor entered the T. p. (M) cell line. Whether the superinfecting T. a. (H) sporozoites developed intracellularly was studied by monitoring daily changes of mean schizont nuclear numbers and by determining electrophoretic mobilities of schizont glucose phosphate isomerase in each cell line using thin-layer starch gel electrophoresis. While the mean schizont nuclear number in freshly-infected PBL underwent a steady increase to the level of those in long standing T. annulata cultures, analysis of variance of similar data in T. a. (H) and T. a. (A) cell lines in which superinfection was demonstrated revealed no significant differences between them and their respective control counterparts, i.e., T. a. (H) and T. a. (A) cultures with no superinfection. Enzyme polymorphism studies showed the formation of uncontaminated species- or strain-specific bands of glucose phosphate isomerase (GPI) isoenzyme activity in the T. p. (M) and in the superinfected T. annulata cell lines.     

Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.     

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。     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

Immunization of cattle with a Theileria parva bovis stock from Zimbabwe protects against challenge with virulent T.p. parva and T.p. lawrencei stocks from Kenya

Following inoculation of 34 Bos indicus (Boran) cattle with a Theileria parva bovis (Boleni) stock from Zimbabwe, 18 animals underwent mild theilerial reactions, 12 underwent moderate reactions, three suffered severe reactions and one died. When these animals were subsequently challenged with different virulent stocks of either T.p. parva (Muguga, Marikebuni or Mariakani) or T.p. lawrencei (Ngong 1 or Nanyuki) from Kenya, all except two animals resisted challenge. The two reactors were part of the group challenged with the T.p. parva (Mariakani) stock. All 12 susceptible control animals underwent severe reactions and 11 died. The results of these experiments suggest that T.p. bovis (Boleni) may be used in some situations to immunize cattle against East Coast fever without the need to provide concomitant chemotherapy as in the infection and treatment method of immunization.     

Induction of immunity against infection with Theileria parva (East Coast fever) in cattle using plasma membranes from parasitized lymphoblasts

Protection against challenge with Theileria parva was conferred on three of four calves given three or four inocula of plasma membranes prepared from 6 to 12 X 10(8) autologous parasitized lymphoblasts from cultured cell lines. In contrast, calves remained susceptible to infection following immunization with membranes prepared from allogeneic parasitized lymphoblasts. Similarly, calves vaccinated with either gamma-irradiated autologous or allogeneic infected cells also died of East Coast fever after challenge. The results raise the possibility of vaccination against T. parva using subcellular material from infected lymphoblasts.     

Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria

Using qPCR, standard PCR and/or sequencing, we investigated the presence of tick-associated microorganisms in ticks and blood from sheep and goats from Souk Ahras, Algeria.Borrelia theileri, was detected in (7/120, 5.8%) blood from sheep and (13/120, 10.8%) goats. Anaplasma ovis was screened in (38/73, 52%) Rhipicephalus bursa and (5/22, 22.7%) R. turanicus and in (74/120, 61.7%), (65/120, 54.2%) blood of sheep and goats respectively.Coxiella burnetii tested positive in R. bursa (4/73, 5.5%) and (7/120, 5.8%) blood of sheep and (2/120, 1.7%) goats. Theileria ovis was detected in (50/147, 34%) R. bursa and (3/22, 13.6%) R. turanicus and in (64/120, 53.3%) blood of sheep and (25/120, 20.8%) goats. Babesia ovis was screened positive in (23/147, 15.6%) R. bursa and (7/48, 14.6%) R. turanicus.Our findings expand knowledge about the repertoire of tick-borne microorganisms present in ectoparasites and/or the blood of small ruminants in Algeria.     

Immunization of cattle against hypodermatosis (Hypoderma lineatum (deVill.) and H. bovis (L.)) using H. lineatum antigens

The feasibility of immunizing calves against Hypoderma lineatum and H. bovis with a crude H. lineatum larval extract or culture-derived antigen was investigated. Larval survival was followed through pupation. Grub appearance in the backs of both of the immunized groups was found to be 50% of that in the control groups. First appearance of H. bovis was significantly retarded in both immunized groups; however, H. lineatum was unaffected. Duration of H. bovis dropping was also shorter in animals receiving crude extract than in controls while duration of dropping of H. lineatum was not influenced by immunization. Survival of H. lineatum larvae to pupation was significantly reduced in animals immunized with crude larval extract, but not in those receiving culture-derived antigen. H. bovis survival was reduced by both treatments.     

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.     

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.     

Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer).

Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10(-1.0) dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-1 body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg-1 bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10(-2.0) dilution, buparvaquone treatment at 5 and 2.5 mg kg-1 bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10(-1.2), 10(-1.4) and 10(-1.6) dilutions of stabilate and were treated with 2.5 mg kg-1 bwt of buparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg-1 bwt and 10(-1.6) dilution of the stabilate would have to be carried out before such a system could be used in the field.