Molecular Detection of Seafood-Borne Human Pathogenic Bacteria - Aquaculture Microbiology and Biotechnology - page 123

Molecular Typing of C. botulinum Type E Strains
The distribution of C. botulinum serotypes A, B, E, and F in Finnish trout
farms was assessed using PCR by Hielm et al. (1998). The PCR primers
from Hielm et al. (1996) including those for botnE were used ( Table 5.2 ) .
A total of 333 samples were tested with neurotoxin gene specifi c PCR
assays. C. botulinu m type E was found in 68% of farm sediment samples,
in 15% of fi sh intestinal samples, and in 5% of the fi sh skin samples. No
other serotypes were found. The average spore count in sediments, fi sh
intestines, and skin were 2 x 10 3 , 1.7 x 10 2 , and 3 x 10 2 per kg respectively.
PFGE with Sma I of 42 Finnish isolates plus 12 North American reference
strains generated 28 PFGE profi les indicating extensive genetic diversity.
The genetic diversity of 92 type E. C. botulinum strains was assessed
by Hyytiä et al. (1999). Sixty-seven were of Finnish seafood and fi shery
origin, 15 were from German farmed fi sh, and 10 from North American
seafoods. PFGE performed with Sma I- Xma I resulted in 75 typeable strains
which yielded 33 profi les. PFGE performed with Xho I allowed 91 strains
to be typed yielding 51 profi les. All 92 strains were typeable with RAPD
primers OPJ-6 and OPJ-13 ( Table 5.2 ) , which yielded 27 and 19 banding
patterns respectively. The frequent occurrence of small fragments and faint
bands made RAPD interpretation diffi cult. A high level of genetic diversity
among the isolates was observed regardless of their source, presumably
because of the absence of strong evolutionary selection factors.
Wang et al. (2000) analyzed type E botulinum toxin producing strains
isolated from botulinum cases or soil specimens in Italy and China using
sequencing of the bontE gene, RAPD ( Table 5.2 ) PFGE, and Southern blot
hybridization for the bontE gene. The deduced amino acid sequences of
the BoNTE's of 11 C. butyricus isolates from China were identical and
exhibited 95.0 and 96.9% identity with those of the Italian BoNTE strain
of C. butyricum BL6340 and C. botulinum type E respectively. The results
indicated that BoNTE-producing C. butryricum is clonally distributed
globally.
Leclair et al. (2006) undertook a comparative typing study involving
the PFGE, RAPD, and automated ribotyping of C. botulinum type E strains
derived from clinical and food sources associated with four botulinum
outbreaks that occurred in the Canadian Arctic. All type E strains previously
untypeable by PFGE, even with the use of a formaldehyde fi xation step,
could be typed by the addition of 50 mM thiourea to the electrophoresis
running buffer. Digestion with Sma I and Xho I followed by PFGE was used
to link food and clinical isolates from the four different type E botulinum
outbreaks and to differentiate them from among 31 recently isolated
Arctic environmental group II C. botulinum strains. Sma I PFGE typing
yielded 18 profi les while Xho I PFGE typing yielded 23 profi les. Strain
differentiation was unsuccessful with the automated ribotyping system