Characterization of a surface antigen ofEimeria nieschulzi (Apicomplexa,Eimeriidae) sporozoites

A monoclonal antibody (McAb 3C3) reacting with a pellicular antigen ofEimeria nieschulzi sporozoites has been selected among hybridomas produced against this organism by immunofluorescence assay. This antigen has been shown to be located on the zoite surface by immunofluorescence on living organisms. Capping and shedding of antigen-monoclonal antibody immune complexes was observed upon incubation at 37°C. On western immunoblotting, two polypeptides at 22 and 26 kDa were recognized by McAb 3C3, whereas only one polypeptide of 22 kDa was immunoprecipitated by the same antibody after lactoperoxidase surface radio-iodination of sporozoites.     

Fine structure of nuclear division and microgametogony ofEimeria nieschulzi Dieben, 1924

Nuclear division and microgametogony ofEimeria nieschulzi were studied by transmission electron microscopy. All nuclear divisions occurred in close proximity to the gamont surface with four centrioles situated between the nuclear envelope and the gamont plasmalemma. During early nuclear divisions, each nucleus had a rod or hourglass-shaped nucleolus, indicating that the nucleolus probably pinched in two during nuclear division. Nuclear divisions occurred by nearly centrally located intranuclear spindles. Two centrioles were associated with each centrocone. Spindle microtubules, which originated from each centrocone, either traversed to the other centrocone or terminated at kinetochores. Four to 6 chromosome-like structures were present in each dividing nucleus. The formation of 50 to 100 biflagellate microgametes occurred at the margin of the microgamont by a process similar to that described for other coccidian species. Microgametes were limited by a single membrane with a prominent glycocalyx, 13 nm thick.Based on a thesis submitted by G.J. Sibert to the Graduate School of the University of Montana in partial fulfillment of the requirements for the Master of Science degree     

Identification of surface membrane proteins and surface membrane antigens of plasmodium chabaudi merozoites

Surface membrane proteins of viable merozoites of Plasmodium chabaudi were iodinated by the Iodogen method and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirteen surface membrane proteins ranging from 22 to 270 kDa were thus identified. Most of these proteins could be immunoprecipitated by sera of mice immunized with extracts of P. chabaudi. A few, however, were precipitated only by sera of mice challenged with living parasites after immunization.     

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).     

Identification of immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites

Immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites were identified by 125I-labeling and immunoblotting studies. Using these methodologies 60% of the immunodominant sporozoite antigens and 90% of the immunodominant merozoite antigens were observed to be 125I-surface labeled. However, several major 125I-labeled sporozoite and merozoite proteins did not represent prominent antigens as measured by immunoblotting. Immunodominant surface antigens were found over a wide size range for sporozoites (21-110 kDa) and for merozoites (20-250 kDa). In order to relate these findings to a 'natural' infection, two groups of 3-week old chickens were inoculated 5 times over a 2.5 week period with either a low or high dose of E. acervulina oocysts. The serum response to sporozoites and merozoites, indicated by enzyme-linked immunosorbent assay titers, was rapid; less than or equal to 7 days post-infection with 10(4) oocysts and less than or equal to 3 days with 10(5) oocysts. Many of the antigens identified by immunoblotting of sera from sporozoite- and merozoite-immunized animals were recognized by sera from both high dose and low dose E. acervulina-infected chickens. Furthermore, the sporozoite and merozoite antigens could be grouped into those constituents which induced a serum response early or late in the infection.     

Biochemical and immunological characterization of major surface antigens ofSarcocystis muris andS. suicanis cyst merozoites

Surface proteins ofSarcocystis cyst merozoites were labeled by biotinylation or radioiodination and identified on Western blots after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The major labeled proteins ofS. muris andS. suicanis have relative molecular masses of 31 and 33 kDa, respectively. Immunoblots performed with the 31-kDa protein and sera of experimentally infected mice or with monoclonal antibodies toS. muris revealed that this protein is immunogenic. Indirect fluorescent antibody tests (IFAT) executed usingSarcocystis cyst merozoites and polyclonal monospecific antibodies obtained from rabbits immunized with homogeneous major surface antigens gave additional evidence for the localization of the identified antigens in the pellicle. Analysis ofS. muris andS. suicanis proteins by two-dimensional gel electrophoresis revealed multiple isoelectric forms.Supported by the Deutsche Forschungsgemeinschaft and dedicated to Prof. Dr. Johannes Eckert (Zürich) on the occasion of his 60th birthday     

Identification of surface and internal antigens from spontaneously releasedPlasmodium falciparum merozoites by radio-iodination and metabolic labelling

Spontaneously released merozoites from synchronousPlasmodium falciparum cultures were isolated in the presence of protease blocker. 1–5×10¹⁰ merozoites were obtained in each experiment. The isolated merozoites possessed a thick surface coat and about 80% were invasive to human erythrocytes although they did not subsequently develop into ring stages. Tests using several analytical methods showed the merozoite preparations to be free of any erythrocyte contamination. Six labelled proteins were identified after surface radio-iodination, the largest with a molecular weight of 82000. All six proteins were precipitated with various immune sera. Four other proteins with molecular weights of 200000 and 160000–145000 (a triplet) were identified by precipitation with the same immune sera after metabolically labelling the merozoites. The six surface proteins were not prominent in the metabolically labelled preparations. Using these methods it is possible to identify and differentiate between surface and internal merozoite antigens.     

Characterization of immunodominant surface antigens of Haemophilus somnus.

An immunodominant Haemophilus somnus outer membrane protein with an apparent molecular mass of 40 kDa on Western blots (immunoblots) of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was characterized because a monospecific antibody against this antigen was protective. This monospecific antibody was used for immunoaffinity purification of the antigen. The immunoaffinity-purified antigen reacted with a polyclonal antibody to the 40-kDa antigen but not with a monoclonal antibody (3G9) which reacted with the 40-kDa antigen in gradient gels. On 8 or 10% gels, the approximately 40-kDa antigen was resolved as two bands, a 40-kDa band which reacted with the protective monospecific polyclonal antibody (p40) and a band of lower molecular mass which reacted with monoclonal antibody 3G9. The latter antigen was designated p39. Both antigens were conserved in all H. somnus isolates tested. The specific antibodies were also used to detect cross-reacting antigens in other gram-negative bacteria. Antibody to p40 reacted with proteins of 55 to 28 kDa, with the greatest intensity shown among proteins from other members of the family Pasteurellaceae. Antibody to p40 was reduced by absorption with live H. somnus or other members of the family Pasteurellaceae, so the antigen appears to be surface exposed. Antibody to p39 only cross-reacted with a broad band (38 to 40 kDa) in Haemophilus agni. Since H. agni is not a bovine pathogen and since convalescent-phase serum from H. somnus-infected animals did recognize p39, the latter may be a good immunodiagnostic antigen, if the lack of cross-reactivity with antigens in other gram-negative bacteria is confirmed with a polyclonal antibody to p39. The cross-reactivity of antiserum to p40 with antigens of members of the family Pasteurellaceae and the ability of this antiserum to protect against H. somnus pneumonia indicate that p40 may be a useful vaccine antigen for H. somnus disease and perhaps even diseases caused by other members of the family Pasteurellaceae.     

Isolation and partial characterization of rhoptries and micronemes fromEimeria nieschulzi zoites (Sporozoa,Coccidia)

Homogenization and subcellular fractionation of sporozoites ofEimeria nieschulzi have allowed the recovery of highly enriched fractions of rhoptries and micronemes. The isolated organelles kept their in situ morphological characteristics. Their apparent densities in sucrose solutions were approximately 1.18 g/cm³ for rhoptries and 1.14 g/cm³ for micronemes. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the microneme fraction showed two major polypeptides at 220 and 94 kDa. The rhoptry fraction contained at least three predominant peptides at 200, 150, and 63 kDa. Micronemes were also isolated from third-genaration merozoites of the same species; their characteristics were identical to those of the organelles isolated from sporozoites.     

Insight into the ultrastructural organisation of sporulated oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa)

Sporulated oocysts of Eimeria contain four sporocysts with two sporozoites each and a sporocyst residuum. The developing sporozoites are protected by the sporocyst wall and the robust double-layered oocyst wall. Because of problems with conventional fixatives, high-pressure freezing, followed by freeze substitution was used to achieve optimal ultrastructural preservation of oocysts, sporocysts and sporozoites. After embedding in Epon?, ultrathin sections were examined by electron microscopy to select specific oocyst regions for further investigation by electron tomography (ET). ET allows high-resolution three-dimensional views of subcellular structures within the oocysts and sporocysts. Analysis of several 300?nm sections by ET revealed a network of small tubular structures with a diameter of 70–120?nm inside the sporocysts which is decribed here for the first time. This network connects the residual body in a sporocyst with the endoplasmic reticulum (ER) of the surrounding sporozoites. The network consists of membrane-bound tubules that contain vesicles but no larger organelles like mitochondria. These tubules, named “sporocord”, may have a function similar to an “umbilical cord” providing the sporozoites with metabolites for long-term survival. Small vesicular structures inside the ER of the sporozoites, multivesicular structures inside the residual bodies and vesicles in the tubules support this hypothesis.     

Allotypic trophoblast—lymphocyte cross-reactive (TLX) cell surface antigens

Evidence is presented for the presence of trophoblast-lymphocyte cross-reactive (TLX) cell surface antigens. Complement-dependent lymphocytotoxicity studies with antitrophoblast sera (Ce) raised to ten separate placentas showed variable reaction patterns on peripheral blood leukocytes isolated from 30 random HLA-typed donors. No correlation with any known antigens of the major histocompatibility complex was evident. Absorption studies with chromatographically prepared fractions of solubilized trophoblast membranes from individual placentas and absorptions with both normal and transformed lymphoid cells removed cytotoxicity from some but not other Ce antisera when tested on a single donor, thus providing serological evidence for allotypic variation among the TLX antigens.     

Coccidia (Apicomplexa: Eimeriidae) of elk (Alces alces) in Poland

Over a 4-year period, we analyzed 128 fecal samples from free-living elk (Alces alces L., 1758) to determine the prevalence of Eimeria infections and identify the species present. Two eimerian species were isolated including Eimeria alces and a morphotype resembling Eimeria catubrina. Overall, two samples from 128 samples collected were positive for Eimeria (prevalence?=?1.6 %), and the oocyst per gram, estimated with the use of the conventional McMaster quantitative technique, ranged from 50 to 100. Also, E. alces has been found in Lithuania and Belarus and is the only known species of eimerian to infect elk. E. catubrina is a parasite typically infecting roe deer (Capreolus capreolus L., 1758). This is the first report of Eimeria spp. in elk in Poland. Results of our investigation indicate that elk may become infected with an eimerian species that is typical for roe deer, but this requires further investigation.     

Characterization of microneme antigens of Cryptosporidium parvum (Protozoa, Apicomplexa).

Two monoclonal antibodies (MAbs) raised against purified excysted oocysts and sporozoites of cryptosporidium parvum reacted in an immunofluorescence assay with antigens located at the anterior pole of the zoites. On Western blots of purified oocysts, these MAbs reacted with a series of bands between 210 and 40 kDa; several of these bands were recognized by both MAbs; others were specific. One MAb (TOU) did not react after periodic acid treatment and was therefore considered to recognize a carbohydrate epitope; as determined by immunoelectron microscopy, this MAb reacted on micronemes of sporozoites and merozoites and also with the peripheral cytoplasm and the parasitophorous vacuole of trophozoites and macrogametes. The other MAb (HAD) reacted with an epitope that was insensitive to periodate treatment but did not react in the immunoelectron microscopy assay. However, the similar labeling pattern obtained with the immunofluorescence assay with both MAbs and the fact that the two antibodies share common bands on Western immunoblots suggest that both MAbs react with molecules located in Cryptosporidium micronemes, one reacting with a glycannic epitope and the other reacting with a peptidic epitope.     

Characterization of antigens and allergens of date palm (Pheonix dactylifera) pollen

Antigenic and allergenic components of date palm (Phoenix dactylifera) pollen were investigated to observe their effects on the skin test reactivity, lymphocyte blastogenesis and cytokine production in atopic and healthy individuals. Date pollen extracts were fractionated using SDS-PAGE and Sephacryl S-200 gel filtration. Western blotting of SDS-PAGE separated components with antiserum raised against whole pollen extract in rabbits revealed at least 22 immunoreactive bands ranging in molecular weight between 12 and 94 kD. The immunogenicity of the pollen extract was further confirmed by strong positive reactions in ELISA and Ouchterlony's double diffusion tests. Immunoblot analyses revealed IgG and IgE reactive components (14-94 kD for IgG and 12-90 kD for IgE) in the skin test-positive patients' sera against whole pollen extract. Fifteen of 60 atopics reacted positively to either whole or some fractions of date pollen extract when skin tested. In response to whole or components of date pollen extract atopic patients showed differential peripheral blood lymphocyte (PBL) proliferative response and cytokine (IL-2, IL-4) production when compared with PBL of normal subjects. Our findings strongly suggest that date palm pollen should be considered a reaginic component and should be included in the battery of allergens for determining the allergic status of atopic patients, particularly in those parts of the world where the date palm is grown commercially.     

Characterization of B cell subpopulations by velocity sedimentation, surface ia antigens and immune function.

An in vitro splenic focus assay for B cell cloning was used to analyze the responses of primary and secondary B cells obtained after fractionation on (1 × g) velocity sedimentation gradients. When nonimmune adult bone marrow cells are fractionated, the small cell (slowly sedimenting) fraction contains the majority of primary B cells specific for the 2,4-dinitrophenyl determinant (DNP). When nonimmune adult spleen cells are fractionated, all of the cell fractions contain B cells specific for DNP or for fluorescein (FL), and the large and medium cell fractions contain approximately 50 % of the activity. There is a differential expression of IgM vs. IgG₁ anti-hapten monoclonal antibody, in that the majority of primary splenic B cells which give rise to clones producing only IgM antibody are found predominantly in the large and medium cell fractions. All of the cell fractions contain B cells which can generate clones producing both IgM and IgG₁, or only IgG₁ antibody. Ia-“negative” B cells which give rise to clones producing only IgM antibody are found in the large and medium cell fractions, whereas all the cell fractions contain la-positive B cells. When secondary spleen cells are fractionated, all of the cell fractions contain secondary B cell activity. The large and medium cell fractions contain half of the DNP-specific secondary B cells, whereas the medium and small cell fractions contain more secondary B cells specific for FL or hemocyanin. The large and medium cell fractions of secondary spleen cells are not enriched for B cells giving rise to clones producing only IgM antibody.