{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T23:39:17Z","timestamp":1790379557592,"version":"4.1.0"},"reference-count":36,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2010,1,15]],"date-time":"2010-01-15T00:00:00Z","timestamp":1263513600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2010,1,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:italic>Clostridium botulinum<\/jats:italic>\n            produces the most poisonous natural toxin known and is a perennial concern to the food industry and to regulatory agencies due to the potential threat of food-borne botulism. To ensure the botulinal safety of foods, rigorous food challenge testing to validate food-processing conditions and food formulations has been routinely performed. Detection of the botulinum neurotoxin is performed by using a mouse bioassay and\/or\n            <jats:italic>in vitro<\/jats:italic>\n            assays. There has been considerable interest by the food industry and regulatory agencies in minimizing or even replacing the use of animals in these challenge studies. In addition, due to stringent select-agent regulations, the testing of various foods using toxigenic\n            <jats:italic>C. botulinum<\/jats:italic>\n            strains requires facilities and personnel that are certified for work with this organism. For this purpose we propose to generate sets of nontoxigenic\n            <jats:italic>C. botulinum<\/jats:italic>\n            strains from proteolytic and nonproteolytic groups that differ from the wild-type strains only by their inability to produce botulinum neurotoxin. In this initial study we describe the generation of a nontoxigenic mutant of\n            <jats:italic>C. botulinum<\/jats:italic>\n            strain 62A using the ClosTron mutagenesis system by inserting a group II intron into the botulinum neurotoxin type A gene (\n            <jats:italic>bont\/A<\/jats:italic>\n            ). The mutant clones were nontoxigenic as determined by Western blots and mouse bioassays but showed physiological characteristics, including growth properties and sporulation, that were similar to those of the parent strain in laboratory media. Additional studies will be required to evaluate comparable characteristics in various food matrices. The availability of suitable nontoxigenic\n            <jats:italic>C. botulinum<\/jats:italic>\n            strains for food challenge studies will be beneficial for enhancing the botulinal safety of foods as well as increasing the biosafety of workers and may eliminate the use of laboratory animals.\n          <\/jats:p>","DOI":"10.1128\/aem.02005-09","type":"journal-article","created":{"date-parts":[[2009,11,21]],"date-time":"2009-11-21T02:38:34Z","timestamp":1258771114000},"page":"387-393","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":32,"title":["Construction of a Nontoxigenic\n            <i>Clostridium botulinum<\/i>\n            Strain for Food Challenge Studies"],"prefix":"10.1128","volume":"76","author":[{"given":"Marite","family":"Bradshaw","sequence":"first","affiliation":[{"name":"Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin\u2014Madison, Madison, Wisconsin 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristin M.","family":"Marshall","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin\u2014Madison, Madison, Wisconsin 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John T.","family":"Heap","sequence":"additional","affiliation":[{"name":"School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William H.","family":"Tepp","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin\u2014Madison, Madison, Wisconsin 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nigel P.","family":"Minton","sequence":"additional","affiliation":[{"name":"School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric A.","family":"Johnson","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin\u2014Madison, Madison, Wisconsin 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1001\/jama.285.8.1059"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2004.07.001"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1541-4337.2003.tb00035.x"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-10-115"},{"key":"e_1_3_2_6_2","unstructured":"Select agent regulations. 2008"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.plasmid.2007.04.002"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.71.11.7542-7547.2005"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0005849"},{"key":"e_1_3_2_10_2","first-page":"53","year":"1989","unstructured":"Dasgupta, B. 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