Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Characterization and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.

Limited heterogeneity between strains of Eimeria tenella isolated from Britain and Bangladesh.

Single-oocyst-derived field strains of Eimeria tenella isolated from Rugby in the United Kingdom (E tenella R) and from Mymensingh and Dhaka in Bangladesh (E tenella M and D, respectively) and a laboratory strain (E tenella, Houghton, H) were compared by isoenzyme electrophoresis, reactivity with antisporozoite monoclonal antibodies and, for some pairs of strains, cross-protection in vivo. The three field strains conformed to one zymodeme with respect to six isoenzymes. For glucose phosphate isomerase (GPI) all field strains were characterised by GPI-9. A panel of six different monoclonal antibodies raised against sporozoites of E tenella H did not discriminate between strains by titration in an immunofluorescence assay against air-dried, acetone fixed sporozoites. In cross-protection experiments involving immunisation and challenge of young chickens, two immunisation schedules were used which, after homologous challenge, provided complete immunity either by the criterion of oocyst output, or by the criterion of weight gain (and more than 94 per cent protection by the criterion of oocyst output). While strain heterogeneity was minimal in the former situation, there was poor cross protection between some strains in the latter case. Under those conditions, heterologous challenge with E tenella M resulted in dysentery and in significantly (P less than 0.05) increased oocyst output and decreased weight gain. The results suggested that E tenella M was immunologically superior to E tenella R and H strains. The results show that a limited degree of immunogenic variability exists between these strains of E tenella and that, unless homologous strain immunity is complete by the criterion of oocyst output, challenge with heterologous strains may result in depressed weight gain.

Isolation of five species of Eimeria from chickens in Bangladesh.

Five species of Eimeria, namely E. acervulina, E. tenella, E. maxima, E. brunetti and E. necatrix were identified in chickens in Bangladesh on the basis of lesions seen at post-mortem examinations of naturally infected birds, and on the dimensions of oocysts and the lesions seen in chicks experimentally infected with single oocyst derived strains. The use of filter top, polycarbonate cages permitted the isolation of strains without sophisticated animal isolators and is appropriate for use in laboratories throughout the developing world. Responses to a questionnaire sent to all known intensive poultry farms suggested that coccidiosis is a major disease. For control, producers rely mostly on management procedures and the tactical use of sulphonamides; in-feed chemoprophylaxis is not widely used. These control measures are unsatisfactory and recent coccidiosis outbreaks were reported from seven of 16 farms. There was evidence of a seasonal incidence in clinical coccidiosis.

Eimeria tenella: local antibodies and interactions with the sporozoite surface.

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)