Immunogenicity of three oocyst extracts of Cryptosporidium baileyi in experimentally infected chickens

Abstract

The immunogenic properties of water-soluble and detergent-extracted components of Cryptosporidium baileyi oocysts were studied. Oocyst cytosol antigen (OCA) containing hydrophilic proteins was obtained by freeze-thaw cycles in liquid nitrogen. This was followed by Triton X-114 extraction of remaining oocyst fragments to dissolve membrane-bound proteins (TRE). The remainder of the pellet was solubilized with sodium dodecyl sulfate and treated with 2-mercaptoethanol to reduce disulfide-linked oocyst wall proteins (BME). The immune recognition of these three extracts was evaluated during the course of experimental cryptosporidiosis in chickens using ELISA, immunoblotting, and the lymphocyte stimulation test (LST). Four groups of chickens were infected at various times with different doses of C. baileyi and one group with the mammalian parasite C. parvum. Analysis of the data revealed that OCA proteins are well recognized by serum antibodies during the infection and to a limited extent by sera from chickens infected with C. parvum. Humoral responses of chicken groups to this antigen did not correlate well with the length of patency in contrast with its cellular recognition in LST. TRE gave lower values than OCA in both ELISA and LST, though it was still specifically recognized by samples from C. baileyi-infected chickens. Antibodies reacted aspecifically with BME, since only samples of birds which were immunocompetent at the time of their infection were able to recognize this extract as antigen. Immunoblotting revealed more specific components in OCA than in TRE or BME.

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

“…Antigens used by other workers (8) were also evaluated for the production of antibodies to the oocyst surface. The mice immunized with OCA and TRE preparations showed no response to the oocyst wall.…”

“…Groups of five BALB/c (ARC) female mice 8 to 12 weeks old were immunized by intraperitoneal injection (i.p.) with either an oocyst wall preparation, whole oocysts, soluble protein extract, or the three preparations described by Hornok et al (8). Each preparation in PBS was emulsified with an equal volume of Freund complete adjuvant.…”

“…To give a comparative measure of the immune response, three extracts of oocysts as described by Hornok et al (8) originally used to orally inoculate chickens were also used for immunization. Briefly, three sequential extraction preparations were prepared.…”

“…Therefore, removal of sporozoites and reduction of antigen size and structure were considered key factors to be taken into account when preparing antigens. In addition, sequentially extracted oocyst antigens (8) were used to give an idea of the immunogenicity of different oocyst wall preparations.…”

Vaccines against the avian enteropathogensEimeria,CryptosporidiumandSalmonella

“…Rather, the cell-mediated response is the predominant form of immunity against cryptosporidiosis. In studying the vaccine potential of an extract of C. baileyi oocysts, Hornok et al (1999) observed that chickens injected intramuscularly with the extract developed partial resistance to oral challenge, but those inoculated orally with live oocysts became completely resistant. However, the antibody responses of these two groups were identical 3 weeks after challenge, suggesting that serum antibodies had negligible effect on the development of protective immunity.…”