M. Gallego

The proliferative capacity of the cells of the avian Harderian Gland

The largest concentration of plasmacytic cells within avian lymphomyeloid tissue appears in the Harderian Gland (HG). Mature plasma cells were the least frequent cell at day old and the most frequent by 5-weeks of age. Failure of phytohemagglutinin-M to stimulate blasting of HG cells may reflect the low number of T cells in the HG. On the other hand, the uptake of tritiated thymidine by HG cells after short term culture in complete medium was significantly greater than that of splenic cells. The proliferating cells were the plasmablast, and mature plasma cell. Our data demonstrated that labeled B-cells, administered intravenously to 2- or 6-week old syngeneic or allogeneic chickens, did not migrate to the HG. The origin of HG plasma cells may be B-cells which entered prior to 2 weeks of age or the HG may possess a special microenvironment which allowed stem cell differentiation. The maintenance of a high concentration of plasma cells in the HG in the presence of low levels of B-cells may reflect the long-lived nature of the resident B-cell and/or the inherent proliferative capacity of the HG plasmacytic cell.

Anti-S-100 antibody recognizes ellipsoid-associated cells and other dendritic cells in the chicken spleen

The chicken spleen was studied immunohistochemically with anti-S-100 protein polyclonal antibody. S-100-positive cells accumulated around the penicilliform capillaries during the first 3 weeks of life. After 2 weeks posthatch the S-100-positive cells appeared in the red pulp, periarterial lymphatic sheath, and subsequently in the germinal center. Their ontogenetic development and intrasplenic distribution strongly suggested that the S-100-positive cells were identical with ellipsoid-associated cells. The S-100-negative cells of the periellipsoidal white pulp gradually transformed to S-100-positive, functionally active cells on the surface of the ellipsoid. The immunohistological findings support the hypothesis that the interdigitating dendritic cells and follicular dendritic cells were not of monocytic origin but belong to a splenic resident, endocytic cell line located on the surface of the ellipsoid.

Immunohistochemical identification of the cells parasitized by second-generation schizonts of Eimeria tenella

Abstract Conflicting reports exist in the literature concerning the type of cells within the lamina propria of the ceca that harbor second-generation schizonts of Eimeria tenella. Most of the previous studies concerning these cells have been performed using routine light or electron microscopy. Consequently, difficulties are evident in precise definition of the type of these cells using normal morphological criteria, since growth of the schizonts of E. tenella alters the morphology of the parasitized cell, making it difficult to recognize the cell type. This has led us to investigate the possibility of precisely identifying the subepithelial cells that are parasitized by mature schizonts. For this purpose we used cytoskeletal markers, namely, keratin and vimentin intermediate filaments, which allow the discrimination between epithelial and mesenchymal cells. Localization of keratin and vimentin on frozen cecal sections was studied immunohistochemically using specific monoclonal antibodies. Sites of antigenicity were detected by the avidin-biotin complex (ABC) immunoperoxidase technique and visualized by the deposition of diaminobenzidine. The identity of the cells was confirmed by the immunodetection of keratin intermediate filaments in the cytoplasm of the cells. Immunoreactivity for vimentin was absent in the parasitized cells. Therefore, we conclude that the development of second-generation schizonts of E. tenella takes place in epithelial cells within the lamina propria, which are presumably crypt epithelial cells that leave the crypts and enter the lamina propria after infection by first-generation merozoites.

A method for the sequential study of eimerian chromosomes by light and electron microscopy.

In the present study, the authors describe a simple method to isolate chromosomes from eimerian oocysts and to submit them to sequential study by light and electron microscopy. This method includes a reliable and reproducible technique for transferring eimerian chromosomes from slides to grid that fulfills the essential requirements for generalized use in cytogenetics. In addition, this method overcomes the difficulty of the resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The observation by the authors of synaptonemal complexes in meiotic chromosomes of different Eimeria species by applying the above-mentioned method to oocysts revealed its importance to future applications.

Immunoglobulin classes synthesised by the chicken Harderian gland after local immunisation.

The immunoglobulin (Ig) levels in tears and sera were compared after antigen administration (salmonella O antigen) by eyedrop and injection into the nictitating membrane, to determine the Ig classes synthesised by the plasma cells in the chicken Harderian gland. Samples of tears and sera were collected from immunised and control birds between 24 hours and 24 days after the antigen or sterile saline was administered. Samples were assayed for IgA, IgG and IgM concentrations using radial immunodiffusion. It is suggested that most of the IgG found in tears after local immunisation has an extraglandular origin.

DIFFERENCES IN HSP70 EXPRESSION IN THE SPOROZOITES OF THE ORIGINAL STRAIN AND PRECOCIOUS LINES OF EIMERIA TENELLA

The levels of expression of Heat shock protein 70 (Hsp 70) in sporozoites of a wild-type parent strain and 2 precocious lines of Eimeria tenella, were compared to investigate the relationship between the heat shock proteins expressed by the parasite and virulence of the strain. Hsp70 expression was analyzed in sporozoites by immunohistochemical techniques, immunoblot, and flow cytometric analyses. One band of 70 kDa was identified and the variation of the Hsp70 expression levels was quantified by optical densitometric analyses. The results showed a significant gradual decrease in the Hsp70 expression in sporozoites of E. tenella as attenuation progressed, suggesting that the Hsp70 expressed in the excysted sporozoites of E. tenella might be involved in parasite pathogenicity. In addition, the cytoplasmic distribution of the Hsp70, which was observed in the entire sporozoites of the wild strain, was reduced to the anterior portion in the precocious lines.

Local immune response in the chicken Harderian gland to antigen given by different ocular routes.

The effectiveness of three ocular routes of antigen administration to produce a local immune response in the Harderian gland was studied. The routes were by eyedrop, injection into the ocular conjunctiva and injection into the nictitating membrane. The antigen was observed in the cytoplasm of macrophages located within the lymphoid tissue only after the injection into the nictitating membrane. The numbers of germinal centres and plaque forming cells found in the gland after injection into the nictitating membrane was higher than the numbers observed following the other two ocular applications. These findings indicate that the injection of the antigen into the nictitating membrane is the most effective ocular route for producing a local immune response in the Harderian gland.

Flotillin-1 localization on sporozoites oF Eimeria tenella.

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin-1 and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.