Cytochemical localization of acid mucosubstances within intestinal tissue stages of Eimeria acervulina and E. necatrix

Synopsis The distribution of acid mucopolysaccharides in the intestinal tissue stages ofEimeria acervulina andE. necatrix has been investigated. The results show that acid mucopolysaccharides are present in the asexual stages of both species and in the sexual stages ofE. acervulina. It is suggested that an esterified hyaluronic acid-like molecule is present in the cytoplasm of schizonts, merozoites, microgametocytes, macrogametocytes and oocysts as well as in the plastic granules of macrogametocytes and the oocyst wall. Sulphated mucopolysaccharides of the chondroitin type occur as additional components in the cytoplasm of the above stages, as well as in large amounts in the perinuclear area of macrogametocytes and oocysts. The functional significance of acid mucopolysaccharides in the coccidial stages is discussed.     

The Migration of Eimeria acervulina Sporozoites to the Duodenal Glands of Lieberkühn

SYNOPSIS. The establishment of Eimeria acervulina sporozoites in the duodenal glands of Lieberkühn of the chicken is described. Sporozoites were found to enter the tips of the villi and pass into the lamina propria, or core, of the villus. Within the lamina propria, sporozoites were engulfed by macrophages and taken to the glandular epithelium.
Data are presented which indicate that macrophages serve as a defense against infection as well as a mode of transportation.     

Sporozoites of mammalian malaria: attachment to, interiorization and fate within macrophages

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozites into the macrohages does occur. Antibodies present in the serum of sporozoite-immunized mice are important in determining the fate of both the intracellular sporozoites and the macrophages containing the parasite. Sporozoites coated with antibodies degenerate within vacuoles of the macrophages, which have no morphologic alteration. Sporozoites incubated in normal serum do not degenerate within macrophages, but the parasitized macrophages become morphologically altered and are destroyed. Preliminary experiments indicate that sporozoites appear to interact with rat Kupffer cells in the same way as with the peritoneal mouse macrophages. It is postulated that Kupffer cells play a dual role in sporozoite-host cell interaction. In normal animals these cells might serve to localize the sporozoites in the immediate vicinity of the hepatocytes. In the immunized animals, macrophages would remove and destroy the antibody-coated parasites, thus contributing to sporozoite-induced resistance.     

Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants

Avian coccidiosis is an intestinal disease caused by protozoa of the genus Eimeria. To investigate the potential of recombinant protein vaccines to control coccidiosis, we cloned 2 Eimeria sp. genes (EtMIC2 and 3-1E), expressed and purified their encoded proteins, and determined the efficacy of in ovo immunization to protect against Eimeria infections. Immunogen-specific serum antibody titers, parasite fecal shedding, and body weight gains were measured as parameters of disease. When administered alone, the recombinant EtMIC2 gene product induced significantly higher antibody responses, lower oocyst fecal shedding, and increased weight gains compared with nonvaccinated controls following infection with E. tenella. Combined embryo immunization with the EtMIC2 protein plus chicken cytokine or chemokine genes demonstrated that all 3 parameters of vaccination were improved compared with those of EtMIC2 alone. In particular, covaccination with EtMIC2 plus interleukin (IL)-8, IL-16, transforming growth factor-beta4, or lymphotactin significantly decreased oocyst shedding and improved weight gains beyond those achieved by EtMIC2 alone. Finally, individual vaccination with either EtMIC2 or 3-1E stimulated protection against infection by the heterologous parasite E. acervulina. Taken together, these results indicate that in ovo vaccination with the EtMIC2 protein plus cytokine/chemokine genes may be an effective method to control coccidiosis.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

Characterization of recombinant scFv antibody reactive with an apical antigen of Eimeria acervulina

The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding V _H and V _L genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l^–1 and exhibited virtually identical antigen reactivity as the original mAb.     

Theophylline effects on normal uveal melanocytes in culture: an ultrastructural study

Theophylline enhances maturation and differentiation of uveal melanocytes. By electron microscopy, we showed that theophylline changes small, dendritic melanocytes into large, platelike cells; it also enhances DOPA reaction as evidenced by increased deposition of DOPA reaction products in dilated cisternae and vesicles around the Golgi region. The effect is partially reversible in choroidal melanocytes but irreversible in iridial cells. It appears that theophylline, in addition to inducing tyrosine activity, accelerates the maturation and/or aging that normally occurs in cultured melanocytes when incubation is prolonged.     

Genetic variability of resistance to Eimeria acervulina and E. tenella in chickens

Summary The genetic variability of 18 sire families of the Athens-Canadian randombred population infected with coccidiosis was assessed by examining the response variables of weight gain, packed red blood cell volume, mortality and coccidial lesions. A significant gain and PCV depression and high lesion scores for Eimeria tenella and E. acervulina were produced in the infected group compared to the noninfected group. Significant variation among the sire families was observed for all of the response variables except E. acervulina lesions and a significant sex x sire interaction was observed for weight gain. The heritability (h²) estimates for the response variables revealed that resistance to coccidiosis in chickens is moderately heritable. The h² estimates for gain and PCV increased with the coccidial infections indicating that maximum progress in selecting for resistance should be made when the population was exposed to coccidial infection. Gain was positively correlated to the other measures of resistance and thus selecting for coccidial resistance should not reduce growth rates. PCV was similarly correlated but had higher positive correlation with E. tenella lesion. Percent mortality which is the selection parameter in most coccidial selection programs was correlated with resistance to coccidiosis. The phenotypic and genotypic correlations demonstrated that chickens susceptible to E. tenella were also susceptible to E. acervulina. Total lesion scores were moderately to highly correlated with the other variables and would be a suitable variable to use in coccidiosis experimentation including a genetic selection program for resistance. This study shows that progress could be made in selecting for resistance to coccidiosis in chickens using one or a combination of these response variables.     

Eimeria acervulina, E. brunetti, and E. maxima: pathogenic effects of single or mixed infections with low doses of oocysts in chickens.

The pathogenic effects of E. acervulina, E. brunetti, and E. maxima were modified when chickens received mixed infections with these species.Six-week-old chickens were inoculated with doses of 20,000 oocysts of E. acervulina, 1250 oocysts of E. brunetti, and 5000 oocysts of E. maxima given as a single or mixed infection.Typical signs of coccidiosis were apparent in chickens infected with a single Eimeria sp. When birds were given infections composed of two species, the weight loss was greater than that due to either given alone but when three species were given, weight loss was slightly less than that due to infection will E. brunetti alone. Oocyst production due to E. acervulina tended to be similar in birds given this species alone or with E. brunetti. Output fell to less than 50% when E. acervulina was administered with E. maxima. Oocyst production due to E. brunetti and E. maxima decreased when these species were inoculated together and when they were administered with E. acervulina. Lesions of E. acervulina and E. brunetti were superimposed on those of E. maxima in birds given mixed infections.Growth retardation was not evident in chickens inoculated with E. acervulina alone, although weight loss increased when this species was administered with either E. brunetti or E. maxima.     

Eimeria acervulina: cloning of a cDNA encoding an immunogenic region of several related merozoite surface and rhoptry proteins.

A cDNA encoding a recombinant Eimeria acervulina antigen, designated EAMZp30-47, that contains an epitope shared among several surface and rhoptry proteins of merozoites was characterized. The respective parasite proteins are between 30 and 47 kDa as revealed by immunostaining of nitrocellulose membrane containing extracts of 125I-labeled merozoites. As indicated by immunofluorescence and immunoelectron microscopic staining, the reactive epitope was localized to both the surface membrane and the internal rhoptries of this asexual stage of the parasite. The recombinant beta-galactosidase fusion protein EAMZp30-47 is 130 kDa, thus representing 15 kDa or 30-50% of the respective parasite protein. Purified EAMZp30-47 stimulates T cells from E. acervulina-immune inbred chickens, but is not recognized by immune chicken serum, suggesting that T cell and not B cell epitopes recognized by the host immune system during a natural infection are present on the recombinant protein. Northern and Southern blot hybridization experiments indicated that expression of EAMZp30-47 is restricted to the merozoite stage of the parasite and the gene occurs as a single copy sequence within the genome.     

Eimeria acervulina and E. tenella:effect on methionine absorption by the avian intestine.

Absorption of l-methionine in the duodenum of intestine of chicks infected with Eimeria acervulina was markedly less than in uninoculated controls or birds infected with E. tenella. Absorption of methionine in the jejunal area of E. acervulina-infected birds was reduced although not as drastically as in the duodenum. There was no difference in the rate of methionine absorption by the ileum. The kinetics of methionine absorption showed that V_max (maximum velocity) in the duodenum and jejunum of E. acervulina-infected birds was reduced when compared with the V_max found in uninoculated controls or E. tenella-infected birds. There was no difference in the K_t (transport constant) regardless of the infection or the region of the intestine examined. No major consistent effect of decreased pH on the rate of methionine absorption could be shown.The broadly spatulate villi seen, using the scanning electron microscope, in control and E. tenella infected duodenum were absent in E. acervulina-infected duodenum. Instead, the villi were reduced in height and noticeably thickened. This reduction in villar height suggests that a portion of the reduction in methionine absorption was related to the change in surface area and loss of transport loci due to damage of the mucosal surface.