11
Vibrio parahaemolyticus—Seafood Safety
and Associations with Higher Organisms
Firdausi Qadri, Nandini Roy Chowdhury, Yoshifumi Takeda,
and G. Balakrish Nair
11.1. INTRODUCTION
Vibrio parahaemolyticus, a halophilic seafood-borne pathogen, is one of the 12 known
pathogenic species of the genus Vibrio. The halophile is one of the major bacterial causes
of food-borne illness that affects people who consume raw or improperly cooked seafood.
This bacterium was first identified from the intestinal contents of a victim of food poisoning
who had consumed half-dried sardines called “shirasu”, in Japan. Fujino (1951) reported the
discovery of this pathogenic bacterium at the 25th Annual Meeting of the Japanese Association for Infectious Diseases in 1951. Since then, V. parahaemolyticus has been recognized
as the cause of sporadic cases of gastroenteritis predominantly in coastal areas of the world,
especially Japan, where consumption of inadequately cooked seafood is common. The past
few years have witnessed V. parahaemolyticus surface as a major public health threat following the emergence of clones in Asia, which have acquired pandemic potential (Matsumoto
et al., 2000). The pandemic caused by V. parahaemolyticus is the first of its kind with the first
pandemic serotype being detected in Taiwan and Indonesia in 1995 (Okuda et al., 1997). Since
then, numerous outbreaks of V. parahaemolyticus caused by the pandemic serotypes have been
reported from various countries extending from Japan to the American continent.
11.2. THE ORGANISM
V. parahaemolyticus is a Gram-negative marine bacterium. This bacterium can exist free
living or attached to submerged, inert, and animate surfaces, such as particulate matter, zooplankton, fish, and shellfish. Therefore, V. parahaemolyticus is a common microflora of marine
and estuarine environments. In laboratory, V. parahaemolyticus is commonly isolated from
Firdausi Qadri and G. Balakrish Nair • International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B),
Dhaka, Bangladesh.
Nandini Roy Chowdhury • Department of Medicine, State University of New York at Stony Brook, Stony Brook,
New York 11794, USA.
Yoshifumi Takeda • Jissen Women’s University, Hino, Tokyo, Japan.
Oceans and Health: Pathogens in the Marine Environment.
Edited by Belkin and Colwell, Springer, New York, 2005.
277
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F. Qadri et al.
Table 11.1. The current O (somatic antigen) and K (capsular antigen) serotype scheme∗ of
V. parahaemolyticus. The table shows the different K serotypes associated with each O serotype.
O
1
2
3
4
5
6
7
8
9
10
11
∗
K
1
3
4
4
15
18
19
20
23
24
19
5
28
5
8
17
46
20
25
26
32
38
41
56
58
60
64
69
6
9
30
7
10
47
29
11
60
31
12
61
33
13
68
37
34
43
42
45
49
48
53
54
55
56
63
21
44
52
36
22
39
70
74
61
40
66
50
71
51
61
57
67
58
73
59
65
72
The serotyping kit of V. parahaemolyticus is commercially available from TOSHIBA KAGAKU KOGYO Co. Ltd., Tokyo, Japan.
clinical samples on thiosulfate citrate bile salts sucrose (TCBS) agar (Kobayashi et al., 1963)
plates on which it grows as large, sticky, bluish-green colonies. A chromogenic agar medium
has recently been developed for effective detection of V. parahaemolyticus from seafood samples (Hara-Kudo et al., 2001). V. parahaemolyticus can be further confirmed by the toxR PCR;
the toxR gene is species specific and can distinguish V. parahaemolyticus from other members
of the genus Vibrio (Kim et al., 1999).
V. parahaemolyticus is classified on the basis of its somatic O antigen and capsular K
antigen; the flagellar H antigens are identical in all strains of this species (Sakazaki, 1992).
From the 13 known “O” types and more than 60 “K” types (Joseph et al., 1982; Hisatsune
et al., 1993), 75 combinations of O:K serovars of V. parahaemolyticus are possible. The current
antigenic schema of V. parahaemolyticus is shown in Table 11.1. Although serovars of most
strains isolated from diarrheal stools can be determined by existing, established O- and K
antisera, they may not be useful in identifying serovars of many isolates from marine sources
(Sakazaki, 1992). V. parahaemolyticus-mediated gastroenteritis is a multi-serotype affliction.
The pathogenicity of V. parahaemolyticus is conferred by the two well-characterized
hemolysin proteins, thermostable direct hemolysin (TDH), and TDH-related hemolysin (TRH),
although the mechanism of action of the two proteins at the cellular level is not yet well defined.
The genome of V. parahaemolyticus consists of two circular chromosomes (Yamaichi et al.,
1999) of 3,288,558 and 1,877,212 bp and contains 4832 genes (Makino et al., 2003). Genes
of the type III secretion system, identified in the genome of V. parahaemolyticus but absent in
V. cholerae, are also a central virulence factor of diarrhea-causing bacteria, such as shigella,
salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or
intimately interacting with intestinal epithelial cells (Makino et al., 2003).
11.3. CLINICAL DISEASE
V. parahaemolyticus causes three major syndromes of clinical illness in humans with the
most common syndrome being gastroenteritis, followed by wound infections and septicemia
(Daniels et al., 2000a; Morris & Black, 1985). Gastroenteritis caused by V. parahaemolyticus
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
279
Figure 11.1. Comparison of stool samples obtained from a patient infected by Vibrio parahaemolyticus (bucket with
red meat-wash stool sample) as compared to a patient infected by Vibrio cholerae O1 (bucket with the rice watery
stool sample).
is characterized by either watery or bloody diarrhea, vomiting, abdominal cramps, headache,
fever, general lassitude, nausea, chills, and tenesmus. The frequency of diarrhea is usually less
than 10 times a day, but some cases have a frequency of more than 21 times a day (Miwatani &
Takeda, 1976). Diarrhea or soft stools persist for 4–7 days or more. Patients usually come to the
hospital seeking help within a median duration of 6 h of onset of the disease (Qadri et al., 2003).
Fever is generally mild and is usually less than 39◦ C and in most cases it does not last for more
than 2 days (Saito, 1967). Stool of patients with diarrhea at the acute stage are of a characteristic
meat washed/reddish watery consistency as shown in Figure 11.1 (Qadri et al., 2003). Dehydration requiring intravenous fluid replacement therapy is needed in about 60% of the patients. Significant decrease in blood pressure in severe cases of food poisoning due to V. parahaemolyticus has also been reported with low blood pressure being observed in patients during the first
and second days of the disease (Saito, 1967). The incubation period of V. parahaemolyticusassociated gastroenteritis varies between 4 and 96 h (Miwatani & Takeda, 1976), the mean
incubation period being 15 h (Centers for Disease Control and Prevention, 1998).
Daniels et al. (2000) reported that 5% of the patients with V. parahaemolyticus infection, whose cases were reported to the CDC between 1988 and 1997, had septicemia.
Epidemiological data on 690 Vibrio infections in Florida during 1981–1993 showed a correlation between preexisting liver disease and occurrence of primary septicemia caused by
V. parahaemolyticus with a fatality rate of 44% for cases with septicemia (Hlady & Klontz,
1996).
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F. Qadri et al.
Wound infections are considered to be the source of infection when a patient incurred
a wound before or during exposure to seawater or seafood drippings and when a culture
of the wound, blood, or of a normally sterile site yielded V. parahaemolyticus. A case of
panophthalamitis caused by V. parahaemolyticus that resulted from the contamination of a
wound with water from a pond in inland Georgia has also been reported (Tacket et al., 1982). A
strain of V. parahaemolyticus serotype O5:K17 was isolated from a case of purulent ear infection
in a child from Burgas (Aldova, 1989). Ear infections, eye infections, and peritoneal infections
caused by V. parahaemolyticus have also been reported by other workers (Hornstrup & GahrnHansen, 1993; Daniels et al., 2000a).
11.4. INCIDENCE AND GEOGRAPHIC DISTRIBUTION
V. parahaemolyticus-associated gastroenteritis has been reported from North America,
Central America, Europe, Asia, and Africa. Numerous studies have shown that this organism is
pathogenic for human populations residing in different geographical locations. In Asia, V. parahaemolyticus is an established enteric pathogen in Japan, where consumption of uncooked
seafood is common. V. parahaemolyticus has been reported to cause about 50–70% of the cases
of bacterial food poisoning in Japan (Miwatani & Takeda, 1976; Obata et al., 2001). Apart
from Japan, various Asian countries, such as Thailand (Echeverria et al., 1983; Sriratanaban &
Reinprayoon, 1982), India (Chatterjee et al., 1978; Bag et al., 1999), Bangladesh (Hughes
et al., 1978; Bhuiyan et al., 2002), Laos (Matsumoto et al., 2000), Vietnam (Neumann et al.,
1972; Van and Tuan, 1974), Tanzania (Mhalu et al., 1982), Hong Kong (Ho & Wong, 1985),
Indonesia (Matsumoto et al., 2000), Philippines (Adkins et al., 1987), and Kuwait (Matsumoto
et al., 2000), have reported the isolation of V. parahaemolyticus from patients suffering from
diarrhea. V. parahaemolyticus has also been isolated from diarrheal cases in Russia (Boiko
et al., 1994), China (Jiang, 1991; Wu et al., 1998), Korea (Matsumoto et al., 2000), and Taiwan
(Wu et al, 1996; Wong et al., 2000).
In Europe, V. parahaemolyticus has been isolated from various areas such as the Baltic Sea,
the North Sea, the Mediterranean Sea (Miwatani & Takeda, 1976), and the Black Sea (Aldova
et al., 1971). V. parahaemolyticus-mediated infection was reported in Spain (Miwatani &
Takeda, 1976; Molero et al., 1989), Greece (Miwatani & Takeda, 1976), Britain (Barrow &
Miller, 1972), Turkey (Miwatani & Takeda, 1976), Denmark (Kristensen, 1974), Yugoslavia
(Muic & Zekic, 1974), and the Scandinavian areas (Hornstrup & Gahrn-Hansen, 1993). A
survey conducted by Ayres and Barrow (1978) showed that this halophile was widely distributed
in the British coastal waters, and during the same year Haghighi and Waleh (1978) reported, for
the first time, the presence of V. parahaemolyticus in the waters of Persian Gulf. The first study
on the incidence of halophilic Vibrio in coastal waters of Guadeloupe was conducted in 1978
and V. parahaemolyticus was isolated from 53 of 100 water samples investigated (Papa, 1980).
The occurrence of V. parahaemolyticus has been reported from marine and estuarine bathing
areas in the Danish coastal waters (Larsen et al., 1981), from the Northumberland Strait of
Nova Scotia (Robertson & Tobin, 1983), and from samples collected from the bathing areas
on the German North Sea coast although a parallel investigation in the German North coast
of fecal specimens from patients suffering from acute enteric disease for this organism gave
negative results (Heinemeyer, 1984). Investigations in the Elbe river flowing through Hamburg
between June 1981 and December 1982 revealed the isolation of 42 Kanagawa-negative strains
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
281
of V. parahaemolyticus from 147 water samples that were screened (Bockemuhl et al., 1986).
The presence of V. parahaemolyticus in seashore water samples collected along the coastline in
Bulgaria and Romania has also been documented (Aldova, 1989). Studies on toxigenic vibrios
in surface water and sediment samples of the freshwater environment of Ohta river showed the
presence of V. parahaemolyticus serotype O4:K34 (Venkateswaran et al., 1989).
In USA, V. parahaemolyticus-mediated outbreak was first identified in 1971 during which
320 people suffered from gastroenteritis in Maryland due to food poisoning (Dadisman et al.,
1972; Molenda et al., 1972). This was the first confirmed outbreak of food poisoning due to
V. parahaemolyticus outside Japan. In 1997, outbreaks of V. parahaemolyticus infections due
to consumption of raw oysters were reported in the Pacific Northwest including California,
Washington, and Oregon in the United States and also from British Columbia (Centers for
Disease Control and Prevention, 1998). V. parahaemolyticus-mediated gastroenteritis has been
reported from Galveston Bay, Texas, New York, New Jersey, and Connecticut (Centers for
Disease Control and Prevention, 1999). The isolation of V. parahaemolyticus from cases of
gastroenteritis in and around Montreal and Quebec City of Canada has also been reported
(Thomson & Thacker, 1974). Bubb (1975) studied the prevalence of V. parahaemolyticus
in the Natal and Eastern Cape coastal water of South Africa and in patients suffering from
gastroenteritis in Togo, West Africa. In Australia, V. parahaemolyticus has been reported from
patients with acute diarrhea in Sydney (Pryor et al., 1987).
11.5. PATHOGENESIS AND VIRULENCE FACTORS
11.5.1. Kanagawa Phenomenon
Clinical strains of V. parahaemolyticus were observed to produce hemolysis on special
blood agar medium (Kato et al., 1965). In 1968, Wagatsuma developed a special medium
for measuring the hemolytic character of V. parahaemolyticus called the Wagatsuma agar, a
high-salt (7%) blood agar (defibrinated human or rabbit blood) medium containing d-mannitol
as the carbohydrate source. The hemolysis observed on Wagatsuma agar medium, referred
to as the Kanagawa phenomenon (KP), has diagnostic as well as pathogenic significance for
V. parahaemolyticus. KP is known to occur due to the expression of TDH that is more frequently detected in clinical strains of V. parahaemolyticus. Only 1–2% of the environmental
strains of V. parahaemolyticus express the hemolytic protein and therefore most non-clinical
isolates of V. parahaemolyticus are KP-negative (Miyamoto et al., 1969). Studies involving
human volunteers have shown that ingestion of 2 × 105 –3 × 107 CFU of KP-positive V. parahaemolyticus can lead to the rapid development of gastrointestinal illness, whereas volunteers
receiving as many as 1.6 × 1010 CFU of KP-negative V. parahaemolyticus exhibited no signs
of diarrhea (Sanyal & Sen, 1974; Oliver & Kaper, 1997).
11.5.2. Thermostable Direct Hemolysin
The pathogenicity of V. parahaemolyticus is well correlated, since a long time, to the
presence of TDH that produces beta-type hemolysis on Wagatsuma agar (Sakazaki et al., 1968;
Miyamoto et al, 1969). The hemolysin is a homodimer protein with a molecular mass of 46
kDa, each peptide being composed of 165 amino acids (Tsunasawa et al., 1987; Honda & Iida,
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F. Qadri et al.
1993). The biologically active hemolysin is formed by non-covalent association of subunits
that are not linked together by disulfide bonds (Tsunasawa et al., 1987). Hemolysis of human
erythrocytes by TDH is a two-step process consisting of adsorption of the hemolysin to human
erythrocytes and the step(s) following adsorption (Sakurai et al., 1975). Trp65 and Leu66 are
essential for the hemolytic property of TDH (Toda et al., 1991; Baba et al., 1992) that acts as
a “pore-forming toxin” in temperature-dependent and independent steps (Honda et al., 1992).
In addition to its hemolytic nature, TDH has been found to be cytotoxic to a variety of
cell types (Takeda, 1983). A study investigating the enterotoxicity of TDH for human colonic
epithelial cells showed that TDH increases Cl− secretion through mechanisms involving cell
binding and Ca++ influx, followed by the elevation of Ca++ concentration in association with
protein kinase C phosphorylation (Takahashi et al., 2000). A dose-dependent increase in the
intracellular free calcium has been reported in Caco-2 and IEC-6 cells (human and rat cell
monolayers) (Raimondi et al., 2000). Recently, TDH was shown to induce cytotoxicity on
cultured rat embryonic fibroblast cells both from outside and inside the cells and could kill the
cells through apoptosis (Naim et al., 2001a,b).
TDH is encoded by the tdh gene located in the chromosome, and all KP-positive
V. parahaemolyticus strains contain two tdh gene copies, tdh1 and tdh2, that are 97% homologous (Nishibuchi & Kaper, 1990). Construction of isogenic mutants defective in either
tdh1 or tdh2 revealed that the KP and >90% of the total TDH protein production were attributable to expression of the tdh2 gene (Nishibuchi & Kaper, 1990; Nishibuchi et al., 1991).
This was possibly due to the increased transcriptional activation of tdh2 gene copy by the
activator protein Vp-ToxRS encoded by the toxRS gene (Lin et al., 1993; Nishibuchi & Kaper,
1995). Although the protein products of the two tdh loci are immunologically indistinguishable, the predicted amino acid sequences of the gene products (mature proteins) vary by seven
amino acid residues (Nishibuchi & Kaper, 1990). Most of the V. parahaemolyticus strains that
showed weak hemolysis on Wagatsuma agar have been shown to possess a single copy of the
tdh gene in contrast to the gene duplication observed in the KP-positive strains (Nishibuchi
& Kaper, 1990). On the other hand, only a few KP-negative strains were found to possess a
single copy of the tdh gene (tdh5) and only one strain was found to carry an additional tdh
copy on a 35 kb plasmid (tdh4) apart from the chromosomal copy (tdh3) (Nishibuchi & Kaper,
1995). Despite variations observed in the nucleotide sequences, the genes of the KP-negative
strains encoded TDH proteins were found to be very similar to the ones encoded by the tdh1
and tdh2 genes of KP-positive strains, i.e., they had hemolytic and other biological activities
and were immunologically indistinguishable (Baba et al., 1991; Honda et al., 1991; Yoh et al.,
1991). These results suggested that low-level expression of the tdh genes in the tdh-bearing
KP-negative strains could be the reason for the manifestation of such a phenotype.
11.5.3. TDH-related Hemolysin
The role of TRH in V. parahaemolyticus pathogenesis was first identified during an outbreak of gastroenteritis in the Maldive Islands; KP-negative isolates of V. parahaemolyticus
associated with the outbreak were found to produce TRH but not TDH (Honda et al., 1988).
Biological, immunological, and physicochemical characteristics of TRH have been found to
be similar but not identical to those of TDH (Honda et al., 1988). TRH is encoded by the trh
gene, two copies (trh1 and trh2) of which are found to be chromosomally located in the V. parahaemolyticus genome. The two trh loci share 84% sequence identity (Kishishita et al., 1992).
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
283
11.5.4. Urease Production
V. parahaemolyticus strains usually do not produce urease. Less than 10% of the strains
were positive for urease activity in most investigations reported between 1963 and 1974
(Sakazaki et al., 1963; Twedt et al., 1969). However, recent reports have shown increased
detection of urease-positive strains from clinical origin isolated from Asia, North America,
and South America (Cai & Ni, 1996; Kelly & Stroh, 1988; Magalhaes et al., 1991; Okuda
et al., 1997; Suthienkul et al., 1995). Studies carried out by different workers have shown
that the relatively rare urease-positive phenotype of V. parahaemolyticus is always associated
with the possession of the trh gene (Okuda et al., 1997; Osawa et al., 1996; Suthienkul et al.,
1995), making urease production a good clinical diagnostic marker for virulent (trh-positive)
V. parahaemolyticus (Suthienkul et al., 1995). The association between urease production and
possession of the trh gene has been shown to be due to a genetic linkage between the structural
gene for urease (ureC) and trh on the chromosome of virulent V. parahaemolyticus strains
(Iida et al., 1997). Recently, it has been shown that the urease gene cluster does not regulate
the expression of the closely associated trh gene or the tdh gene (Nakaguchi et al., 2003).
11.5.5. Other Putative Virulence Factors
Isolation of clinical strains of V. parahaemolyticus that express neither TDH nor TRH has
indicated the possibility of existence of other virulence factors. Studies on the invasive ability
of V. parahaemolyticus indicated that active processes in cells, such as signal transduction by
tyrosine protein kinase, may be involved in the internalization of this bacterium in Caco-2
cells and that actin filaments and cytoskeletal structure may have important roles in this process (Akeda et al., 1997). These results indicate that the disease caused by some isolates of
V. parahaemolyticus could be attributable not only to toxin production but also to invasion into
intestinal epithelium. A serine protease (protease A) has been purified directly from the supernatant of V. parahaemolyticus and identified as a potential virulence factor (Lee et al., 2002).
The protease A was a monomeric protein having a molecular mass of 43 kDa and an isoelectric
point of 5.0. The protease could be inhibited by the serine protease inhibitors, and was found
to have significant effects on the growth of Chinese hamster ovary, HeLa, Vero, and Caco-2
cells. The purified protease-induced tissue hemorrhage and caused death of experimental mice
when injected intravenously and intraperitoneally (Lee et al., 2002).
11.6. SEAFOOD SAFETY
11.6.1. Ecology of V. parahaemolyticus
Occupying a variety of niches, V. parahaemolyticus is a common bacterium in marine
and estuarine environments. It can exist planktonically or attached to submerged, inert, and
animate surfaces, including particulate matter, zooplankton, fish, and shellfish (Kaneko &
Colwell, 1973, 1975). Kaneko and Colwell (1975) have shown that V. parahaemolyticus had
the highest efficiency for adsorption onto chitin as compared to other bacterial strains tested, and
the adsorption effect was one of the major factors determining the distribution of this species
in the estuarine system. V. parahaemolyticus have been isolated from seawater and seafood in
the Hawaiin Archipelago (Yasunaga, 1965), Puget Sound (Baross & Liston, 1968), Washington
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F. Qadri et al.
State estuary (Baross & Liston, 1968; Kaysner et al., 1990), Gulf of Mexico (Ward, 1968), and
southern Atlantic coast (Ward, 1968). Despite its halophilic nature, V. parahaemolyticus has
been isolated from saline-free waters (Joseph et al., 1982), and its occurrence in freshwater
systems has been construed as a fortuitous incidence that is probably related to tidal drift of
the organism from the upper reaches of rivers (Kaper et al., 1981) or to its introduction by
ambulatory cases or carriers (Sarkar et al., 1983). In the same study, it was evident that fishes
provide an ideal substrate for the survival and proliferation of V. parahaemolyticus (Sarkar
et al., 1985). Fishes and perhaps other aquatic animals are reservoirs of V. parahaemolyticus in
freshwater environs. The highest recovery of this halophile in both freshly caught and market
samples of freshwater fishes were from fecal swabs, suggesting that the gastrointestinal tracts
of freshwater fishes provide an excellent microcosm for proliferation of V. parahaemolyticus
in freshwater areas (Sarkar et al., 1985). Consequently, fecal matter of fishes could form a
significant source of inoculum of the halophile into the surrounding medium. The survival of
such cells entering into the environment is likely to be transient and to be dependent on another
host and on the prevailing environmental conditions.
Hemolymph and external carapace of normal blue crabs have been shown to contain
V. parahaemolyticus (Sizemore et al., 1975; Davis & Sizemore, 1982) along with other members of the Vibrio species. The occurrence of V. parahaemolyticus from crabs collected from
Galveston Bay in Texas were higher during the summer months indicating relatedness of its
occurrence to higher water temperatures. The bacterial count was found to be higher in the interior parts of the organism especially in the digestive tract (Davis & Sizemore, 1982). Study of
crab specimens in Lebanon also showed the presence of this halophile (Abdelnoor & Roumani,
1980).
11.6.2. Seafood as a Medium of Survival and Transmission
V. parahaemolyticus infections are usually acquired by persons who eat raw or undercooked seafood or whose wounds are exposed to warm seawaters. Raw, mishandled, improperly stored, or cross-contaminated cooled seafood are generally the source of illness
with shrimp, crab, lobster, and oysters producing the most incidences. A large number of
V. parahaemolyticus-mediated infections occurring in the coastal population have not only
been due to seafood-borne transmission but also due to exposure to seawater. In an outbreak of
gastroenteritis on two Caribbean cruise ships in late 1974 and early 1975, 697 passengers and
27 crew members were affected following consumption of seafood that was contaminated with
V. parahaemolyticus after cooking in seawater from the ships’ internal seawater distribution
systems (Lawrence et al., 1979).
Ingestion of seafood is the most common mode of transmission for V. parahaemolyticus
as evidenced by numerous outbreaks reported from different parts of the world where people
regularly consume improperly processed and undercooked seafood. Various marine organisms
that include marine fish, crustacean shellfish (prawns, lobsters, crabs), and molluscan shellfish
(oysters) have been implicated as vehicles for transmission of this human pathogen.
Person-to-person transmission of infection may occur from contact with an ill individual,
either directly or via secondary contamination of household water, food, or fomites. Duangmani
et al. (1985) determined the frequency of mother-to-infant transmission of enteric pathogens
and documented the isolation of V. parahaemolyticus from the stool of one of the 75 Thai
mothers just prior to delivery. V. parahaemolyticus has been detected from flies in all months
of the year in Calcutta. The mean percentage of isolation was: housefly, 14.1; blue bottle
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
a
Acute stage of infection
285
b
Healthy control
Figure 11.2. (A) Acute inflammation in a rectal biopsy section processed from a patient infected with Vibrio parahaemolyticus. (B) A normal histopathology is seen in the section obtained from a healthy control.
fly, 18.5; external washing, 10.1; viscera, 22.6; flies of sweet shops, 9.7; and those from fish
market, 22.7 (Chatterjee et al., 1978). Flies revealed higher (4.4%) isolation of Kanagawapositive V. parahaemolyticus strains than those of water and fish specimens (0.8%) studied.
11.7. IMMUNE RESPONSE
Until recently, very little was known about the immunological and inflammatory responses
and the immunopathogenesis due to natural infection caused by V. parahaemolyticus. The
damage in the small bowel autopsy of four persons who had died of V. parahaemolyticusinduced food poisoning was first reported about 40 years ago (Okudaira, 1962). In a recent
study done on Bangladeshi patients, both the small intestinal and large bowel tissues were
affected by the pathogen, and epithelial damage and denudation were seen at both sites of the
intestine. The duodenal and rectal sites showed acute inflammatory responses with infiltration
of surface epithelium, crypt, and lamina propria with neutrophil polymorphs (Fig. 11.2). The
lamina propria showed edema, congestion of blood vessels, and hemorrhage with an increase
in circulating macrophages.
TDH-specific mucosal immune responses, reflected by antibody secreting cells in the
circulation and antibodies in the intestinal secretions have been detected after natural infection.
The systemic responses to TDH in the IgA and IgG isotypes were also increased (Fig. 11.3).
A response to the lipopolysaccharide antigen of the homologous infecting strain was also seen
in both local and systemic circulation.
Inflammatory markers were upregulated in the systemic and mucosal compartments.
Increased concentrations of C-reactive protein and the nitric oxide metabolites were seen in the
systemic circulation at the acute stage. Elevated levels of TNF-α and lactoferrin are detected
in the local secretions and in plasma, while the chemokine IL-1β is elevated only in the
intestine. All these results suggest the intensely inflammatory nature of gastroenteritis caused
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F. Qadri et al.
1000
100
♣
♣
10
♣*
♣*
♣*
♣*
♣*
♣*
♣*
♣
d14
d30
♣
♣
1
d2
d7
d0
Figure 11.3. Immune response in sera to the thermostable direct hemolysin in patients infected with V. parahaemolyticus. Responses in the IgA ( r), IgG (), and IgM isotype () are shown at the acute stage (d2: day 2) and at convalescence
(day 7, 14, and 30) after onset of illness. Response is also compared to that seen in healthy controls (d0). Symbols
indicate statistically significant increases in comparison to the acute stage (∗) or to the response in the healthy controls
(♣).
by V. parahaemolyticus. The mucosal and systemic inflammatory responses are, however,
short-lived and revert to baseline levels by convalescence. There is a contrast to the response
seen in non-invasive infections caused V. cholerae or that seen in the overtly invasive Shigella
infections. In the former, the inflammatory response at the mucosal site is of a more subtle
nature, while in the latter, it is, in general, more severe and recovery is not seen as quickly as
observed in V. parahaemolyticus-induced disease. This is not altogether surprising since the
invasion process of the bacteria under in vitro conditions appears to be different from other
invasive enteric pathogens (Akeda, 2002).
Eight types of O:K combination of strains isolated from patients with diarrhea have been
tested for serum bactericidal killing. Strains belonging to different O:K serotypes exhibited
varying resistance to killing by pooled human serum, with the O8:K21 being most sensitive
followed in decreasing order by O3:K6, O5:KUT, O4:K55, O4:K37, O3:K29, O1:K25, and
O1:K56. An inverse relation was seen between bacterial resistance and onset of the mucosal
immune response in patients. Those infected with strains more susceptible to killing showed
a late onset of the antibody secreting cell response and vice versa (Qadri et al., 2003).
11.8. THE FIRST PANDEMIC V. parahaemolyticus
11.8.1. Origin and Global Spread of the Pandemic Strains
Unlike cholera, which is caused by only two virulent serotypes of V. cholerae, multiple serotypes of V. parahaemolyticus are associated with causing gastroenteritis. However,
from 1995 onward, most clinical isolates of V. parahaemolyticus isolated from different
countries belonged to the O3:K6 serotype. The abrupt appearance and rapid spread of the
O3:K6 strains to different countries after 1995 marked the beginning of the first pandemic
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
287
of V. parahaemolyticus. The first isolation of O3:K6 strain belonging to the new group was
reported from Indonesia and Taiwan in 1995 (Chiou et al., 2000; Matsumoto et al., 2000)
following which reports of isolation of strains belonging to the same serotype were obtained
from Japan, Thailand, Bangladesh, India, Vietnam, Laos, and Kuwait. Apart from the Asian
countries, the O3:K6 strains were also isolated from outbreaks in the United States during
1997–1998. Overall, the global incidence of V. parahaemolyticus had shown a marked increase
after 1995 and this increase was due to increased isolation of O3:K6 strains. This was the first
occurrence of a single serotype-associated outbreak of V. parahaemolyticus, and consequent
molecular studies revealed the genesis of a unique pathogenic clone, called the new O3:K6
clone that could spread rapidly and probably establish itself better in the human intestine.
Interestingly, in the following years, strains belonging to serotypes O1:KUT, O4:K68,
and O1:K25 were also associated with the first pandemic of V. parahaemolyticus. The nonO3:K6 serotypes were characterized to have the same genetic makeup as the O3:K6 strains as
discussed below. Therefore, the first pandemic of V. parahaemolyticus was caused by a single
clone that was expressing variant surface traits.
11.8.2. Markers of Pandemicity
With the rapid global proliferation of the pandemic clone along with its propensity to show
varying serovars, it became important to identify markers that would help distinguish pandemic
strains from others. Matsumoto et al. (2000) first reported the use of group-specific PCR
(GS-PCR) for rapid identification of the pandemic clone. GS-PCR is based on the nucleotide
differences at the ToxRS region identified between the pre-1995 and post-1995 group of O3:K6
strains. All post-1995 O3:K6 strains analyzed were 100% homologous at the nucleotide level
of the ToxRS region and differed from pre-1995 isolates by 11–14 bp (Matsumoto et al., 2000).
However, differences between the two groups were found invariably at seven base regions and
two of these seven bases were utilized for developing the group-specific PCR (Fig. 11.4), so
called because of its ability to distinguish the new group of O3:K6 strains (post-1995) from
the old group (pre-1995) of O3:K6 strains, which amplified a 651-bp new O3:K6 specific
fragment. The pandemic properties of serotypes O1:KUT, O4:K68, and O1:K25 were first
identified by GS-PCR analysis.
bp
1
2
3
4
5
6
7
1500
600
651 bp
100
Figure 11.4. Group-specific PCR for identification of pandemic strains of V. parahaemolyticus. PCR products were
loaded on 1% agarose gels and the amplicons visualized by ethidium bromide staining. Lane 1: 100 bp molecular weight
standard (Gibco-BRL, USA); lane 2: negative control (non-pandemic strain AQ3810, an O3:K6 1983 isolate from
Singapore); lanes 3 and 4: O3:K6 pandemic strains (VP294, 1999/Calcutta, and VP362, 2000/Calcutta, respectively);
lane 5: O4:K68 pandemic strain (VP293, 1999/Calcutta); lane 6: O1:KUT pandemic strain (VP356, 2000/Calcutta);
and lane 7: O1:K25 pandemic strain (VP343, 2000/Calcutta).
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F. Qadri et al.
1
2
3
4
5
6
7
388 bp
Figure 11.5. Detection of the ORF8 from the bacteriophage f237 by PCR. PCR products were loaded on 1% agarose
gels and the amplicons visualized by ethidium bromide staining. Lane 1: negative control (non-pandemic strain
AQ3810, an O3:K6 1983 isolate from Singapore); lanes 2 and 3: O3:K6 pandemic strains (VP294, 1999/Calcutta,
and VP362, 2000/Calcutta, respectively); lane 4: O4:K68 pandemic strain (VP293, 1999/Calcutta); lane 5: O1:KUT
pandemic strain (VP356, 2000/Calcutta); lane 6: O1:K25 pandemic strain (VP343, 2000/Calcutta); and lane 7: 100
bp molecular weight standard (Gibco-BRL, USA).
Apart from the GS-PCR assay, the presence of a unique f237 filamentous phage has
been proposed to be a genetic marker for the identification of pandemic strains. The f237
filamentous phage was initially shown to be present only in the new O3:K6 strains isolated after 1995 from different geographical regions (Nasu et al., 2000). The importance
of the phage assumed significance due to the presence of an open reading frame (designated
ORF8) encoding a protein that had no homology with any proteins in the database (Fig. 11.5).
The lower G + C content of this ORF indicated a recent acquisition of the ORF into the
phage, thus indicating the possibility of the association of the presence of the ORF8-encoded
protein and the pandemicity of the new O3:K6 strains. A PCR assay targeting the detection
of the ORF8 region was developed; and in such an assay apart from the O3:K6 strains, other
GS-PCR-positive strains were also found to carry this unique phage (Nasu et al., 2000). Therefore, the GS-PCR assay and the ORF8 assay are now routinely used as rapid diagnostic markers
for distinction of pandemic strains from the non-pandemic V. parahaemolyticus.
11.8.3. Molecular Epidemiology
The pandemic O3:K6 strains possessed only the tdh gene and not the trh gene. In order
to address the question of whether the evolutionary success of the pandemic strains could be
due to (a) increased copy numbers of the tdh gene, or (b) due to over-expression of the tdh
gene resulting in excess secretion of hemolysin, earlier findings reported by Bag et al. (1999)
and Okuda et al. (1997) assume significance. In these studies, restriction fragment length
polymorphisms of the tdh locus obtained were found to be identical in all the O3:K6 strains
isolated during 1996–1998 from Calcutta (Bag et al., 1999), and the amount of hemolysin
produced by the O3:K6 strains isolated in 1996 from Calcutta, as well as from travelers to
Japan (as measured by ELISA), was equal to that of hemolysin-producing strains belonging
to other serotypes (Okuda et al., 1997). Hence, the success of the pandemic strains was not
directly related to the possession of the tdh gene.
The molecular analysis of the pandemic V. parahaemolyticus strains involved techniques
that could identify macro as well as micro variations among the circulating strains. Thus,
289
Vibrio parahaemolyticus—Seafood Safety and Associations with Higher Organisms
kb
1 2 3 4 5 6 7 8 9 10
kb
10.0
5.0
1
2 3 4
5
6
10.0
5.0
3.0
3.0
2.0
2.0
1.0
1.0
0.5
kb
10.0
5.0
3.0
1 2 3 4 5 6 7 8 9 10
kb
1
2
3
4
5
6
10.0
5.0
3.0
2.0
2.0
1.0
1.0
0.5
Figure 11.6. Arbitrarily Primed PCR profiles of representative pandemic and non-pandemic isolates obtained with
primers 1281 (upper panel) and 1283 (lower panel). Upper panel left: AP-PCR profiles of representative pandemic
strains with the primer 1281. Lane 1: 1 kb molecular weight standard (Gibco-BRL); lanes 2–4: O3:K6 pandemic
strains; lanes 5 and 6: O4:K68 pandemic strains; lanes 7 and 8: O1:KUT pandemic strains; and lanes 9 and 10:
O1:K25 pandemic strains. Upper panel right: AP-PCR profiles of some randomly chosen non-pandemic strains. Lane
1: 1 kb molecular weight standard (Gibco-BRL); lane 2: O2:K3 (VP191, 1997, India); lane 3: O4:K13 (VP206, 1997,
India); lane 4: O3:K29 (VP263, 1998, India); lane 5: O1:K25 (TdP10, 1998, Thailand); and lane 6: O4:K9 (TdP16,
1998, Thailand). Lower panel left: AP-PCR profiles of representative pandemic strains with the primer 1283. Lane 1:
1 kb molecular weight standard (Gibco-BRL); lanes 2–4: O3:K6 pandemic strains; lanes 5 and 6: O4:K68 pandemic
strains; lanes 7 and 8: O1:KUT pandemic strains; and lanes 9 and 10: O1:K25 pandemic strains. Lower panel right:
AP-PCR profiles of some randomly chosen non-pandemic strains. Lane 1: 1 kb molecular weight standard (GibcoBRL); lane 2: O2:K3 (VP191, 1997, India); lane 3: O4:K13 (VP206, 1997, India); lane 4: O3:K29 (VP263, 1998,
India); lane 5: O1:K25 (TdP10, 1998, Thailand); and lane 6: O4:K9 (TdP16, 1998, Thailand).
the pandemic V. parahaemolyticus strains were subjected to analysis by AP-PCR, ribotyping,
PFGE, and MLST. The post-1995 O3:K6 strains were analyzed by AP-PCR and were shown
to exhibit an identical profile that differed considerably from each of the different profiles
exhibited by the pre-1995 O3:K6 isolates (Fig. 11.6). A comparative molecular analysis of
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pandemic strains belonging to four different serotypes by AP-PCR, ribotyping, and PFGE
showed that all the strains belonging to different serotypes were similar and had probably
originated from the same parent strain. The AP-PCR, ribotype, and PFGE profiles of the
pandemic strains were unique and distinct from the profiles of pre-1995 O3:K6 strains and
non-pandemic strains (Chowdhury et al., 2000a, 2000b). These molecular fingerprinting data
strongly supported the hypothesis that a single clone of V. parahaemolyticus had given rise
to this pandemic. This clone first appeared as the O3:K6 serotype and later disseminated
into other serotypes, namely O1:KUT, O4:K68, and O1:K25, in consecutive years. This phenomenon of relaying the pandemic genotype into strains of different serotypes could be a
survival mechanism of the pandemic clone to evade identification by the host immune system
to be a successful pathogen. However, the likely progenitor of the pandemic clone could not
be identified by any of the molecular techniques that have been applied so far to characterize
the V. parahaemolyticus strains. Further, multi-locus sequence analysis of four housekeeping
genes, such as gyrB, recA, dnaE, and gnd, of pandemic and non-pandemic strains irrevocably supported the hypothesis that the pandemic V. parahaemolyticus strains were clonal
in nature (Chowdhury et al., 2004). For each gene, the pandemic strains belonging to the
four different serotypes showed no allelic variation, unlike the multiple alleles that were observed for non-pandemic strains. Although the old O3:K6 strains shared a common allele
in the gyrB (encoding B subunit protein of DNA gyrase) and gnd (encoding 6-phospho gluconate dehydrogenase) genes with the pandemic strains, this data could not be extrapolated
to speculate that any of the pre-1995 isolates were indeed responsible for giving rise to the
pandemic strains. It may seem plausible that a pre-1995 O3:K6 strain could have given rise
to the new clone, however, the likely progenitor for the pandemic clone could not be clearly
defined.
11.9. IMPACT OF THE PANDEMIC ON THE SEAFOOD INDUSTRY
The sudden emergence of the V. parahaemolyticus pandemic and a consequent rise in the
number of outbreaks related to seafood consumption called for an immediate monitoring of
seafood safety procedures that are applied to different seafood industries. V. parahaemolyticus
concentration in associated shellfish and oysters are known to increase in summer and therefore water temperature monitoring during harvest became important to prevent outbreaks. In
addition, the US and Canadian officials allowed the sale of oysters that had less than 10,000
CFU of V. parahaemolyticus per gram of oyster that was a log less than the infectious dose
of 100,000 CFU or more (CDC, 1997). However, the above measures were unable to stop
V. parahaemolyticus-mediated outbreaks, and oyster beds around the outbreak regions were
closed down until the pathogen was no longer detected or the water temperature became
unsuitable for proliferation of the pathogen.
In a recently published risk assessment of V. parahaemolyticus in seafood by WHO,
it has been proposed that during harvest, water salinity and temperature contribute to the
concentration of V. parahaemolyticus in seafood and post-harvest numerous factors, which
include time of refrigeration, air temperature, cool-down time, and storage time, contribute
significantly to the final concentration of V. parahaemolyticus in the food that is consumed
(www.who.int/fsf/Micro/index.htm). Much more needs to be learned in these areas before the
burden of V. parahaemolyticus disease can be alleviated.
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291
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