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.     

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.     

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.     

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.     

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.     

Subgroup-A Rous sarcoma virus-induced growth stimulation of chick embryos infected via the chorioallantoic membrane.

Chicks that hatch from eggs containing group specific antigen (gs antigen) of lymphoid leukosis virus (LLV) subgroups, grow poorly. In our laboratory for more precise identification of LLV-of subgroup A (LLV-A) resistant and susceptible genotypes by progeny testing, the chorioallantoic membrane (CAM) assay in complemented by liver tumour (LT) assay, wherein Rous sarcoma virus (RSV) of subgroup A (homologous to LLV-A) was used. The present study was conducted in a light breed (White Leghorn) and also in a heavy breed (Rhode Island Red) to ascertain the effect of infection on embryonic growth by RSV subgroup A. Mean relative body weight (rbw) of infected LT negative chicks of either breed exceeded the control highly significantly (P < 0.01) by 2%. However, neither the dose of virus inoculated per embryo, nor egg size influenced the relative body weight of day old chicks (P > 0.05). No difference in relative body weight of LT positive and control chicks was observed.     

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.     

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.     

Hepatotropism of Tyzzer bacteria in experimentally infected chick embryos]

Tyzzer's organism from mice propagated more remarkably in hepatocytes than in yolk-sac epithelial cells producing confluent necrosis of the liver after intravenous as well as intravitelline inoculation. Some lesions with bacterial growth were also produced in the heart muscles and kidneys. The organisms from mouse, rat, hamster and kitten were shown to be equally pathogenic for chick embryos.     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.