Cryopreservation of Leucocytozoon caulleryi Sporozoites

Leucocytozoon caulleryi sporozoites that had been stored at - 196° C or -80° C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae , which had been stored at -80° C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.     

Cryopreservation of Leucocytozoon caulleryi sporozoites

Leucocytozoon caulleryi sporozoites that had been stored at -196 degrees C or -80 degrees C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae, which had been stored at -80 degrees C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.     

Development of Second Generation Merozoites of Leucocytozoon caulleryi In Vitro

Sporozoites of Leucocytozoon caulleryi were inoculated into specific-pathogen-free (SPF) chickens intravenously. Fourteen days after inoculation, the infected blood, parasitized with second generation merozoites, was collected from the chickens. The blood was then suspended in SPF chicken serum or RPMI-1640 culture medium supplemented with 20% SPF chicken serum in petri dishes, which were cultured at 37°C or 41°C in a humidified atmosphere of 5% CO₂. Second generation merozoites in parasitized, immature erythrocytes developed continuously through several substages and finally became micro- and macro-gametocytes after 6 days of incubation.     

Observations on First-Generation Schizogony of Leucocytozoon caulleryi in Chickens

ABSTRACT. First and second generation schizogony of Leucocytozoon caulleryi occurred in chickens infected with sporozoites. First generation schizogony was studied by light and electron microscopy. First-generation schizonts were first detected in capillary endothelial cells in the spleen, lung, liver, and bursa of Fabricius between 3 and 6 d post-sporozoite inoculation (DPI). The schizonts ranged from 15 to 65 μm in diameter and were surrounded by a thin pellicle. Early schizonts contained numerous round or oval nuclei, endoplasmic reticulum, and mitochondria. The schizonts reached maturity 5 DPI and produced first-generation merozoites which were released into the peripheral bloodstream. The merozoites. which were infective to chickens, measured 7.1 μm in length. They were slender and had a large nucleus, a mitochondrion, and an apical complex consisting of three polar rings, rhoptries, numerous micronemes. The morphology of first-generation merozoites was different from that of second-generation merozoites.     

Development of second generation merozoites of Leucocytozoon caulleryi in vitro

Sporozoites of Leucocytozoon caulleryi were inoculated into specific-pathogen-free (SPF) chickens intravenously. Fourteen days after inoculation, the infected blood, parasitized with second generation merozoites, was collected from the chickens. The blood was then suspended in SPF chicken serum or RPMI-1640 culture medium supplemented with 20% SPF chicken serum in petri dishes, which were cultured at 37 degrees C or 41 degrees C in a humidified atmosphere of 5% CO2. Second generation merozoites in parasitized, immature erythrocytes developed continuously through several substages and finally became micro- and macro-gametocytes after 6 days of incubation.     

Pathogenicity of Leucocytozoon caulleryi for specific pathogen-free laying hens.

The pathogenicity of Leucocytozoon caulleryi against specific-pathogen-free laying hens was investigated. Many large schizonts (second-generation schizonts) of L. caulleryi were seen in the ovary and oviducts of chickens. Edema and pressure atrophy of the adjacent tissues were associated with these schizonts. The eggshell-secreting portion of the uterus exhibited the most severe damage in the oviduct. This experiment reconfirms that L. caulleryi may stop egg production in laying hens, presumably as a result of damage to ovaries and oviducts.     

Infectivity of Leucocytozoon caulleryi sporozoites developed in vitro and in vivo

Morii T., Matsui T., Iijima T. and Fiotnaoa F. 1984. Infectivity of Leucocytozoon caulleryi sporozoites developed in vitro and in vivo. International Journal for Parasitology14: 135–139. Infectivity of Leucocytozoon caulleryi sporozoites isolated from various sites in Culicoides arakawae and from the midguts and the salivary glands which had been cultured in vitro after the infective blood meals was studied. Sporozoites isolated from the midguts, the abdominal and thoracic hemocoel and the salivary glands of biting midges on the 2nd day after feeding did not show infectivity to any of the chickens inoculated. Sporozoites obtained from the salivary glands on the 3rd day after feeding caused infection in all the inoculated chickens. The results indicated that sporozoites which had been just released from oocysts or had just reached the salivary glands cannot induce infection in chickens. Sporozoites were produced in the midguts which had been cultured in vitro in Medium 199 or Grace's medium after the infective blood meals, but they showed lower infectivity than those isolated from the salivary glands which had been cultured by the same methods as the midgut cultivation. The development of infectivity of L. caulleryi sporozoites seems to be site-dependent rather than time-dependent. High infectivity of sporozoites develops during their residence in the salivary glands of biting midges.     

Observations on the Taiwanese Strain of Leucocytozoon caulleryi (Haemosporina) in Chickens

ABSTRACT. The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25°C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected witt the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the strain isolated in Japan.     

Detection of gametocytes in chickens recovered from natural infection with Leucocytozoon caulleryi

Gametocytes of Leucocytozoon caulleryi were found again in two chickens from the peripheral blood of which they had been detected in the previous year. It was discussed from the results of agar gel precipitation tests whether these chickens were involved in the reinfection or the relapse of this organism.     

Observations on the Taiwanese strain of Leucocytozoon caulleryi (Haemosporina) in chickens

The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25 degrees C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected with the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the the strain isolated in Japan.     

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.     

Development of Leucocytozoon caulleryi in chick embryos infected by biting of Culicoides arakawae through shell membrane.

Chick embryos were infected with Leucocytozoon caulleryi by biting of the midge, Culicoides arakawae, through the shell membrane. Schizonts of L. caulleryi were detected in the chorioallantoic membrane and most of the internal organs of embryos and of chicks after hatching. The development of schizonts was slower in embryos than in chickens. Soluble antigens of L. caulleryi were demonstrated by the precipitation test in the allantoic fluid and blood from the embryos and chicks. No erythrocytic stage of L. caulleryi, however, was observed in any embryo or chick.     

Rapid detection by counterimmunoelectrophoresis of antigens and antibodies in the sera of chickens infected with Leucocytozoon caulleryi

The method of counterimmunoelectrophoresis was used to detect rapidly soluble antigens and antibodies in the sera of chickens infected with Leucocytozoon caulleryi. This method retained the specificity revealed by the Ouchterlony gel-diffusion technique, and induced no false positive reactions. Therefore, it was applicable to the serological diagnosis of chicken leucocytozoonosis, as well as the Ouchterlony technique which is used in Japan at present.     

Gametocytogenesis of Leucocytozoon caulleryi in In Vitro Culture: Effect of Human Red Blood Cells on the Development of Second-Generation Merozoites

For investigating development of second-generation merozoites of Leucocytozoon caulleryi into mature gametocytes, infected erythrocytes from chickens at 15 d after sporozoites inoculation were cultured in RPMI-1640 modified medium supplemented with 10% horse serum and 0.5 ml of human erythrocytes (type O). When culture was carried out at 37°C in a humidified atmosphere of 5% CO₂ for 7 d, the very small number of second-generation merozoites developed into morphologically mature gametocytes. However, in the high carbon dioxide and low oxygen condition, mature gametocytes weren't observed in cluture. The role of human erythrocytes added has not been clarified yet.     

Gametocytogenesis of Leucocytozoon Smithi

ABSTRACT. Development of young gamelocytes of Leucocytozoon smithi into morphologically mature forms was studied using electron microscopy. Gametocytogenesis began on day seven post inoculation when merozoites, released from ruptured hepatic schizonts, developed into gametocytes within mononuclear phagocytes or leukocytes (monocytes or lymphocytes). No gametocytes were observed in any erythrocytes or polymorphonuclear leukocytes. Two gametocyte forms, round and elongate, were observed. Immature round gametocytes occurred on days 7-10 post inoculation in the deep vasculature of liver, lung and spleen. Mature elongate gametocytes were observed beginning on day 12 post inoculation in both the deep tissue vasculature and peripheral circulation of the turkey host. Growth and elongation of the gametocyte resulted in distortion of the host cell and its nucleus. the host cell nucleus initially was elongated and displaced to one side or indented by the growing parasite. Eventually, the nucleus was laterally compressed or split into two or three fragments. the compressed host cell cytoplasm was displaced longitudinally and stretched over the parasite to form hornlike cytoplasmic extensions from each end. the potential role of microtubules in the elongation of the gametocyte and its host cell, and possibly in the indentation and splitting of the host cell nucleus, is discussed.     

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.     

Schizogonic Development of Leucocytozoon smithi

ABSTRACT The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.