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.

Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites.

Besnoitia besnoiti is an economically important tissue cyst-forming apicomplexan of cattle in Africa and Israel. Tissue cysts and bradyzoites of B. besnoiti from the skin of a naturally infected bull were studied by transmission electron microscopy. Tissue cysts enclosed host cell and bradyzoites. Bradyzoites were 6-7.5 x 1.9-2.3 microm in size and contained organelles found in coccidian merozoites including numerous micronemes, rhoptries, amylopectin granules, and a posteriorly located nucleus. Enigmatic bodies, characteristically found in Besnoitia sp. bradyzoites, were not observed. Therefore, enigmatic bodies should be removed as a generic character of the bradyzoites of Besnoitia species.

Babesia bovis and B. bigemina: Persistence of infection in friesian cows following vaccination with live antibabesial vaccines.

The persistence of Babesia bovis and B. bigemina infection in Friesian cows, following vaccination with attenuated live vaccines, was assessed by subinoculation of blood into splenectomized calves. Subinoculation tests showed that B. bigemina persisted in two out of 20 cows vaccinated 10 and 46 months previously, and that B. bovis persisted in 11 out of 22 cows vaccinated 10 to 47 months previously. Antibody was detected in five B. bigemina - and 15B. bovis -vaccinated cows. Parasites of both species persisted among the serologically negative cows, whereas blood obtained from serologically positive cows failed to transmit infection. It is concluded that in the absence of reinfection Friesian cattle may spontaneously eliminate B. bigemina and B. bovis infection after various periods following vaccination.

Susceptibility of Psammomys obesus and Meriones tristrami to tachyzoites of Neospora caninum.

The susceptibility of Psammomys obesus (sand rat) and Meriones tristrami (Tristram's jird) to Neospora caninum was investigated by subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10-fold doses of culture-derived tachyzoites. Groups of 5 animals were inoculated with doses of 10-10(7) parasites via each route of inoculation. All but 2 of the sand rats inoculated with doses of 10-10(4) parasites succumbed to the infection by 7-18 days postinfection. All jirds inoculated with 10(7) tachyzoites succumbed by 5-16 days postinfection and those inoculated with 10(6) tachyzoites by 9-25 days. A considerable proportion of the jirds inoculated with 10-10(5) tachyzoites survived. Fibrinous peritonitis with ascites containing numerous tachyzoites was observed in the i.p.-inoculated sand rats and jirds that succumbed to the infection. In the jirds, tachyzoites were also found in pleural exudate. A considerable number (42.8%) of the jirds inoculated s.c. or i.p. exhibited neuromuscular symptoms, expressed in ataxia, head tilt, circling movement, and posterior paralysis. Seven successive passage of tachyzoites were achieved in sand rats with doses of 10(5) parasites and in jirds with doses of 10(7) parasites. All surviving jirds became seroconverted and were immune to lethal challenge.

Culture-derived parasites in vaccination of cattle against tick-borne diseases.

The major economically important tick-borne diseases of cattle are theileriosis, babesiosis, anaplasmosis, and cowdriosis. Culture-derived attenuated schizonts of Theileria annulata have proved to be safe for all types of cattle and they protect against tick-borne theileriosis. T. parva was also successfully grown in vitro; however, inoculation of cattle with allogeneic schizont-infected cells resulted in rejection and destruction of the parasites together with the host cells. The number of schizont-infected cells needed for immunization is greater than for T. annulata theileriosis. Culture-propagated Babesia bovis and B. bigemina were used for large scale vaccination in the field. An avirulent population of Babesia spp. was obtained by in vitro cloning; inoculation of cattle did not induce clinical babesiosis, but produced specific antibodies. Culture-derived exoantigens of Babesia spp. proved to be completely safe for cattle, however, they conferred less protection than live parasites. Cell-cultured Cowdria ruminantium was highly infective for susceptible animals but, attenuated in vitro, could offer a potential source for vaccination. Anaplasma marginale, successfully grown in tick cell culture, may be developed for vaccines. Factors that should be considered in the developing of vaccines against tick-borne diseases include: the protective immune response to the pathogenic parasite developmental stages, virulence, immunological strain differences, and antigenic variations in cattle and in culture.

Vaccination against tropical theileriosis.

Theileria annulata, the cause of tropical theileriosis is propagated in cattle with stage-to-stage transmission by Hyalomma ticks. Three stages in the life cycle of the parasite--tick-derived sporozoites, intramononuclear schizonts, and erythrocytic merozoites--infect cattle. When cattle are inoculated with schizont-infected cells, the parasite is transferred from the donor cell to the recipient. The main pathological damage in cattle is induced by the schizont stage. Each development stage of T. annulata elicits a specific immune response. Schizont-infected lymphoid cells can be grown indefinitely in culture and prolonged cultivation results in loss of virulence. Blood-derived schizonts induce stronger immunity than culture-derived schizonts, which suggests that restrictions on the parasite population or antigenic variation occur during prolonged cultivation. The duration of immunity following sporozoite or schizont infections has not yet been determined, but does not appear to be lifelong. The attenuated, culture-derived anti-theileria vaccine proved to be safe and effective in prevention of field theileriosis in large enzootic areas.

Seroprevalence of Babesia equi among horses in Israel using competitive inhibition ELISA and IFA assays.

Sera from 361 horses were tested by indirect immunofluorescence antibody test (IFA) and by competitive inhibition ELISA (cELISA), to detect antibodies to Babesia equi. The concordance between the assays was 95.7%. Application of a cutoff based on a calculated percent inhibition of < 20% gave a total of 22 discrepant results, while only 8 sera negative by the cELISA were found positive by the IFA when a cutoff of > 20% inhibition was used. Approximately one-third of all the horses tested were found serologically positive to B. equi, with more horses testing positive from northern Israel. Among horses raised with access to pasture there was a significant difference in the percentage of seropositive reactors (76.6% in the north and 20.1% in the central region), compared with horses without access to pasture (14.3 and 10.3%, respectively). Nineteen percent of stallions were found to be positive, which was significantly less than the proportions of seropositive mares and geldings: 38 and 42%, respectively. No significant association was found between the mean age of horses and seroreactivity to B. equi.

Phenotypic and genotypic alterations associated with the attenuation of a Theileria annulata vaccine cell line from Turkey.

Attenuated vaccines, produced by prolonged in vitro culture of the macroschizont stage of the life-cycle, are the main method of controlling Theileria annulata infections. Little is known about the mechanism(s) of attenuation. Here we present data from a Turkish cell line demonstrating that attenuation is associated with reduced ability to differentiate into microschizonts and a reduction in matrix metalloproteinase activity. We also show that attenuation results in a change in the structure of the parasite population. Using the technique of differential mRNA display, we demonstrate that gene expression profiles differ between non-attenuated and attenuated macroschizont infected leucocytes. One differentially expressed gene is of parasite origin. These data are discussed in the context of a multifactorial model for virulence.

Antibody response to Hepatozoon canis in experimentally infected dogs.

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon canis. Five puppies were inoculated by ingestion of Rhipicephalus sanguineus ticks experimentally infected with H. canis, and all became infected with H. canis: gametocytes were detected in blood smears from four dogs and schizonts were observed in the spleen and bone marrow of the fifth. Antibodies reactive with H. canis gametocytes were detected by the indirect fluorescent antibody test (IFA), with IgM detected initially in all dogs 16 to 39 days post infection (PI) and IgG 22 to 43 days PI. The presence of gametocytes was first observed within peripheral blood neutrophils in Giemsa-stained blood smears between days 28 and 43 PI. Gametocyte-reactive antibodies were detected before the appearance of blood gametocytes in three of the four parasitemic dogs and also in a dog with no observed parasitemia. The detection of serum antibodies prior to the detection of blood gametocytes, or without apparent parasitemia, suggests that antibodies reactive with gametocytes may be formed against earlier forms of the parasite developing in the parenchymal tissues. Sera of dogs experimentally infected with Babesia canis, Babesia gibsoni and Ehrlichia canis exhibited no reactivity when tested with H. canis antigen. Additionally, sera positive for H. canis were not reactive with antigens of Toxoplasma gondii, Neospora caninum, Leishmania donovani and E. canis. In conclusion, incoculation of dogs with ticks infected with H. canis results in production of antibodies reactive with peripheral blood gametocytes. Detection of IgG titres would be beneficial for the diagnosis of progressive infections with undetectable parasitemia, for seroprevalence studies, and as an adjunct to IgM titres in early infections.

Vaccines against hemoparasitic diseases in Israel with special reference to quality assurance.

Four vaccines against hemoparasitic diseases (anaplasmosis, babesiosis and theileriosis) and a vaccine against besnoitiosis are currently used in Israel. These vaccines contain live attenuated parasites derived from cell culture (Theileria annulata and Besnoitia besnoiti) or from blood of infected, splenectomized calves (Babesia bigemina, B. bovis and Anaplasma centrale). Cryopreserved master seed is used to initiate production of the vaccines. Quality control performed during the preproduction period is particularly important with blood-derived vaccines. Post production quality control comprises sterility, potency (viability of immunizing organisms), safety (degree of attenuation) and efficacy (capacity to protect against virulent parasite stock). All vaccines are stored and dispatched to the field in a concentrated frozen state. The culture-derived vaccines are safe for all varieties of cattle, regardless of age or physiological condition, whereas the blood-derived vaccines are recommended mainly for young cattle, the age limit varying with the type of vaccine and breed of cattle. The viability of T. annulata infected cells in the anti-theilerial vaccine is tested after thawing by in vitro plating efficiency and the infectivity of blood-derived vaccines is tested by titration in susceptible cattle.

Theileria annulata: in vitro cultivation of schizont-infected bovine lymphocytes.

Experimental parameters of optimization of the in vitro growth conditions for Theileria annulata schizont-infected bovine lymphoid cells are presented. Of the nine different media tested in the course of 14 successive passages, Leibovitz L-15 (L-15) and a combination of McCoy and Leibovitz L-15 (ML) were preferable to McCoy, Dulbecco (DMEM), RPMI 1640 or Eagles's minimum essential medium (MEM) based on either Hank's or Earle's salts. The lowest multiplication rate was obtained with M-199 medium based on Hank's or Earle's salts. The highest yield of cells was obtained when L-15 was supplemented with 20% newborn bovine serum, while lowering the serum concentration by half resulted in a 25% decrease in cell yield. There was no effect on multiplication rate observed during ten passages when 2-mercaptoethanol and a mixture of oxaloacetate, sodium pyruvate and insulin were added to the growth medium. On substitution of conditioned medium for 20 or 50% of the growth medium, the yield of cells decreased by 12 or 20%, respectively, a factor which might be considered in calculations of the cost of anti-T. annulata vaccine production. When cells were grown in stationary cultures in Roux bottles for 7 days without change of medium, the highest yield resulted from seeding at 10(7) cells per vessel (100 ml) in L-15 supplemented with 20% serum. In Roller bottles, with the same type and volume of medium and cultivation for 7 days without medium change, best yield resulted from seeding with the highest inoculum size of 4 x 10(7) cells per vessel.

Hepatozoon canis: the prevalence of antibodies and gametocytes in dogs in Israel.

A survey for the prevalence of antibodies to Hepatozoon canis and for intraneutrophilic H. canis gametocytes in the peripheral blood neutrophils of dogs in Israel showed that 33.1% were seropositive, while only 1% of the dogs sampled had detectable parasites in their blood smears. Exposure to H. canis is widespread but it appears that most infected dogs undergo a subclinical infection and only a small proportion develop clinical disease.

Live vaccines against hemoparasitic diseases in livestock.

Live vaccines against hemoparasitic diseases in livestock are based on parasites derived from culture (Theileria annulata), from blood of infected animals (Babesia bovis, Babesia bigemina, Anaplasma centrale, (attenuated) Anaplasma marginale and Cowdria ruminantium), and from ticks (Theileria parva). The T. annulata attenuated cultured schizont vaccine is safe for all varieties of cattle. Blood derived vaccines are recommended mainly for young cattle, the age limit varying with the different vaccines and breeds of cattle. In older animals, monitoring of the individual response is needed. Immunization against T. parva requires simultaneous or postinoculation chemotherapy. The potential for accidental transmission of disease agents exists with all blood derived vaccines. Various degrees of resistance to field infection have been reported in animals immunized with live vaccines. Nevertheless, all of them engender a level of protection against natural challenge that justifies their use in field vaccination. Chemotherapy or chemoprophylaxis may prevent establishment of infection with the vaccinal parasites, and thus may interfere with elaboration of immunity. Outbreaks of disease in vaccinated herds, caused by antigenic variants among the tick-transmitted parasites, have been observed mainly in Babesia infections. In recent years, the main efforts towards improvement of live vaccines have been in the direction of replacing blood- and tick-derived parasites by those cultured in vitro under controlled standardized conditions.

Activity of a monoclonal antibody against Besnoitia besnoiti endozoites.

Besnoitia besnoiti multiplication in vitro was inhibited by a specific organelle complex-directed monoclonal antibody (MoAb) raised against the endozoite stage. Multiplication rates in antibody-treated cultures were lower at parasite/cell ratios of 1:1, 1:2 and 1:10, than in untreated cultures, after 4 or 8 d of incubation. At 1:100 ratio, there was no difference between test and control cultures irrespective of the incubation period. In in vivo experiments, the anti-B besnoiti MoAb had no neutralizing effect on the infectivity of endozoites. Inoculation of gerbils with antibody-preincubated endozoites, followed by treatment with these MoAb through 5 successive days ultimately failed to prevent death in any of them. In Western blots the specific anti-B besnoiti MoAb of the IgG1 subclass recognized a 70 kDa endozoite protein in the cytosolic and the insoluble membrane fraction of endozoites.

Differentiating between Besnoitia besnoiti from cattle and Sarcocystis hoarensis from rodents.

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogen Besnoitia besnoiti, we compared salient features of this pathogen in cattle with those of Sarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast to S. hoarensis, which is well adapted to rodents, B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens. B. besnoiti, therefore, can unambiguously be differentiated from S. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.

Bovine besnoitiosis: transfer of colostral antibodies with observations possibly relating to natural transmission of the infection.

Colostral antibodies to B. besnoiti were detected by immunofluorescence in four calves born to two Besnoitia-infected dams, with titres ranging from 1:64 to 1:1024. A specific antibody titre of 1:1024 was found in colostrum collected from one of the dams. Two of the newborn calves, when sampled immediately after birth, were serologically negative to B. besnoiti, but became positive on the next day. In all the calves, antibodies were detectable up to the age of 4 months. Observations concerning passive transfer of antibodies from Besnoitia-infected dams to offspring, and transmission of the infection among infected and non-infected closely kept cows, are discussed.

Cross-protective immunity induced by Babesia bovis clones with antigenically unrelated variable merozoite surface antigens.

Babesia bovis merozoite surface antigen 1 (MSA-1) induces antibodies capable of neutralizing merozoites in vitro. Both MSA-1 and the co-expressed MSA-2 are encoded by a polymorphic multigene family and are antigenically variant among strains isolated from widely separated geographic regions. In this study, cross-protective immunity between two B. bovis clones, Mexico Mo-7 and Israel-C, that have antigenically unrelated MSA-1 and MSA-2 surface proteins was assessed. Cattle immunized by infection with either clone were significantly protected against challenge with the uncloned Israel Bbv strain. This indicates that epitopes capable of inducing partial protection are shared among different strains and that immunity is not solely dependent upon MSA-1 or MSA-2. However, cattle immunized with the Israel-C clone, derived from the Israel Bbv strain, were significantly better protected against BbV challenge than were cattle immunized with the Mexico Mo-7 clone bearing antigenically unrelated MSA-1 and MSA-2. The significant difference in immunity induced by the homologous strain versus an antigenically variant strain indicates that epitope variation among strains is relevant to immunity against babesiosis.

The route of immunization of gerbils with live Besnoitia besnoiti as a factor in protection against lethal challenge.

Gerbils (Meriones tristrami) were infected subcutaneously or intraperitoneally with live culture-grown Besnoitia besnoiti at doses ranging from 10(1) to 10(7) endozoites. All animals injected subcutaneously survived the infection and were refractory to a lethal challenge dose of 10(7) endozoites given intraperitoneally 6 weeks later. By contrast, gerbils surviving primary intraperitoneal inoculation with 10(2) to 10(6) endozoites showed a variable survival rate to challenge. All gerbils developed antibodies to B. besnoiti regardless of the route of inoculation, except for those given 10(1) endozoites intraperitoneally. There was no statistical difference between the immunofluorescent antibody titres developed in groups vaccinated subcutaneously or intraperitoneally (P = 0.556).

Intrastadial and interstadial transmission of Anaplasma marginale by Boophilus annulatus ticks in cattle.

The 1-host tick Boophilus annulatus was found to transmit anaplasmosis in cattle transstadially. Anaplasma marginale was invariably transmitted when ticks that had been pulled off Anaplasma-infected calves either after 7 days (as fully engorged larvae) or after 14 to 15 days (as fully engorged nymphs) were transferred within 4 days to susceptible calves. Three morphologically different A marginale isolates, 1 round (tailless) and 2 with different types of appendages (tailed) were transmitted by the ticks. These findings might explain the overlap of the geographic distribution of the disease and that of Boophilus spp in some areas of the world.