E. Pipano

Recognition of conserved surface protein epitopes on Anaplasma centrale and Anaplasma marginale isolates from Israel, Kenya and the United States

Palmer G. H., Barbet A. F., Musoke A. J., Katende J. M., Rurangirwa F., Shkap V., Pipano E., Davis W. C. and Mcguire T. C. 1988. Recognition of conserved surface protein epitopes on Anaplasma centrale and Anaplasma marginale isolates from Israel, Kenya and the United States. International Journal for Parasitology18: 33–38. Field isolates of A. marginale from Israel, Kenya and the United States and Anaplasma centrale were shown to be antigenically distinct using a panel of 1:1 isolaterestricted monoclonal antibodies. One monoclonal antibody reacted with all A. marginale isolates but not A. centrale and may be species-specific. A second panel of seven antibodies detected epitopes common to all isolates and both species. These common epitopes were present on two major Anaplasma surface proteins of Mr 36,000 and Mr 105,000. Monoclonal antibodies capable of neutralizing heterologous A. marginale isolate infectivity recognized 100% of the organisms in each isolate including A. centrale. In addition, immune sera from cattle recovered from infection with each of the isolates contained high titer antibodies to the isolated surface proteins bearing the common epitopes. These results demonstrate that antigenically distinct Anaplasma isolates and species contain common surface epitopes that may be important in developing effective immunoprophylaxis.

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.

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.

Immunological relationship between Neospora caninum and Besnoitia besnoiti

Neospora caninum is a coccidian parasite identified as a major cause of abortion in cattle. A combined infection of N. caninum with another taxonomically related parasite of cattle, Besnoitia besnoiti can occur in geographical areas endemic for both species. Both infections are routinely diagnosed serologically, and incorrect diagnosis could occur if immunological cross-reactivity exists between the two parasites. To investigate the possible degree of cross-reactivity, we compared results obtained with two serological techniques, immunofluorescent antibody test (IFA), and Western blot analysis on known positive and negative sera. The test sera were derived from naturally infected cattle and from experimentally infected Mongolian gerbils. In IFA of bovine sera, no cross-reactvity was detected at the commonly used serum dilution cutoffs of 1:200 for N. caninum and 1:256 for B. besnoiti. However, at 1:64 dilution of both cattle and gerbil sera, anti-N. caninum sera reacted with B. besnoiti antigen in some individual samples. Anti-B. besnoiti serum did not react with N. caninum antigen at any dilution. This low level one directional cross-reactivity was confirmed by Western blot analysis. B. besnoiti antigen showed two immunoreactive bands when probed with anti-N. caninum serum, while no bands appeared when N. caninum antigen was probed with B. besnoiti antiserum. Immunization and challenge experiments in the highly susceptible Mongolian gerbil (Meriones unguiculatus) showed essentially no cross-protection between N. caninum and B. besnoiti.

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.

Circulating Antibodies to Hepatozoon Canis Demonstrated by Immunofluorescence

Hepatozoon canis is a protozoan parasite of dogs that has a worldwide distribution. Dogs become infected by ingesting the infected tick vector, Rhipicephalus sanguineus. Natural clinical infections range in severity from asymptomatic to fatal. 6,14 The life cycle in the canine host includes the development of macroand microschizonts in various tissues, with gamonts appearing in the peripheral blood. Diagnosis is made by detecting the appropriate stages in blood smears, tissue impressions, or sections from biopsy materials. Humoral antibody maybe stimulated by H. canis infection, but a serologic test has not been developed because of the unavailability of a satisfactory antigen. This paper describes the detection of antibodies in infected dogs by the indirect fluorescent antibody (IFA) test. Results of a limited survey indicate that this technique may be of diagnostic value in canine hepatozoonosis. Blood from a dog heavily infected with H. canis was drawn into 0.4% sodium citrate, The titrated blood was centrifuged at 600 x g for 10 minutes, and the plasma and buffy coat were removed. The erythrocyte pellet was discarded. The plasma and buffy coat were recentrifuged, and the buffy coat was removed and washed 3 times with phosphate-buffered saline (PBS), (pH 7.2) by successive 10-minute centrifugations at 700 x g. The plasma was saved for use as a possible positive serum control in the IFA test. The final pellet was resuspended in a mixture of equal volumes of PBS and 3% bovine serum albumin (BSA), and thin blood smears were prepared on glass slides. After drying at room temperature, the slides were immersed for 10 minutes in acetone, or 80% acetone/PBS containing 1% formalin, or methanol. The slides were wrapped individually in filter paper and stored at -80 C. Some of the slides were wrapped and frozen before fixation. Twenty-nine sera were tested: 12 were from healthy, tickfree, laboratory colony-bred dogs in which H. canis was not detected in Giemsa-stained blood smears; 10 were from dogs showing gametocytes in blood smears; and 7 were from dogs negative for gametocytes at the time of blood sampling but with a confirmed history of H. canis infection up to 4 months previously. Antigen slides were removed from the freezer and warmed to 37 C in a container with silica gel for about 30 minutes. Dry slides that were not fixed before freezing were immersed in 1 of the 3 fixatives for 10 minutes. Circles were marked on the smears with cosmetic nail polish to prevent intermixing of serum dilutions. Two-fold dilutions of serum in PBS, from 1:16 to 1:1,024, were applied to the smears, and

Cultivation of Besnoitia besnoiti and evaluation of susceptibility of laboratory animals to cultured parasites

Cultivation of Besnoitia besnoiti on five mammalian cell types showed that Vero (green monkey kidney), L929 (mouse fibroblasts) and BEK (bovine embryo kidney) cells were highly susceptible and were almost destroyed after 5 days of incubation, while MDBK (Madin-Darby bovine kidney) and BL (Theileria annulata-infected bovine lymphoid) cells were less affected. Intraperitoneal infection with culture-derived endozoites was fatal for gerbils (Meriones tristrami) and sand rats (Psammomys obesus). Rabbits showed fever, conjunctivitis and orchitis, but survived the infection. Mice, rats, guinea pigs, hamsters and Microtus guentheri did not show signs of illness, but developed specific antibodies to B. besnoiti. B. besnoiti subcultivated for 2 years in BL cells remained virulent for gerbils.

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

Abstract: 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.

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.

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.