Botulinum Toxin Gene Research Articles

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

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.

Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A- hyper, and NCTC 2916

The kinetics of botulinum toxin gene expression have been investigated in Clostridium botulinum type A strains 62A, Hall A- hyper, and type A(B) strain NCTC 2916 during the growth cycle. The analyses were performed in TPGY and type A Toxin Production Media (TPM). The mRNA transcript levels encoding the proteins of the neurotoxin complex were determined using Northern analyses. Neurotoxin concentrations in culture supernatants and lysed cell pellets were assayed using ELISA, Western blots, and mouse bioassay. Proteolytic activation of botulinum neurotoxin during the growth cycle was evaluated by Western blots. For all three strains, mRNA transcripts for the toxin complex genes were initially detected in early log phase, reached peak levels in early stationary phase, and rapidly decreased in mid-to-late stationary phase and during lysis. Toxin expression varied depending on the strain and growth medium. Toxin production was highest in strain Hall A- hyper, followed by NCTC 2916 and 62A. For C. botulinum strain Hall A- hyper, cell lysis and toxin release into the supernatant occurred rapidly for cells grown in TPM, while cells grown in TPGY remained in stationary phase with minimal lysis and toxin release through 96 h of growth. In contrast, strains 62A and NCTC 2916 lysed more extensively than Hall A- hyper in TPGY. TPM supported higher toxin production and activation than TPGY in strains 62A and Hall A- hyper. These data support that the genes of the botulinum neurotoxin complex are temporally expressed during late-log and early stationary phase and that toxin complex formation depends on the strain and growth medium. Botulinum toxin synthesis and activation appears to be a complex process that is highly regulated by nutritional and environmental conditions. Further research is needed to elucidate the sensing mechanisms and genetic regulatory factors controlling these processes.

Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.

Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism.

Neurotoxigenic Clostridium butyricum was isolated from the food implicated in an outbreak of clinically diagnosed type E botulism in China. PCR assay showed that the isolate (LCL 155) contained the type E botulinum toxin gene. This appears to be the first report of neurotoxigenic C. butyricum causing food-borne botulism.

Genetic characterisation of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916

An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5′ to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames. Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex. The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutans, UviA from C. perfringens and Orftxe1 located upstream of the C. difficile toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins. Primer extension and Northern blotting analysis demonstrate that the genes are expressed as two divergent opérons [HA34, HA17, HA70] and [NTNH/A, type A toxin gene], with the OrfX gene expressed singly. Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (5′-ATTTTagGTTTACAAAA-3′ and 5′-ATGTTATATgTaA-3′), separated by 12 bp, that span the putative − 35 and −10 promoter regions. Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci. It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C. botulinum strains and in that of the tetanus toxin gene.