118 Aquaculture Microbiology and Biotechnology
virulence-associated factors are present in clusters within at least three
regions in the V. cholera genome. The fi rst consists of the ctxA/ctxB genes
which reside on a lysogenized phage (Waldor and Mekalanos, 1996). The
second is a large pathogenicity island that encodes a toxin co-regulated
pilus gene cluster involving a type IV pilus that is the primary adhesion
and colonizing factor (Taylor et al., 1987) and acts as a phage receptor. The
third gene cluster, the RTX gene cluster, encodes a cytotoxin active against
cultured Hep-2 cells in V. cholera El Tor strains (Lin et al., 1999).
PCR Detection, Identifi cation and Characterization of V. cholerae
In January 1991, an outbreak of cholera started in Peru and rapidly spread
throughout most of Latin America. Within 15 mon over 450,000 cases
occurred with about 4,000 deaths. The causative organism was toxigenic
V. cholerae O1 of the El Tor biotype which is distinct from the U.S. Gulf
Coast strains. Fields et al. (1992) reported on the use of primers CTX2/
CTX3 (Table 5.2) that amplifi ed a 564-pb sequence of the ctxA gene for its
detection in 150 V. cholerae isolates derived from patients, food, and water
from the 1991-1991 outbreak. One hundred forty isolates were found to be
toxigenic both by PCR and immuno assay.
Koch et al. (1993) reported on the development of a PCR assay for
detection of V. cholerae seeded onto oysters, crab meat, shrimp, and lettuce.
The primers P1/P3 (Table 5.2) amplifi ed a 778-bp sequence of the ctxB
gene from a V. cholerae O1 strain. Seeded foods were homogenized or
rinsed with APW followed by a 6-8 h enrichment incubation at 37°C. One
ml enrichments were boiled and 2 to 5 ml added to 100 ml PCR reaction
volumes. A detection limit of 1 CFU/10g of food was obtained.
DePaola and Hwang (1995) determined the optimum conditions of
enrichment for detection of V. cholerae by the PCR. Recovery and PCR
detection was signifi cantly greater from oyster homogenates diluted 1:100
in alkaline peptone water and incubated at 42°C for 18-21 h. The primers
used (Table 5.2) were from Fields at al. (1992) and amplifi ed a 564-bp of
the ctxA gene.
Ripabelli et al. (1999) reported on the occurrence of various pathogenic
Vibrio species from mussels harvested from approved shellfi sh waters in
the Adriatic Sea. V. cholerae O1 and O139 serotypes were not detected.
However, V. cholerae non-01/non-O139 was found in only 1.6% of the
samples compared to 32.2% for V. alginolyticus. PCR with primers STO-F/
STO-R (Table 5.2) were used to amplify a 238-bp sequence of the sto gene
that encodes the thermotolerant enterotoxin of V. cholerae and revealed the
absence of the sto gene in all of these environmental isolates of V. cholerae.
Chow et al. (2001) developed a PCR assay for detection of the rtxA,
rtxC, (encoding the RTX repeat in toxin) and the ctxB toxin genes among
virulence-associated factors are present in clusters within at least three
regions in the V. cholera genome. The fi rst consists of the ctxA/ctxB genes
which reside on a lysogenized phage (Waldor and Mekalanos, 1996). The
second is a large pathogenicity island that encodes a toxin co-regulated
pilus gene cluster involving a type IV pilus that is the primary adhesion
and colonizing factor (Taylor et al., 1987) and acts as a phage receptor. The
third gene cluster, the RTX gene cluster, encodes a cytotoxin active against
cultured Hep-2 cells in V. cholera El Tor strains (Lin et al., 1999).
PCR Detection, Identifi cation and Characterization of V. cholerae
In January 1991, an outbreak of cholera started in Peru and rapidly spread
throughout most of Latin America. Within 15 mon over 450,000 cases
occurred with about 4,000 deaths. The causative organism was toxigenic
V. cholerae O1 of the El Tor biotype which is distinct from the U.S. Gulf
Coast strains. Fields et al. (1992) reported on the use of primers CTX2/
CTX3 (Table 5.2) that amplifi ed a 564-pb sequence of the ctxA gene for its
detection in 150 V. cholerae isolates derived from patients, food, and water
from the 1991-1991 outbreak. One hundred forty isolates were found to be
toxigenic both by PCR and immuno assay.
Koch et al. (1993) reported on the development of a PCR assay for
detection of V. cholerae seeded onto oysters, crab meat, shrimp, and lettuce.
The primers P1/P3 (Table 5.2) amplifi ed a 778-bp sequence of the ctxB
gene from a V. cholerae O1 strain. Seeded foods were homogenized or
rinsed with APW followed by a 6-8 h enrichment incubation at 37°C. One
ml enrichments were boiled and 2 to 5 ml added to 100 ml PCR reaction
volumes. A detection limit of 1 CFU/10g of food was obtained.
DePaola and Hwang (1995) determined the optimum conditions of
enrichment for detection of V. cholerae by the PCR. Recovery and PCR
detection was signifi cantly greater from oyster homogenates diluted 1:100
in alkaline peptone water and incubated at 42°C for 18-21 h. The primers
used (Table 5.2) were from Fields at al. (1992) and amplifi ed a 564-bp of
the ctxA gene.
Ripabelli et al. (1999) reported on the occurrence of various pathogenic
Vibrio species from mussels harvested from approved shellfi sh waters in
the Adriatic Sea. V. cholerae O1 and O139 serotypes were not detected.
However, V. cholerae non-01/non-O139 was found in only 1.6% of the
samples compared to 32.2% for V. alginolyticus. PCR with primers STO-F/
STO-R (Table 5.2) were used to amplify a 238-bp sequence of the sto gene
that encodes the thermotolerant enterotoxin of V. cholerae and revealed the
absence of the sto gene in all of these environmental isolates of V. cholerae.
Chow et al. (2001) developed a PCR assay for detection of the rtxA,
rtxC, (encoding the RTX repeat in toxin) and the ctxB toxin genes among