{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/76516"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/76516","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Purification and characterization of Clostridium perfringens iota toxin","abstract":"Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting \"iota-like\" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i<sub>b</sub>), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i<sub>a</sub>) antigen, had little to no biological activity. When fractions containing i<sub>a</sub> and i<sub>b</sub> were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i<sub>a</sub> or i<sub>b</sub>, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i<sub>a</sub> and i<sub>b</sub>, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i<sub>a</sub> and i<sub>b</sub> were mixed. Monospecific antisera against purified i<sub>a</sub> and i<sub>b</sub> neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i<sub>a</sub> and i<sub>b</sub> proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i<sub>a</sub> protein is heat stable (85° C/15 min) while i<sub>b</sub> lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i<sub>a</sub>, but not i<sub>b</sub>, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.","abstract_html":"Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting &quot;iota-like&quot; toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i&lt;sub&gt;b&lt;/sub&gt;), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i&lt;sub&gt;a&lt;/sub&gt;) antigen, had little to no biological activity. When fractions containing i&lt;sub&gt;a&lt;/sub&gt; and i&lt;sub&gt;b&lt;/sub&gt; were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i&lt;sub&gt;a&lt;/sub&gt; or i&lt;sub&gt;b&lt;/sub&gt;, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i&lt;sub&gt;a&lt;/sub&gt; and i&lt;sub&gt;b&lt;/sub&gt;, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i&lt;sub&gt;a&lt;/sub&gt; and i&lt;sub&gt;b&lt;/sub&gt; were mixed. Monospecific antisera against purified i&lt;sub&gt;a&lt;/sub&gt; and i&lt;sub&gt;b&lt;/sub&gt; neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i&lt;sub&gt;a&lt;/sub&gt; and i&lt;sub&gt;b&lt;/sub&gt; proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i&lt;sub&gt;a&lt;/sub&gt; protein is heat stable (85° C/15 min) while i&lt;sub&gt;b&lt;/sub&gt; lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i&lt;sub&gt;a&lt;/sub&gt;, but not i&lt;sub&gt;b&lt;/sub&gt;, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.","abstract_has_math":false,"creators":["Stiles, Bradley G."],"institution":"Virginia Polytechnic Institute and State University","degree_name":"Ph. D.","degree_level":"doctoral","degree_discipline":"Microbiology","degree_department":"Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Wilkins, Tracy D."],"committee_members":["Johnson, J.L.","Smibert, R.M.","Gregory, Eugene \"Mick\"","Chen, Jiann - Shin"],"year":1987,"date_issued":"1987","date_published":"1987","updated_at":"2026-07-22T22:19:12Z","subjects":[],"languages":["en_US"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10919/76516","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Wilkins, Tracy D."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Johnson, J.L.","Smibert, R.M.","Gregory, Eugene \"Mick\"","Chen, Jiann - Shin"]},{"key":"dc:contributor.department","label":"Department","values":["Microbiology"]},{"key":"dc:creator","label":"Author","values":["Stiles, Bradley G."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2017-03-10T21:55:00Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2017-03-10T21:55:00Z"]},{"key":"dc:date.issued","label":"Date","values":["1987"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Polytechnic Institute and State University"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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A crossreacting \"iota-like\" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i<sub>b</sub>), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i<sub>a</sub>) antigen, had little to no biological activity. When fractions containing i<sub>a</sub> and i<sub>b</sub> were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i<sub>a</sub> or i<sub>b</sub>, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i<sub>a</sub> and i<sub>b</sub>, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i<sub>a</sub> and i<sub>b</sub> were mixed. Monospecific antisera against purified i<sub>a</sub> and i<sub>b</sub> neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i<sub>a</sub> and i<sub>b</sub> proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i<sub>a</sub> protein is heat stable (85° C/15 min) while i<sub>b</sub> lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i<sub>a</sub>, but not i<sub>b</sub>, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Purification and characterization of Clostridium perfringens iota toxin"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Wilkins, Tracy D."],"dc:contributor.committeemember":["Johnson, J.L.","Smibert, R.M.","Gregory, Eugene \"Mick\"","Chen, Jiann - Shin"],"dc:contributor.department":["Microbiology"],"dc:creator":["Stiles, Bradley G."],"dc:date.accessioned":["2017-03-10T21:55:00Z"],"dc:date.available":["2017-03-10T21:55:00Z"],"dc:date.issued":["1987"],"dc:description.abstract":["Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting \"iota-like\" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i<sub>b</sub>), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i<sub>a</sub>) antigen, had little to no biological activity. When fractions containing i<sub>a</sub> and i<sub>b</sub> were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i<sub>a</sub> or i<sub>b</sub>, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i<sub>a</sub> and i<sub>b</sub>, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i<sub>a</sub> and i<sub>b</sub> were mixed. Monospecific antisera against purified i<sub>a</sub> and i<sub>b</sub> neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i<sub>a</sub> and i<sub>b</sub> proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i<sub>a</sub> protein is heat stable (85° C/15 min) while i<sub>b</sub> lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i<sub>a</sub>, but not i<sub>b</sub>, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor."],"dc:description.degree":["Ph. D."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10919/76516"],"dc:language.iso":["en_US"],"dc:publisher":["Virginia Polytechnic Institute and State University"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["Purification and characterization of Clostridium perfringens iota toxin"],"dc:type":["Dissertation"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Ph. D."],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:12Z"}