Vibrio parahaemolyticus- An emerging foodborne pathogen-A Review

Vibrio parahaemolyticus is a halophilic gram negative, motile, oxidase positive, straight or curved rod-shaped, facultative anaerobic bacteria that occur naturally in the marine environment. They form part of the indigenous microflora of aquatic habitats of various salinity and are the major causative agents for some of the most serious diseases in fish, shellfish and penacid shrimp. This human pathogen causes acute gastroenteritis characterized by diarrhea, vomiting and abdominal cramps through consumption of contaminated raw fish or shellfish. V. parahaemolyticus is the leading cause of gastroenteritis due to the consumption of seafood worldwide. The incidence of V. parahaemolyticus infection has been increasing in many parts of the world, due to the emergence of O :K serotype carrying the tdh gene which is responsible for most outbreaks worldwide. The pathogenicity of 3 6 this organism is closely correlated with the Kanagawa phenomenon (KP +) due to production of Kanagawa hemolysin or the thermostable direct hemolysin (TDH). The TDH and TRH (TDH-related hemolysin) encoded by tdh and trh genes are considered to be important virulence factors.


Introduction
microflora of aquatic habitats of various salinity (Colwell et. al., 1984) and are the major causative Vibrio parahaemolyticus is a gram-negative agents for some of the most serious diseases in bacterium that occurs naturally in the estuarine fish, shellfish and penacid shrimp (Lee et.al., environment.This human pathogen is frequently 1996; Sugumar et.al., 1998).found in seawater, sediments, plankton, finfish Shrimp culture has grown into a major and shellfishes (Pavia et. al., 1989) and 30 different economic industry worldwide.The rapid marine species, including eel, crab, clams, expansion of commercial culture of shrimp is oysters, lobsters, scallops, sardines, shrimp, and threatened by vibrio infection affecting its squid (Fishbein et. al., 1974) and can cause acute survival and growth (Vandenberghe et. al., 1999).gastroenteritis characterized by diarrhea, vomiting The occurrence of this organism in fresh water and abdominal cramps through consumption of has been construed as a fortuitous incidence that contaminated raw fish or shellfish (Rippey, is probably related to tidal drift of organism from 1994).It also causes traveller's diarrhea, wound upper reaches of the river or its introduction by infection, ear infection and secondary septicemia ambulatory cases or carriers (Sarkar et. al., 1983). in humans (Pavia et. al., 1989).This organism The haemolytic activity of pathogenic was first identified as a causative agent of foodstrains of this organism on Wagatsuma's agar is borne gastroenteritis after a large outbreak (272 popularly known as kanagawa phenomenon, illnesses and 20 deaths) associated with which is due to Thermostable Direct Haemolysin consumption of sardines in Japan in 1951 (Fujino (Honda and Iida, 1993).Some of the kanagawa et.al., 1953).It forms part of the indigenous negative V. parahaemolyticus produces a toxin where seafood is often consumed (Pan et. al., named TDH-related haemolysin (TRH) also 1997).causing gastroenteritis (Honda et. al., 1988).The Following a social get-together some staff TDH and TRH encoded by tdh and trh genes are members of Christian Medical College and considered to be important virulence factors.
Hospital, Vellore involved in gastroenteritis due Enumeration of this organism from seafood to V. parahaemolyticus and this has emphasized is important in the context of current FDA the public health hazard of this organism in India guidelines, which stipulate that seafood should (Lalitha et. al., 1983).V. parahaemolyticus accounts contain less than 10,000 cells per gram for about 70% of the gastroenteritis cases (McCarthy et. al., 1999), but inadequacy of this is associated with seafood in Japan (Kaneko and indicated by the outbreaks that occurred in the Colwell, 1975), whereas Nishibuchi (2004) United States, despite the V. parahaemolyticus reported that this food poisoning are most number being lower than the permissible limit common in Japan and Southeast Asia, although (Nair and Hormazabal, 2005 (2000) observed that the of contaminated raw or partially cooked fish or incidence of V. parahaemolyticus infection in shell fish, particularly oysters or exposure to a recent years has been increasing in many parts of marine environment (Khan et. al., 2002).
the world, due to the emergence of O :K serotype 3 6 V. parahaemolyticus was first isolated carrying only the tdh gene that is responsible for following food poisoning outbreaks in Japan in the early 1950s.In India V. parahaemolyticus was most out breaks worldwide since, 1996.In recent first isolated from a case of gastroenteritis by years, outbreaks of V. parahaemolyticus infection Chatterjee et.al. (1970) and about 10% of the have increased in Japan, Taiwan (Infectious cases of gastroenteritis in patients admitted to the Disease Surveillance Center, NIID, 1999; Chiou Infectious Disease Hospital in Kolkata are due to et. al., 2000) and United States, due to consumption V. parahaemolyticus (Deb et. al., 1975).Etiological of raw sea food contaminated with O :K serovar  enterotoxins produced by human pathogens, 2x10 to 3x10 CFU.The mechanism of whose action is mediated by intracellular pathogenicity of has not been defined or fully calcium.TDH also raises the cytosolic free understood, but it was closely correlated with the 2+ calcium concentration [ca ] in non transformed Kanagawa phenomenon (KP +) due to production rat intestinal IEC-6 cells (Fabbri et. al., 1999). of Kanagawa hemolysin or the thermostable TDH has also cardiotoxic (Honda et. al., 1976a) direct hemolysin (TDH) (Takeda, 1983;and cytotoxic (Sakazaki et. al., 1974) (Miwatani and Takeda, 1976;Joseph et. al., diarrhea. 1983) and Vp-TRH (Honda et. al., 1988;1989).Clinical isolates of V. parahaemolyticus Nakayama et. al. (1995) described the fourth type most often produce either the TDH or TRH of hemolysin-Vp-TDH/II produced by a KP encoded by tdh and trh genes respectively clinical isolate of V. parahaemolyticus (O13: K, (Nishibuchi and Kapar, 1995).A molecular study untypable), which was bio-physico-chemically with tdh-and trh-specific DNA probes and immunologically similar, but not identical to demonstrated a strong association of two genes Vp-TDH, Vp-TRH and Vp-TDH/I.with clinical strains, suggesting that TRH as well ii.
Thermostable direct hemolysin-related as TDH is an important virulence factor (Shirai et.outbreak of gastroenteritis in the Republic of

I. Thermostable direct hemolysin (TDH):
Maldives in 1985, which produced TRH.Honda TDH was the first recognized virulence factor for and Iida (1993) reported that purified Vp-TRH V. parahaemolyticus and has been used as an showed various biological activities, such as fluid important marker for identifying virulent strains accumulation in rabbit ilea loops, increase of (Okuda et. al., 1997a;Cook et. al., 2002).TDH rabbit skin vascular permeability, and was produced from the tdh2 gene rather than the cardiotoxicity on cultured myocardial cell.Vp-tdh1 gene.The nucleotide homologies of tdh2 TRH played roles similar to Vp-TDH in the with tdh1, tdh3 and tdh4 were 97%, 98 Primary septicemia is reported in the 84% homologous to the trh gene (newly named individuals with chronic illness (Cook et. al.,trh1) and 54.8 -68.8% homologous to the tdh 2002) and becomes life-threatening to people gene, indicated that both the trh1 and trh2-having underlying medical conditions such as carrying strains should be considered potentially liver disease or immune disorders.In addition, V. virulent.Similar to TDH, TRH can induce parahaemolyticus also can cause wound chloride secretion in human colonic epithelial infection in people exposed to contaminated cells and is, therefore, considered a virulence seawater (Bonner et.al. 1983 Lecithin dependent hemolysin (LDH), which is one of the species specific gene fragments of V.
The incidence of food poisoning in Japan parahaemolyticus.Taniguchi et. al. (1990) cloned caused by this organism is restricted to the a new thermostable hemolysin (ä -VPH) gene summer months, very likely because of from a KP negative V. parahaemolyticus strain sensitivity of the organism to low temperatures into vector pBR 322 in E. coli K12.
(Kaneko and Colwell, 1973) and it can survive A number of possible virulence factors the winter in sediment, in scavenger fish and shell including a haemolysin, a Chinese hamster ovary fish (Noguchi and Asakava, 1967), but the (CHO) cell elongation factor and factors number of organisms isolated are less.responsible for cytotoxicity, invasiveness and Kaneko and Colwell (1975) reported that adherence (Blake et.al., 1980).V. parahaemolyticus when the temperature of seawater is below 13-0 produce a novel siderophore named vibrioferrin 15 C, V. parahaemolyticus is rarely isolated and under conditions of little or no iron (Yamamoto probably exists in a viable but non-culturable et.al., 1994;Yamamoto et. al., 1995).Yamamoto state (VBNC) and is not culturable on common et.al., (1999) reported higher levels of media.Many researchers reported the abundance vibrioferrin in the nutrient-depleted culture of of V. parahaemolyticus during summer in clinical isolates of V. parahaemolyticus than by temperate zone, when temperature was above o isolates from food or environmental sources, 25 C (CDC 1998; 1999; Khan et.al., 2002), when they were grown in a medium containing where as the organism is expected to be prevalent limited iron.They also reported that vibrioferrin throughout the year in the tropical zone like under iron-limited conditions might contribute to Malaysia (Elhadi et.al., 2004).the pathogenesis.
Deepanjali et.al. (2005) observed high levels of V. parahaemolyticus during the dry Symptoms season between January and May and decreased Although most oftenly it induces a selfduring post monsoon months.In tropical limiting watery diarrhea, it occasionally causes countries the seasonal cycle of the organism is bloody diarrhea and rarely sudden cardiac correlated with rainy and dry seasons; the lowest arrhythmia (Honda et. al., 1976b).Clinical numbers are found in rainy months, and the manifestations of V. parahaemolyticus include highest numbers are found in the dry season diarrhea, abdominal cramps, nausea, vomiting, (Neumann et. al., 1972).head ache, fever and chills (Takeda, 1983).

Dehydration, collapse, and abnormality on electrocardiograms have occurred in individual
Vibrio spp.have been isolated from cases (Joseph et.al., 1983;Honda and Iida, 1993; seawater, sea mud, or sea foods in Asia, North America, Australia, New Zealand, Africa, and causative agent in mortalities of blue crabs in Europe.It has been reported that vibrios are the Chesapeake Bay (Krantz et.al., 1969) and gulf predominant bacteria in the digestive tracts of shrimp (Vanderzant et. al., 1969) and has been oysters, clams and mussels (Sugita et. al., 1981; isolated from sea water and sediment (Bartly and Kueh and Chan, 1985), prawns (Yasuda and Slanetz, 1971).Kitao, 1980), and Artemia (Puente, et. al. 1992).
The first outbreak of V. parahaemolyticus in the United States was recorded in 1971 in Fish: ICMSF has recommended an acceptability Maryland, where in 425 cases of gastroenteritis limit of 100/g for V. parahaemolyticus organism associated with consumption of improperly in some fishery products (ICMSF, 1974).Quadri cooked crabs.(Molenda et. al., 1972 moderately halophilic organism in fresh water Crabs: V. parahaemolyticus was identified as the has been construed as a fortuitous incidence that is probably related to tidal drift of the organism Other products: Sanjeev and Stephen (1995) from the upper reaches of rivers or to its reported that all the strains of V. parahaemolyticus introduction by ambulatory cases or carriers.isolated from cooked, shucked clams were Sarkar et.al. (1983) reported that the incidence kanagawa negative and 50% of the isolated from and counts of V. parahaemolyticus were mussels were kanagawa positive.consistently higher in association with plankton Lhafi and Kuhne (2007) analyzed 90 blue than with water and sediment samples (Sarkar et. mussels  ferment sucrose and produce green colonies.So, Clinical samples: Epidemiological studies this medium is widely used for isolation of V. revealed the high incidence of human carriers of parahaemolyticus.Detection of pathogenic V. V. parahaemolyticus in Kolkata (Deb, et. al. parahaemolyticus is traditionally done by the 1975).Lee et.al., (2003) described the isolation Wagatsuma agar test for the Kanagawa reaction, and characterization of two V. parahaemolyticus which require fresh human or rabbit blood and strains isolated either from a patient's stool tends to give false positive reaction also sample or a diseased abalone with withering (Raghunath et. al., 2008).Production of TDH is syndrome (Huang et. al., 2001) and their implication responsible for phenomenon (Sakurai et. al., as the first evidence of a laboratory acquired 1974), which is manifested as â-type haemolysis zoonotic infection.Nithya Quintoil et.al. on a special blood agar called wagutsuma agar (2008b) reported that 13.33% of the stool (Miyamoto et. al., 1969).samples collected from ailing fish handlers gave positive results for V. parahaemolyticus in ii.Special media: In order to provide results Calcutta.
more rapidly with sensitivity similar to or greater than the conventional methods, several workers Processed foods: Anonymous (1998) studied the used different special media as detailed in food borne outbreaks due to various vibrio Table .41 C on the selective agar, followed by in-situ and 1.7% egg products.They also reported that testing for the fermentation of galactose and the causative organisms account to V.

iv. Latex agglutination test:
The presence of modified Elek test and immunohalo test (Honda TDH can be detected using a commercial latex et.al. (1982).agglutination kit.Rapid anti-V.parahaemolyticus Tomoyasu (1992) developed a method that involved Immunomagnetic separation to isolate outer membrane protein antibodies should be the specified K serovar of V. parahaemolyticus produced and latex particles sensitized with the from a mixture of a large no. of bacteria with affinity purified antibodies should be used as a other K serovars by using antisera (anti-rabbit reagent for the rapid identification of the immunoglobulin G) against K antigens coated on bacterium (Chang et al., 1994).
super para magnetic polysterene beads and v. Enzyme Linked Immunomagnetic Sorbent harvested using a magnetic concentrator.Assay (ELISA): Honda et. al. (1985) developed Chen and Chang (1996) developed immuno-ELISA for detection of tdh of V. parahaemolyticus.fluorescence microscopy method to target two The method was sensitive but preparation of antispecific outer membrane proteins 34 and 34KDa.tdh-alkaline phosphatase conjugate may be All V. parahaemolyticus tested produced strong needed.Chen and Chang (1995) developed an fluorescence under fluorescence microscopy, ELISA for detection of two outer membrane while only six of the other 63 bacteria generated proteins of V. parahaemolyticus.The detection weak to moderate fluorescence.limit of this assay was 10 ng/ml and need 18 hr for the enrichment step.
vii.Pulsed Field Gel Electrophoresis (PFGE): A PFGE method was developed for a molecular vi.Immunological methods: Honda and Finkelstein typing of V. parahaemolyticus (Wong et. al., (1979) and Honda et. al. (1980) developed the 1996), later the method has been used to type modified Elek test by adding anti haemolysin highly genetically diverse V. parahaemolyticus serum to a well cut in agar medium near a colony strains in sea food imported from Asian countries grown on the medium and the immunohalo test (Wong, et. al. 1999).was carried out by inoculating bacteria onto agar medium containing anti haemolysin antiserum or viii.Colony hybridization: Toxin gene elements in V. parahaemolyticus can be detected by colony anti haemolysin immunoglobulin G.
hybridization (Nishibuchi et. al., 1985; Banerjee Selective media for V. parahaemolyticus, et. al., 2002) or bot hybridization (Yamamoto et.BTB-teepol agar and modified arabinoseal., 1992) using appropriate nucleic acid probes.ammonium sulphate-cholate agar, were modified Non-radioactive labeled oligonucleotide probes for use in immunological detection of the TDH were used for binding tlh and tdh genes for produced by V. parahaemolyticus by using detection of total and pathogenic V. parahaemo-toxRS/new sequence.lyticus respectively (Deepanjali et. al., 2005).
Random Amplified Polymorphic DNA-PCR (RAPD-PCR): RAPD analysis is a commonly used ix.Polymerase Chain Reaction: The PCR represents method in PCR for typing and differentiation of a rapid procedure with both high sensitivity and bacteria and increasingly for the study of genetic specificity for the immediate detection and relationships between strains and species of identification of specific pathogenic bacteria microorganisms, plants and animals (Oakey et.from different food materials (Lantz et. al., 1994;al., 1998).Hill, 1996) et. al., 2003) and methods for the identification of V. parahaemolyticus tlh gene (Kaufman et.al., 2004) in V. parahaemoto the species level by using 25 Vibrio reference lyticus using specific primer sets and fluorogenic strains and 163 isolates from fishery products, probes.environmental sources and clinical samples using Reverse transcriptase PCR: The development of target genes of toxR, gyrB, tlh (tested with two reverse transcriptase PCR allows the detection of protocols) and the fragment PR72H and they mRNA with advantage over other methods, such reported that the PCR assay targeting the toxR as high sensitivity, rapid turn-around time and use and tlh gene achieved the highest performance, of total RNA instead of just poly (A)+ mRNA where as the genes gyrB and PR72H fragments (Rappolee et. al., 1988).were less reliable.
The mRNA is not expected to be detected in Tada et.al. (1992) developed a polymerase a VBNC bacterial cell because there is no growth chain reaction (PCR) protocol using DNA probes activity of the cell under the VBNC stage.This for specific detection of the tdh and trh genes of makes the detection of mRNA a suitable means of pathogenic V.parahaemolyticus.The procedure identifying VBNC V.parahaemolyticus.A could detect both tdh and trh genes in 400 fg of sample containing VBNC V.parahaemolyticus cellular DNA derived from 100 cells.However, will produce PCR assay targeting tlh gene and a this PCR protocol requires enrichment as a negative results by mRNA analysis (Siebert and pretreatment to detect pathogenic strains in fecal Larrick, 1995).samples and cannot be used to detect nonpathogenic V. parahaemolyticus strains.
Other PCR based methods: Wong   and aquatic foods should not be harvested form Appl. Environ.Microbiol., heavily contaminated water bodies. of this infection, V. prahaemolyticus should be (1999).Detection of total and hemolysingiven considerable preference in diagnosing the producing Vibrio parahaemolyticus in shell infections and eliminating the transmission fish using multiplex PCR amplification of tl, tdh and trh.J. Micrbiol. Meth., through aquatic products.O :K serotype carrying the tdh gene is responsible 3 6 caused by vibrios.Annu.Rev. Microbiol., for most out breaks worldwide O3:K6 and is products should be strictly followed.

(KP). Usually it induces a self-limiting watery
hemolysin (TRH): Honda et.al. (1988) isolated a al., 1990; Okuda et.al., 1997b).KP-negative V. parahaemolyticus strain from an Virulence factors of Vibrio Parahaemolyticus: cooking of marine and other aquatic foods prior 3. Banerjee, S.K., Pandian, S., Todd, E.C. and to consumption and hygienic handling of Farber, J.M. (2002).A rapid and improved products to avoid cross-contamination between method for the detection of Vibrio parahaemoraw and cooked products.Proper environmental lyticus and Vibrio vulnificus strains grown on hygienic measures should also be taken to avoid hydrophobic grid membrane filters.J. Food this infection i.e. the untreated sewage should not Prot., 65(6): 1049-1053.bedischarged in the sea/rivers near coastal areas4.Bartley, C.H. and Slanetz, L.W. (1971).Occurrence of Vibrio parahaemolyticus in since most of the catch is made from these areas estuarine waters and oysters of New Hampshire.
isolates from seawater are not of the human Random Amplified Polymorphic DNA-PCR pathogenic kind (they are KP-).The pathogenic typing of Vibrio parahaemolyticus isolated from strains can be detected by Kanagawa Phenomenon local cockles (Anadara granosa).American J.
.6% and pathogenesis.A survey of 285 strains of V. 98.6%, respectively (Nishibuchi and Kaper, parahaemolyticus revealed that the trh-positive 1985; Nishibuchi and Kaper, 1995).strains had a strong association with Yoh et.al. (1991) studied the characteristics gastroenteritis (Shirai et.al., 1990). of TDH encoded by four representative tdh genes Nishibuchi et.al. (1989) studied the gene and showed different Electrophoretic mobilities encoding TRH (trh gene) and proposed that the under non-denaturing condition.All the gene nucleotide sequence of trh gene, like tdh gene, products had hemolytic activities for various animal encoded the hemolysin subunit and the trh gene erythrocytes, stimulated vascular permeability in had significant nucleotide sequence homology the rabbit skin, and were lethal to mice, although with the tdh gene (68.4% with the tdh1 gene copy their potencies were slightly different.and 68% with the tdh2 gene copy).Nishibuchi et.al. (1992) demonstrated the Subsequently, Kishishita et.al. (1992) identified Carpenter, 1995).a variant of the trh gene (named trh2), which was ; Murray et.al. factor of V. parahaemolyticus (Takahashi et.al., 1998) and two deaths were reported among three 2000a; b).cases of wound infections in Louisiana and Mississippi after Hurricane Katrina in 2005 ). and Zuberi, (1977) reported high percentage of kanagawa positive isolates (52.5%) from fish and Oysters: Vanderzant et.al. (1973) recovered V. parahaemolyticus from 70% of oyster samples shell fish samples of Karachi.Abraham et.al. (1997) and Sanjeev (2002) from Galveston Bay collected over a 12-months 4 reported that the incidence of V. parahaemolyticus period with counts as high as 10 CFU/g.Vibrio in fresh, marine and brackish water fish in India bacteria concentrate in the gut of oysters and varied from 35 to 55%.The incidence of this other filter feeding molluscs (including clams, organism was reported high in faeces, least in mussels, scallops) where these adhere and external surface and moderate in gills of the fish multiply, making them resistant to depuration and (Sarkar et.al., 1985; Sanjeev and Stephen, 1995).protect from external disinfectants (Tamplin and Nithya Quintol et.al. (2008a) observed that the Capers, 1991).From 1997 to 1998, more than incidence of V. parahaemolyticus contamination 700 cases of illness associated with eating raw ranges from 15 to 46.66% of fish samples oysters contaminated with V. parahaemolyticus collected from different fish markets of West were reported in California, Oregon, Washington, Bengal, India.Connecticut, New Jersey, New York, and British Columbia of Canada (CDC, 1998; 1999).
samples from German WaddenSea and  al. 1985).reportedthat 74.4% of the samples contained Sanjeev (1999) reported that the counts of V. vibrio species, among these 39.5% contain V. parahaemolyticus were 460 MPN/ml and parahaemolyticus.
. The advantage of PCR over Bilung et.al. (2005) demonstrated that conventional isolation is its ability to distinguish genotyping V. parahaemolyticus isolates by using virulent and avirulent strains, saves time and RAPD-PCR is feasible for detection of various hence molecular methods are valuable in such strains and genetic relatedness among V. cases (Deepanjali et.al., 2005).parahaemolyticus strains.Okura et.al. (2003) developed PCR based assay to identify pandemic group of V. parahae-Real-time PCR: Real time PCR shows the ability molyticus by using oligonucleotide primer pair to process many samples with speed and derived from the group specific sequence of an consistency and single-tube amplification and arbitrary primed-PCR fragment by yielding a confirmation of target sequences (McKillip and 235-bp specific amplicon product.Drake, 2000).Real-time PCR can be used to Croci et.al. (2007) evaluated five PCR detect the tdh gene (Black stone Bej et.al.(1999)developedV.parahaemolyticus that is RS-PCR, REP-PCR multiplex PCR to detect total and pathogenic V. and ERIC-PCR to avoid the use of random parahaemolyticus targeting tlh, tdh and trh genes.primers.Further they reported that REP-PCR isOkura et.al.(2003)developed a multiplex PCR better over ERIC-PCR due to greater reproducibility.