Quantitative Polymerase Chain Reaction Assay Used to Measure the Prevalence of Clostridium botulinum type E in Fish in the Lower Great Lakes

Abstract

AbstractA quantitative polymerase chain reaction (QPCR) assay for the type E Clostridium botulinum toxin gene was developed and used in a survey of apparently healthy, moribund, and freshly dead fish from the lower Great Lakes. The focus was on species that had died in recent fish kills, such as freshwater drum Aplodinotus grunniens, smallmouth bass Micropterus dolomieu, and round goby Neogobius melanostomus. Samples of these species were obtained during scheduled collections and during die‐offs. A 139‐base‐pair fragment of DNA, located near the junction of the light and heavy chains of the type E toxin gene, was cloned for the real‐time assay and was used as a standard to quantify the number of C. botulinum type E bacteria present in the liver and intestinal content samples tested. Samples that tested positive for C. botulinum type E were then assayed for toxin by the traditional house mouse Mus musculus bioassay and with a botulinum toxin rapid detection kit designed for qualitative environmental screening. We tested a total of 736 fish from Lakes Erie and Ontario during 2002, and 678 fish were tested in 2003. This work has demonstrated the presence of the type E C. botulinum toxin gene and, by inference, the presence of vegetative cells in dying freshwater drum, apparently healthy smallmouth bass and round goby, and a dead lake sturgeon Acipenser fulvescens. The freshwater drum were found either in a moribund condition with signs of botulism or freshly dead during die‐offs in July 2002 and late August 2003 at several locations in the eastern basin of Lake Erie; no fish from Lake Ontario tested positive by means of QPCR. Our research has begun to clarify the role moribund fish may play in the C. botulinum type E intoxications that have recently struck the live‐fish‐eating birds of the lower Great Lakes.