A Review of the Current Status of Cultural and Rapid Detection Of

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Invited review

A review of the current status of cultural and rapid detection of

Vibrio parahaemolyticus

Bledar Bisha,1* Janet Simonson,2 Marlene Janes,2 Kellie Bauman1 & Lawrence D. Goodridge1

1 Department of Animal Sciences, Center for Meat Safety and Quality, Food Safety Cluster, Colorado State University, Fort Collins, CO 80523,

USA

2 Department of Food Science, Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, USA

(Received 13 July 2011; Accepted in revised form 29 December 2011)

Summary Vibrio parahaemolyticus is ubiquitous in estuarine environments and can be commonly found in seafood

products. This bacterial pathogen continues to emerge as an important cause of foodborne illness, and

several foodborne disease outbreaks caused by V. parahaemolyticus have been linked to the consumption of

contaminated seafood, in particular those consumed raw such as oysters. In response to these outbreaks,

especially during the 1990s, several cultural, immunological-based and molecular detection methods have

been developed, which allow for rapid detection and quantification of total and pathogenic V. parahae-molyticus. The development of molecular methodology has allowed for clinical and environmental isolates of

V. parahaemolyticus to be subtyped, thus providing the framework for risk-based strategies aimed at

controlling foodborne outbreaks cause by this pathogen. It is important that the detection and typing

methods strive to accomplish detection and differentiation of the pathogenic strains from environmental

(non-pathogenic) ones, as well as to detect the presence of the organism and not just the presence of

V. parahaemolyticus produced toxins, which can also be produced by closely related species. This review

covers the current status of detection and typing methodology for identification and characterisation of

V. parahaemolyticus from seafood.

Keywords Cultural methods, detection, immunological methods, molecular methods, Vibrio parahaemolyticus, Vibrio spp.

Introduction

Vibrio parahaemolyticus is a member of the familyVibrionaceae, genus Vibrio, comprising 30 species ofGram-negative, straight or curved, non-sporeformingrods, 0.50.8 lm wide by 1.42.6 lm long, including 13human pathogenic species (McLaughlin, 1995; Drakeet al., 2007). Vibrio parahaemolyticus ferments glucosewithout gas production and is oxidase positive. Theorganism is ornithine and lysine decarboxylase positive,and urease variable, depending on strain and serotype(Ray et al., 1978; Kaysner & DePaola, 2004; Martinez-Urtaza et al., 2006), and it is facultatively anaerobic

displaying polar flagellation (Levin, 2010). It is ahalophilic foodborne pathogenic bacterium that isubiquitous in estuarine environments and commonlyisolated from various seafoods, including oysters, scal-lops, octopus, shrimp, clam, crab, mackerel, sardines,codfish and more, and is recognised as the leading

causative agent of seafood-borne gastroenteritis in theUS (Kaysneret al., 1990; Wong, 2003; Su & Liu, 2007).

Large variations in V. parahaemolyticus presence andconcentrations in the environment and seafood aresignificantly dependent on the season, location andsample type (Earle & Crisley, 1975; Kaneko & Colwell,1975; DePaola et al., 1990, 2000; Kaysner et al., 1990;Cook et al., 2002). Vibrio parahaemolyticus requirespresence of salinity with optimum levels of 13% tosurvive and multiply, which is in the range of 0.83%levels commonly found in marine environments (De-Paola et al., 2000; Yeung & Boor, 2004). AlthoughV. parahaemolyticus presence in estuarine waters is

independent of faecal contamination, Watkins & Cabelli(1985) have reported that faecal contamination mayhave indirect effect on the increase in the concentrationof the bacterium by biostimulation of the zooplankton.

Vibrio parahaemolyticus disease outbreaks are sea-sonal and highly dependent on water temperature, withfewer outbreaks observed in the colder winter monthsand in temperate areas; the pathogen is rarely isolatedwhen the water temperature is below 1315 C (Kaneko

*Correspondent: Fax: (970) 491 5326;

e-mail: [email protected]

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& Colwell, 1975; DePaola et al., 2000, 2003). Outbreaksof V. parahaemolyticus associated gastroenteritis areusually attributed to the consumption of contaminatedseafood, especially raw seafood, such as raw oysters(Ellison et al., 2001). Daniels et al. (2000a) report thatbetween 1973 and 1998, forty outbreaks have been

notified from 15 US states and territories. After the mid1990s, serovar O3:K6 termed pandemic because of itsglobal dissemination (Nairet al., 2007) as well as relatedserotypes O4:K68 and O1:KUT have become increas-ingly more often associated with foodborne vibriosis(Yeung & Boor, 2004). Gastrointestinal illness caused byV. parahaemolyticus is typically accompanied by symp-toms including vomiting, diarrhoea, headache, nausea,low-grade fever and abdominal cramps and is com-monly self-limiting (Jandaet al., 1988; Nairet al., 2007).The infectious dose is believed to be approximately 105

cells, even though there is significant variance betweenreports (Yeung & Boor, 2004).

A thermolabile haemolysin that does not determine

pathogenicity is produced by all strains ofV. parahae-molyticus, and is encoded by the lht gene, giving rise topolymerase chain reaction (PCR) detection methodstargeting this gene for determination of total V. para-haemolyticus. By contrast, pathogenic strains have beenfound to contain thetdh gene encoding the thermostabledirect haemolysin (TDH) and thetrh gene encoding theTDH-related haemolysin (TRH), even though the trhgene is not found in all clinical isolates (Levin, 2010).TDH is responsible for the Kanagawa phenomenon(KP), confirmed via -haemolytic action on Wagatsu-mas Agar (Wagatsuma, 1968), which designates path-ogenic strains as KP+ and environmental strains as

KP- (Chun et al., 1975). Only about 12% of theenvironmental isolates are KP+, while the overwhelm-ing majority of clinical isolates are KP+ (Sakazakiet al., 1968; Miyamotoet al., 1969; Nishibuchi & Kaper,1995; Drake et al., 2007; Levin, 2010). A number ofvirulence factors have been determined for V. parahae-molyticus and include toxins TDH, TRH, cellularadhesion promoters such as flagella and capsule, ureaseproduction, which has a strong correlation with thepresence of trh, a siderophore termed vibrioferrin, aswell as a pathogenicity island and a type III secretionsystem (Yeung & Boor, 2004; Drakeet al., 2007; Levin,2010; Broberg et al., 2011; Dziejman & Yildiz, 2011).For a recent review of pathogenicity determinants in

V. parahaemolyticus, see Broberg et al. (2011).Several subtyping methods have been developed to

characterise isolates of V. parahaemolyticus, and thesemethods are useful during epidemiological studies ofclinical isolates. Such methods also provide a frameworkfor development of risk-based approaches for control-ling the presence of V. parahaemolyticusin seafood. Thedevelopment of several cultural and molecular methods,enabling rapid detection and quantification of total and

pathogenic V. parahaemolyticus in oysters occurred inresponse to oyster-associated outbreaks ofV. parahae-molyticus in several states during the late 1990s (CDC,1998; CDC, 1999; Danielset al., 2000a,b; DePaola et al.,2003). Many molecular assays have been designed totarget the tdh and trh genes. The main concern with

molecular assays and immunoassays that target only thetdh and trhgenes or their products as an indication of thepresence of V. parahaemolyticus is the fact that otherorganisms harbour genes and proteins that share geneticand amino acid homology with these toxins. Forexample, DNA hybridisation experiments conductedwith a tdh1 probe led to the discovery of homologousgenes in three Vibrio species, including Vibrio hollisae(since reclassified as Grimontia hollisae) (Thompsonet al., 2003), Vibrio mimicus and Vibrio cholerae non-O1 (Nishibuchi et al., 1985, 1990; Honda et al., 1986).While it is thought that all strains ofV. hollisae containthetdh gene (Nishibuchi et al., 1985, 1988, 1996; Curtiset al., 2007), only some strains of V. mimicus and

V. cholerae non-O1 isolated are known to containhomologoustdhsequences (Hondaet al., 1986; Nishibu-chiet al., 1990). In addition, a trh-like gene sharing 99%sequence homology with trh2 gene of V. parahaemolyt-icus has been reported in Vibrio alginolyticus isolatedfrom Alaskan oysters (Gonza lez-Escalona et al., 2006).Furthermore, a non-functional gene sharing 99% and84% homology with the trh1and trh2genes ofV. para-haemolyticus, respectively, was described in clinicalisolates of Aeromonas veronii(Raghunath et al., 2010).

Molecular detection assays have been applied inconjunction with typing methods, to determine theclinical importance of V. parahaemolyticus as this path-

ogen continues to emerge as an important aetiologicalagent of seafood-borne disease in several countries.

Typing methods

Several typing methods have been described for V. para-haemolyticus, and serotyping and molecular-based typ-ing have been used extensively and continue to be themethods of choice for routine characterisation ofV. parahaemolyticus isolates. Protein and biochemicalprofiling have also been explored as typing methods forV. parahaemolyticus (Rahman et al., 2006; Bhowmicket al., 2008). Protein profiling was observed to have lessdiscriminatory power than molecular-based typing

methods but may be useful in identifying new proteinswhich might play a role in establishment in the host orvirulence of the organism (Bhowmick et al., 2008).

Strain and serotype variations in biochemical pheno-types have been exploited to develop a biochemicaltyping system based on the commercially availablePhenePlate system. Rahman et al. (2006) evaluatedthe biochemical typing method and observed thatpandemic isolates showed a high level of phenotypic

Detection methods forV. parahaemolyticus B. Bishaet al.

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homogeneity and a majority of the pandemic isolatesbelonged to the same biochemical phenotype, while non-pandemic V. parahaemolyticus isolates were more het-erogeneous. The authors concluded that biochemicalfingerprinting of V. parahaemolyticus can be used as apreliminary method to differentiate between pandemic

and non-pandemic isolates of V. parahaemolyticus.Nevertheless, neither protein nor biochemical typinghas received much attention as alternatives for typing ofV. parahaemolyticus.

Vibrio parahaemolyticus can be differentiated byserotyping with commercially available antisera. Thir-teen O groups and 71 K types have been identified. AllV. parahaemolyticus strains share a common (flagellar)antigen. There are conflicting reports in the literature asto the number of V. parahaemolyticus O groups. Forexample, according to Kaysner & DePaola (2004), 12 Oantigen types and over 70 K antigen types have beendescribed (see Table 1, adapted from Kaysner & De-Paola, 2004). The O antigen types have been further

divided into nine chemotypes, with each O antigenbelonging to an individual chemotype, with the excep-tion of the O3, O5 and O11 antigens that belong to thesame chemotype, and the O7 and O12 antigens thatbelong to another chemotype (Hisatsune et al., 1980).Earlier, however, a thirteenth O group was identifiedand characterised (Hisatsune et al., 1993; Iguchi et al.,1995). The reason for this discrepancy may arise fromthe fact that the O-serotypes of many strains have notbeen identified because they are not agglutinated bydiagnostic antisera against known O-serotype strains(Hashii et al., 2000), and many strains remain untype-able (Kaysner & DePaola, 2004). Compositional sugar

analysis was evaluated by Hashii et al. (2000) tocharacterise the lipopolysaccharide of six untypeable(OUT) strains, and the study found that the O-serotypeof two of the OUT strains employed was not apreviously known O-serotype and represented a newserotype that had not been earlier reported.

Serotyping studies have indicated that the epidemiol-ogy and environmental distribution ofV. parahaemo-lyticus continue to evolve (Wright & Schneider, 2009).Until 1992, the majority of the outbreaks had occurredin Japan and were related to consumption of uncookedseafood (Fujino et al., 1953), while the most frequent

serovars clinically isolated between 1992 and 1995 inTaiwan were O5:K15 (18.5%), O4:K8 (16.2%), O3:K29(12.5%), O1:K56 (8.3%), O2:K3 (6.5%) and O4:K12(6.0%) (Wong et al., 2000). The epidemiology ofV. parahaemolyticus changed dramatically in 1996 withthe appearance of a new serovar, designated as O3:K6.New strains belonging to the O3:K6 serovar appearedfor the first time in February 1996 in Calcutta, India,and were indistinguishable from other O3:K6 isolatesobtained between 1995 and 1996 from Southeast Asiancountries. Since then, the new O3:K6 strains have beenconsidered to be the first pandemic strains of V. para-haemolyticus and are involved in a high proportion offoodborne outbreaks in several Asian countries (Arak-

awa et al., 1999; Chiou et al., 2000; Matsumoto et al.,2000). Foodborne outbreaks attributable to the pan-demic O3:K6 infections also occurred in the UnitedStates and were associated with the consumption ofoysters (CDC, 1998, 1999; Khan et al., 2002). Thepandemic O3:K6 serotype was implicated in twooutbreaks in Chile in 1998 and 2004 (Gonza lez-Escalonaet al., 2005), in France (Quilici et al., 2005) and Spain(Martinez-Urtaza et al., 2005), allowing for the deter-mination that O3:K6 had been disseminated to Europe.Ansaruzzaman et al. (2005) reported the presence ofpandemic serovars of V. parahaemolyticus in sub-Saha-ran Africa, with 42 cases of illness in Beira, Mozam-

bique, from February to May 2004. The globaldissemination of V. parahaemolyticus serotype O3:K6has been recently reviewed (Nair et al., 2007).

In addition to serotyping, several molecular ap-proaches have been developed and are effective fortyping V. parahaemolyticus strains. Wong et al. (1996)developed a pulsed-field gel electrophoresis (PFGE)protocol to subtype V. parahaemolyticus. In this meth-od, genomic DNA was digested with the restrictionenzyme SfiI, and the fragments were resolved on a 1%agarose gel at 190 V with a pulse time of 380 s. Later,Wonget al. (1999a) used the PFGE method to subtypeV. parahaemolyticus strains in seafood imported fromAsian countries, and strains isolated during a nosoco-

mial outbreak at a hospital were also subtyped usingPFGE (Lu et al., 2000). The PFGE method was alsoused to distinguish between V. parahaemolyticus O3:K6pandemic and non-pandemic isolates. For example,Wong et al. (2000) examined a total of 371 isolates ofV. parahaemolyticus collected from patients involved infoodborne outbreaks in Taiwan between 1992 and 1995.The isolates were separated into 57 patterns, with mostdomestic isolates clustering into five types representing

Table 1 Antigenic scheme ofVibrio parahaemolyticus(from Kaysner &

DePaola, 2004)

O group K type

1 1, 25, 26, 32, 38, 41, 56, 58, 64, 69

2 3, 28

3 4, 5, 6, 7, 27, 30, 31, 33, 37, 43, 45, 48, 54, 57, 58, 59, 65

4 4 , 8, 9, 10, 11 , 12 , 1 3, 34, 42, 49, 53, 55, 63, 6 7

5 5, 15, 17, 30, 47, 60, 61, 68

6 6, 18, 46

7 7, 19

8 8, 20, 21, 22, 39, 70

9 9, 23, 44

10 19, 24, 52, 66, 71

11 36, 40, 50, 51, 61

12 52


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76% of the isolates. In contrast, the pandemic O3:K6strains were genetically distant from the main domestictypes (Wong et al., 2000). Also, a large number ofO3:K6 strains collected before 1996 and post-1996O3:K6 strains from India, Japan, Korea and Taiwanwere studied using PFGE. The results indicated that the

O3:K6 strains were separated into two geneticallyunrelated groups, the old (pre-1996) O3:K6 group andthe new (post-1996) O3:K6 group. The old groupincluded O3:K6 strains isolated before 1996 and wasdivided into six patterns (A1, A2, A3, A8, B2 and R) inthe PFGE typing scheme. The recently isolated (post-1996 O3:K6) strains were placed into the new O3:K6group (group I), which included eight closely relatedpatterns of which I1 (81%) and I5 (13%) were the mostcommon patterns.

In addition to PFGE, ribotyping has been investi-gated as a subtyping tool to differentiate V. parahaemo-lyticus isolates. Wong et al. (1999b) used ribotyping toinvestigate 121 isolates obtained from outbreaks in

Taiwan between 1992 and 1994 and showed that theribotyping method grouped the isolates into 30 differentribotypes. The discriminatory ability of the method wassimilar to that of the previously reported PFGE method(Wong et al., 1999a). Ribotyping was also used tosubtype 28 new O3:K6 strains into five patterns (Baget al., 1999).

Molecular typing using PCR techniques have beendeveloped and used to subtype V. parahaemolyticus. Forexample, a novel, random amplified polymorphic DNA(RAPD) method was developed for the molecular typingof this pathogen. Unlike PCR, RAPD does not require aknown target sequence for its operation. A 10-mer

primer (5-CAG GCG CAC A-3

) was selected togenerate polymorphic amplification profiles ofV. para-

haemolyticus genomic DNA of 308 clinical and envi-ronmental isolates of V. parahaemolyticusby the RAPDmethod, generating 41 patterns that were grouped into16 types (AP) (Wong et al., 1999c). Several groupshave used a variation of RAPD known as arbitrarilyprimed PCR to type urease-positive strains ofV. para-haemolyticus (Okuda et al., 1997a) and the pandemicO3:K6 strains (Okuda et al., 1997b). However, thediscriminatory ability of both methods appears to beless than that of PFGE and ribotyping, as demonstratedby Wong et al. (2000). One disadvantage of RAPDmethods is the lack of reproducibility because of

variations in band intensity and the presence of someminor bands, requiring the use of other PCR methodsfor typing V. parahaemolyticus, which target variousgenetic sequences including the conserved ribosomalgene spacer sequence (RS-PCR), the repetitive extra-genic palindromic sequence (REP-PCR) and the entero-bacterial repetitive intergenic consensus sequence(ERIC-PCR) (Wong & Lin, 2001). Such methods thatdo not require the use of random primers were found to

be more robust than the RAPD methods and closelyapproached or even exceeded the performance of PFGEand ribotyping (Wong & Lin, 2001). REP-PCR wasshown to exhibit greater reproducibility than ERIC-PCR, while RS-PCR generated fewer amplificationbands and patterns than the other PCR-based typing

methods (Wong & Lin, 2001).Bacterial isolates from patients (38 isolates) and

environmental sources (16 isolates) collected during anoutbreak of V. parahaemolyticus gastroenteritis in Brit-ish Columbia, Canada, in 1997 were used to compareERIC-PCR, ribotyping, PFGE and restriction fragmentlength polymorphism (RFLP) analysis of the geneticlocus encoding the polar flagellum (Fla locus RFLPanalysis) in an attempt to determine the discriminatorypower of each method (Marshall et al., 1999). ERIC-PCR and ribotyping were the most discriminatory typingmethods, especially when combined, while Fla locusRFLP analysis was the least discriminatory. PFGEexhibited good discrimination, but the DNA of isolates

subjected to PFGE following digestion with ApaI wasoften degraded. Later, Wong & Lin (2001) observed thatapproximately 7% of the V. parahaemolyticus strainsthat were evaluated by PFGE exhibited DNA degrada-tion and were not typeable when the restriction enzymeSfiI was used during DNA digestion. Therefore, thechoice of restriction enzyme used during PFGE analysisof V. parahaemolyticus isolates may affect the typingresults, and it may be better to independently typeisolates using both enzymes. Collectively, these studiesindicate that the advantages and disadvantages of eachtyping method should be carefully considered, takinginto account the objectives of the respective study, before

a suitable method is chosen for analysis.Recently, several groups have used subtyping methodsto characterise V. parahaemolyticus isolates from sea-food and the environment. For example, Chakraborty &Surendran (2009) used RAPD, ERIC-PCR, REP PCRand RS-PCR methods to assess the genetic diversity ofV. parahaemolyticus isolates obtained from shrimp andenvironmental samples in India. RAPD analysis re-vealed clustering of toxigenic strains into a single group.RS, ERIC and REP-PCR methods were more discrim-inatory and placed the isolates into two, five and sevenmajor groups. RS-PCR generated fewer amplified bandsthan the REP and ERIC-PCR methods, as previouslydescribed (Wong & Lin, 2001). The authors observed

that RS-PCR patterns were more visually discerniblethan patterns produced by the other methods, suggest-ing the applicability of RS-PCR as a practical method toroutinely subtype V. parahaemolyticus (Chakraborty &Surendran, 2009). Bilunget al. (2005) used RAPD-PCRto examine the genetic relatedness among sixty-twoisolates of V. parahaemolyticus isolated from Anadara

granosa (cockles) obtained from a harvesting site inKuala Selangor. The RAPD profiles revealed a high





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level of DNA sequence diversity within the strainstested, leading the authors to conclude that the cocklesin the study area were populated by genetically poly-morphic strains of V. parahaemolyticus. Other groupshave also confirmed the genetic diversity ofV. parahae-molyticusin seafood and environmental samples (Sarkar

et al., 2003; Bhowmick et al., 2008; Chao et al., 2009).A summary of typing methods employed for V. para-

haemolyticus is shown in Table 2.

Cultural methods

Vibrio parahaemolyticus enrichment media and selectivity

Several selective enrichment broths employed in isola-tion and detection of V. parahaemolyticus have beendeveloped as a consequence of its recognition as animportant agent of foodborne illness linked to seafood

consumption (Donovan & van Netten, 1995). Takinginto consideration the halophilic nature and tolerance ofV. parahaemolyticus to alkaline pH, selectivity to mediaemployed in enrichment of this microorganism is oftenconveyed by application of alkaline pH (8.69.4) andaddition of l7% NaCl, while additional selectivity can

be supplied by addition of surfactants such as sodiumdodecyl sulphate (SDS) and alkylbenzene sulphonate,bile salts, dyes such as metachrome yellow II RD, andantibiotics such as colistin or polymyxin B (Donovan &van Netten, 1995). As pathogenic V. parahaemolyticushave been found in


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plating method should be able to identify knownvirulence factors to distinguish non-pathogenic (envi-ronmental or food) from pathogenic (clinical) isolates.The KP, defined as -haemolytic activity observed onWagatsuma blood agar (Chunet al., 1975), has been fora long time the gold standard for identifying pathogenic

isolates, but it has now been superseded by molecular-based detection methods, including DNA hybridisationand PCR, which target genes encoding known virulencefactors such as tdh or trh (Nishibuchi & Kaper, 1995).

Currently, V. parahaemolyticus isolation strategiesemploy one or more culture-based methods for speciesidentification, followed by a molecular method fordetection of virulence factors. Various selective mediahave been developed for isolation and identificationof V. parahaemolyticus. Frequently used enrichmentbroths include alkaline peptone water (APW), saltpolymyxin broth (SPB), salt colistin broth and glucosesalt teepol broth, which can also be modified to includeSDS instead of teepol (Donovan & van Netten, 1995).

Addition of bile salts to enrichment media has beenproposed to selectively isolate pathogenic isolates,taking into the consideration the fact that, while theseisolates are found at low ratios in the environmentcompared to the non-pathogenic isolates, they survivebetter in the gastrointestinal tract of the host and areable to cause disease (Pace et al., 1997; Raghunathet al., 2009).

An enrichment broth supplemented with the bile saltsodium taurocholate (ST broth) was compared to APWfor its ability to isolate pathogenic V. parahaemolyticusisolates from seafood harvested from the southwestcoast of India. The enrichment was followed by PCR

identification directly from broth after enrichment for18 h, or following streaking onto thiosulfate citrate bilesalts sucrose (TCBS) agar, or by colony hybridisationfollowing enrichment (CFE) or streaking on T1N3 (1%tryptone, 3% NaCl) agar plates (Raghunath et al.,2009). The study found that improved isolation andsubsequent identification of pathogenic V. parahaemo-lyticusfrom seafood samples could be achieved by usingST compared to APW broth, with 6.9% and 3.4% ofsamples containing V. parahaemolyticuscarrying thetdhgene isolated following enrichment in ST and APW,respectively. Samples containing V. parahaemolyticuscarrying thetrh gene were isolated following enrichmentin ST (20.7%) and APW (13.8%), respectively. Signif-

icantly higher numbers of pathogenic V. parahaemolyt-icus were detected by either CFE or PCR followingenrichment in ST broth as compared to APW broth(Raghunathet al., 2009).

Cultural detection and enumeration

Standard procedures set forth by the FDA Bacteriolog-ical Analytical Manual (BAM) for isolation and

enumeration of V. parahaemolyticus from raw shellfishemploy the most probable number (MPN) technique, ahydrophobic grid membrane filter filtration protocol ordirect plating on non-selective media combined withconfirmation via DNA colony hybridisation (Kaysner &DePaola, 2004). MPN is performed in triplicate in APW

following 10-fold dilutions of samples in phosphate-buffered saline and then incubated for 1824 h at3537 C with tubes showing growth confirmed bystreaking onto TCBS agar followed by biochemicalconfirmation (Drake et al., 2007). A schematic repre-sentation of the FDA-MPN method is shown in Fig. 1.Large numbers of background microorganisms that canpossibly grow even on selective media may pose issues inregard to effective isolation and enumeration ofV. para-haemolyticusfrom environmental samples. For example,Hara-Kudo et al. (2001) observed that when seafoodwas evaluated for the presence of V. parahaemolyticus,colonies on TCBS agar were difficult to distinguishvisually from other bacterial colonies because of the

presence of a yellow pigment produced by sucrosefermenting bacteria. To counteract for these phenom-ena, a plethora of alternative enrichment broths andplating media have been developed (Oliver, 1981; Oliveret al., 1992; Hagenet al., 1994; Azanza et al., 1996; Hiet al., 1998a,b; Cerda -Cue llar et al., 2000; Alam et al.,2001). For example, CHROMagar Vibrio is a differ-ential medium that represents an alternative to TCBS.This medium contains colorimetric substrates for-galactosidase and was developed specifically to differ-entiate ortho-nitrophenyl-b-galactoside-positive V. para-haemolyticus from other closely related bacteria.Hara-Kudo et al. (2001) developed a procedure that

combined enrichment and plating on CHROMagar

Vibrio for detecting V. parahaemolyticusin seafood. Theprocedure consisted of a non-selective enrichment stepin salt trypticase soy broth, followed by a selectiveenrichment step in SPB. The two-step enrichmentprocedure was found to be more effective than theone-step enrichment in SPB alone. The enrichmentcultures were plated onto CHROMagar Vibrio orTCBS agar, and higher isolation frequencies fromnaturally contaminated seafood samples were observedon CHROMagarVibrio than on TCBS agar. Blanco-Abad et al. (2009) described a two-step enrichmentprotocol that employed APW broth as the first enrich-ment step and SPB as the selective secondary broth,

followed by isolation on CHROMagar Vibrio. Theauthors reported that the use of CHROMagarVibrioled to increased isolation of V. parahaemolyticus incomparison to plating on TCBS agar. Other groupshave also incorporated CHROMagarVibrio into theirV. parahaemolyticus isolation schemes (Hara-Kudoet al., 2003; Miyasaka et al., 2006). Canizalez-Romanet al.(2011) compared a protocol involving plating ontoCHROMagar Vibrio followed by PCR with a culture





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method comprising plating onto TCBS followed byconfirmation via biochemical tests and plating ontoWagatsuma agar for isolation of pathogenic and envi-ronmental V. parahaemolyticus from a total of 131shrimp, water, sediment and stool samples. The authorsreported a significantly (P < 0.05) improved sensitivityof the chromogenic agar when combined with PCR, ascompared to the conventional method, which impartedlow sensitivity (65.4% and 75.6% at 24 and 48 h,respectively) and specificity (52.4% at 48 h).

The development of other selective media for differ-entiation of V. parahaemolyticus from other Vibriospecies has been reported. For example, the use oftrypticase soy agar supplemented with sucrose, sodiumchloride, bile salts and triphenyltetrazolium chloride forthe isolation and differentiation of V. parahaemolyticusfrom V. alginolyticus has been described, with V. para-haemolyticus producing dark red colonies and V. algi-nolyticus white colonies with occasional pink centres(Kourany, 1983).

Molecular-based detection methods

One of the greatest challenges to detection ofV. para-

haemolyticus is the low number of organisms present,especially in cooler waters (Su & Liu, 2007). Moleculartechniques allow for more rapid analysis than culturalmethods, and provide the sensitivity and specificityneeded to detect low numbers of V. parahaemolyticus.Molecular-based methods employed to detect V. para-haemolyticus in food and environmental samples haveprimarily consisted of DNA hybridisation and PCR,with loop-mediated isothermal amplification (LAMP)

detection techniques recently attracting increased inter-est. Also, PCR or DNA hybridisation methods withalkaline phosphatase-labelled probes targeted at the tlhgene are set forth by the BAM as alternatives to selectiveand differential plating, or biochemical identification(Kaysner & DePaola, 2004).

DNA hybridisation

Hybridisation techniques have been developed that

target species-specific (tlh) or virulence (tdh, trh) genes,and these assays are generally considered to offeradvantages compared to cultural methods. For instance,Gooch et al. (2001) were able to obtain comparableresults to the BAM-MPN method by using directplating on T1N3 media combined with hybridisationwith alkaline phosphatase (AP) or digoxigenin-labelledprobes targetingtlh.Another study (Ellison et al., 2001)evaluated direct plating followed by detection with thetlh-targeted alkaline phosphatase-labelled DNA probe(VPAP) for detection of V. parahaemolyticus in retailoysters over a 9-month period and found a goodcorrelation with the BAM-MPN method (r = 0.78).Direct-VPAP offered increased rapidity and accuracy

compared to the BAM-MPN method, which on theother hand was found to be more sensitive and allow forbetter recovery of stressed cells. Alternative targets forhybridisation probes have included the toxin genes tdhandtrh. In a study by Lee et al. (1992), a Southern blottechnique employing a 26-mer DNA probe targeting tdhdetected 89 of 95 V. parahaemolyticus but none of theother 48Vibriospp. or non-Vibriospp. strains employedin the screening, ultimately being able to detect V. para-

Figure 1 Simplified schematic representation of

the FDA-most probable number method

(Kaysner & DePaola, 2004). Sample prepara-

tion and details will vary. For example, surface

tissue, gut and gills are recommended for sam-

pling of fish; for molluscan shellfish pooling of

12 animals is recommended, while for whole

crustaceans (such as shrimp), the whole animal

is to be used. PBS, phosphate-buffered saline;

APW, alkaline peptone water; TCBS, thiosul-

fate citrate bile salts sucrose; AGS, arginine

glucose slants; T1N0, tryptone broth with no

NaCl; T1N3, tryptone broth with no 3% NaCl.

See Kaysner & DePaola (2004) for more detailsand biochemical identification tests.





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haemolyticus from artificially spiked oysters in


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the minimum detection limit was 102 CFU mL)1 for allthree broths. The authors observed that there was nodifference between different enrichment media withrespect to tdh gene detection of V. parahaemolyticus inpure culture or artificially inoculated shrimp samples.

Loop-mediated isothermal amplification

LAMP-based assays have been developed as an alter-native to PCR (Notomi et al., 2000). One advantage ofLAMP-based assays as compared to PCR is that duringLAMP, nucleic acid amplification occurs at a singletemperature, eliminating the need for thermal cyclers.Nemotoet al. (2009) used LAMP to detect tdh-positiveisolates of V. parahaemolyticus. Their assay targeted sixregions of the tdh gene and was compared to PCR fordetection of tdh and reverse passive latex agglutinationfor TDH detection. The specificity of the LAMP assaywas evaluated using 32 strains of TDH-producingV. parahaemolyticus, one strain of TDH-producing

G. hollisae, 10 strains of TDH-V. parahaemolyticus and94 strains of TDH- non-Vibrio bacteria. Upon testingartificially spiked oysters, 32 of 32 TDH-producingV. parahaemolyticus strains and one TDH-producingstrain ofG. hollisae produced positive results with boththe LAMP and PCR procedures. No false-positiveresults were observed with any TDH-non-producingV. parahaemolyticus or non-Vibrio species.

Yamazakiet al. (2008) developed a LAMP assay fordetection of tlh and tested both pure V. parahaemolyt-icus cultures and artificially inoculated shrimp. In pureculture, 143 of 143 V. parahaemolyticus strains werepositive by LAMP, while no LAMP product was

detected from any of 33 non-parahaemolyticus or 56non-Vibrio isolates. LAMP consistently identified2.0 CFU per reaction, while PCR required approxi-mately 10-fold more bacteria for detection. Later,Yamazaki et al. (2010) followed up on their previouswork by developing a LAMP assay to detect the tdh andtrh genes in V. parahaemolyticus and related Vibriospecies. In this work, the LAMP assay was designed forboth the combined and individual detection of the tdh,trh1 and trh2 genes and combined detection of the trh1and trh2 genes. When the assay was evaluated againsttdh-positive, andtrh1- andtrh2-positive isolates belong-ing to Vibrio spp. and G. hollisae, and bacterial strainslacking these genes, similar results were achieved in

comparison to PCR and DNA hybridisation. Forexample, all 125 strains of V. parahaemolyticus, threestrains of G. hollisae and two strains of V. mimicuscarrying the tdh, trh1 and trh2 genes in variouscombinations were positive by LAMP, PCR andDNA hybridisation. In contrast, no LAMP productswere detected from any of the 20 bacterial strains thatlacked these genes. When V. parahaemolyticus wasartificially spiked into shrimp samples, the detection

sensitivity of tdh, trh1- and trh2-positiveV. parahaemo-lyticus was observed to 0.8, 21.3 and 5.0 CFU perLAMP reaction tube, respectively. The assay requiredbetween 27 and 60 min, including DNA extraction, forcompletion from pure cultures and less than 80 minwhen shrimp samples were analysed. Nemoto et al.

(2011) targeted the rpoD and toxR genes of V. para-haemolyticus via LAMP. Positive detection of all 78V. parahaemolyticusstrains was achieved within 40 min,while the assay did not cross-react with another 69strains of other microorganisms. Detection sensitivity ofthe LAMP assays targeting rpoD and toxR was deter-mined to be 3.7 and 450 CFU per test in pure culture.The authors combined their rpoD-LAMP method withan MPN protocol for detection of V. parahaemolyticusin spiked short-necked clams and compared it to anMPN method with a culture method using agarmedium, achieving better sensitivity using the rpoD-LAMP method.

Fluorescencein situ hybridisation

Fluorescence in situ hybridisation, a method thatemploys fluorescently labelled short nucleotides tospecifically hybridise with target rRNA in whole per-meabilised cells, has received little interest as a methodof detection for V. parahaemolyticus. That is primarilybecause of the scarce rRNA sequence variability of thetarget microorganism, which makes differentiation fromclosely related species difficult at this point in time.Sawabeet al. (2009) employed a multi-probe approach(using designed probes VP437, VP612 and VP1253);however, the assay was only species specific, which

would only allow for employment of this method todetect total V. parahaemolyticus. A recognition of indi-vidual gene fluorescence in situ hybridisation (RING-FISH) method using polynucleotides targeting the tlhgene has also been applied (Griffitt et al., 2011).Although the method was deemed to be highly specificand sensitive by the authors and was also used incombination with a protocol to determine cell viability,the problem of differentiation of pathogenic V. para-haemolyticus from environmental isolates remains.Table 3 shows a summary of nucleic acid methodsemployed in detection of V. parahaemolyticus.

Antibody-based assaysAntibody-based assays are still considered the goldstandard of affinity-based detection methods, and anumber of these assays have been developed to detectV. parahaemolyticusor its toxins. Immunoprecipitation-based assays have been developed to affect detection ofV. parahaemolyticus. An example of work conducted inthis direction is given by the study by Honda et al.(1980), who developed a modified Elek test and an





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immuno-halo test for detection of TDH in V. parahae-

molyticus, as an alternative to the KP assay on Wagat-suma media, which may produce false-positive results attimes because of the instability of blood used in themedium. The authors evaluated both immunologicalmethods using 29 strains of KP-positive V. parahaemo-lyticus, 14 strains of KP-negative V. parahaemolyticus,and two strains of V. parahaemolyticus with indetermi-nate KP reactions on Wagatsuma medium. The sensi-tivity of the modified Elek test was similar to that of the

KP on Wagatsuma medium, and the results of both

reactions correlated very well. For instance, all of the 29KP-positive strains gave positive Elek tests and all 14Kanagawa KP-negative strains gave negative Elek tests.Of the two indeterminate KP strains, one was Elek-positive while the other produced a negative result. Theimmuno-halo test was slightly less sensitive than themodified Elek test. One KP-positive and one indetermi-nate KP strain, as determined by the Elek test, wereobserved to be negative by the immuno-halo assay. The

Table 3 Summary of nucleic acid-based detection of Vibrio parahaemolyticus (illustrated by representative studies)

Method Target Sensitivityspecificity Advantagesdisadvantages

DNA hybridisation tlh(Ellison et al., 2001;

Goochet al., 2001;),

tdh (Lee et al., 1992;

Nordstrom& DePaola, 2003)

Sensitivity: Higher than culture methods

alone

Specificity: Based on the target, it can be

used to detect environmental,pathogenic or both V. parahaemolyticus

Advantages: Widely accepted method for

identification of V. parahaemolyticus, as

evidenced by inclusion in FDA-BAM

methods. Imparts enhanced rapidity andspecificity compared to culture methods

alone

Disadvantages: It is dependent on culture

methods, which affects the rapidity of

detection

Polymerase chain

reaction (PCR)

tdh, trh, toxR(Bhuiyan

et al., 2002) tlh, tdh, trh,

(Nordstromet al., 2007),

ORF8 (Myers et al., 2003;

tlh, tdh, trh, ORF8 (Ward

& Bej, 2006)

Sensitivity: High (theoretically one copy

of target), however, dependent on

effective target separation and

enrichment

Specificity: Highly specific. Can be used

to detect total, environmental,

pathogenic V. parahaemolyticusor even

specific serovars (e.g. pandemic O3:K6)

and can be multiplexed

Advantages: Rapid, specific, sensitive,

allows for multiplexing, allows for

differentiation of pathogenic vs.

environmental isolates and can be

optimised to detect specific serovars

Disadvantages: Can only be performed

by highly skilled personnel, theoretical

high sensitivity can be hindered by non-

optimised separation and enrichment

protocols. Targets of the assay believedto be unique in pandemic strains can

detect closely related serovars

Loop-mediated

isothermal

amplification

(LAMP)

tdh(Nemoto et al., 2009),

tlh (Yamazaki et al.,

2008; tdh, trh1 and trh 2

(Yamazakiet al., 2010),

rpoDand toxR(Nemoto

et al., 2011)

Sensitivity: Higher than culture,

comparable to PCR

Specificity: Based on the target, it can

be used to detect environmental,

pathogenic or both

V. parahaemolyticus

Advantages: Rapid, sensitive, simpler to

perform than PCR, can be performed at

one single temperature and does not

require cycling

Disadvantages: Similar to PCR, it is greatly

affected by methods of target separation

and enrichment. It is also not very simple

to perform by non-specialised personnel

Fluorescence in situ

hybridisation (FISH)

and recognition of

individual gene

fluorescence in situhybridisation

(RING-FISH)

rRNA (Sawabe et al.,

2009), tlh (Griffitt

et al., 2011)

Sensitivity: Theoretical sensitivity is high

(as few as one cell). Practically sensitivity

can be negatively affected by a number

of factors, including target cell

physiological status and separation andconcentration methods

Specificity: It is highly specific, however,

at this point in time; differentiation can

be made only at the species level

Advantages: Rapid and specific and if

coupled with adequate sample preparation

can be very specific. Easier to perform

than LAMP or PCR

Disadvantages: The most importantdisadvantage is the fact that the method

cannot differentiate pathogenic

V. parahaemolyticusfrom environmental

isolates. Owing to the fact that the

ubiquitous nature of the environmental

isolates demands detection of pathogenic

strains, FISH and its variations can only

be deemed as emerging technologies for

detection of V. parahaemolyticus

BAM, Bacteriological Analytical Manual.

Detection methods forV. parahaemolyticus B. Bishaet al.0




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authors recommended the use of both immunoassays toscreen V. parahaemolyticus for the presence of TDH(Hondaet al., 1980).

Vibrio parahaemolyticus can also be identified sero-logically through the detection of unique H antigensexpressed in the core protein of the polar flagellum

(Terada, 1968; Tassin et al., 1983). Simonson & Siebel-ing (1986) coated Staphylococcus aureus Cowan 1 cellswith V. parahaemolyticus anti-flagellar polyclonalantibodies and assessed this reagent for its abilityto agglutinate V. parahaemolyticus isolates. The anti-V. parahaemolyticus H coagglutination reagent aggluti-nated 96% of 395 isolates identified bacteriologically asV. parahaemolyticus. The anti-H antibodies were alsoshown to cross-react with Vibrio alginolyticus,Vibrio natriegens, Vibrio harveyi and Vibrio campbellii.Datta et al. (2008) produced monoclonal anti-H anti-bodies reactive with purified V. parahaemolyticus flagel-lar cores which agglutinated 100% of the 41 isolatesidentified genetically as V. parahaemolyticus. The mono-

clonal anti-H antibodies still exhibited cross-reactionswith V. alginolyticus, V. harveyiand V. campbelliiindi-cating that these vibrios must share flagellar antigens.

Honda et al. (1985) have also reported the develop-ment of three enzyme-linked immunosorbent assays(ELISA) assays, including a ganglioside ELISA, a directELISA and a sandwich ELISA to detect the presence ofTDH. The ganglioside ELISA was unable to detectTDH, and the direct ELISA, while effective at detect-ing TDH in pure culture, was unable to do so in crudeculture supernatants. In contrast, the sandwich ELISAwas effective at detecting TDH in crude mixtures, andeven produced positive results from V. parahaemolyti-

cus isolates that had been observed to be KP-negativeusing the traditional Wagatsuma assay. Later, thesame research group (Honda et al., 1995) chemicallyimmobilised antibodies on nylon as an alternativemethod to simultaneously and differentially detectTDH and TRH in V. parahaemolyticus. The resultswere easy to evaluate visually and were similar to thoseobtained by using DNA probes. The assay could detecttoxin in culture as well as clinical (diarrhoeal stool)samples.

To address the issues related to using TDH toxindetection to definitively detect V. parahaemolyticus,Changet al. (1994) also developed a latex agglutinationimmunoassay for detection of V. parahaemolyticus. Two

V. parahaemolyticus outer membrane proteins wereisolated, purified and subsequently used as antigens forproducing polyclonal antibodies in rabbits. Latex par-ticles sensitised with the affinity purified antibodies wereemployed to rapidly identify V. parahaemolyticus. Whenthe assay was evaluated against 173 strains (including 94isolates of V. parahaemolyticus, 40 isolates of othervibrios and 39 strains of other bacteria), the false-negative and false-positive rates were 1.4% and 3.1%,

respectively. While several strains of three Vibriospecies(V. alginolyticus, V. harveyi, and V. mimicus) producedfalse-positive results, no false-positive results wereobserved among the 39 strains (33 species) of otherbacteria. Kumar et al. (2011) developed a monoclonalantibody-based sandwich ELISA, using monoclonal

antibodies against purified TRH recombinant protein,ultimately detecting pathogenic V. parahaemolyticus in14 out of 34 (41%) seafood samples tested as opposed to64% of positive samples obtained by PCR targeting theToxR gene. The authors noted that the method cannotdifferentiate trh and tdh, basically pointing out alimitation of immunoassay-based techniques for detec-tion of V. parahaemolyticus; however, they concludedthat these types of methods may be appropriate inapplications such as routine screening by non-highlytrained personnel.

An immunological-based biosensor to detect V. para-haemolyticushas also been described (Zhaoet al., 2007).

Conclusions

Vibrio parahaemolyticus continues to emerge as anincreasingly important human pathogen implicated inoutbreaks of seafood-borne illness. Many cultural,molecular and immunological-based methods thatdetect environmental and clinical isolates have beendeveloped, and it is likely that modifications of theseassays will be described in the future. It will beimperative to develop methods that identify the presenceof the organism, and not just the presence of toxin insamples, because other microorganisms such as G. hol-lisae share sequence and protein homology with the

TDH toxin of V. parahaemolyticus (Nishibuchi et al.,1998). Although detection methods for V. parahaemo-lyticus are continually evolving, they are not yet usedroutinely to test seafood for the presence of thispathogen. The development of robust assays for detec-tion of V. parahaemolyticus that integrate samplepreparation from different seafood matrices shouldmake routine testing for this pathogen more feasible.Phenotypic and molecular subtyping will continue tobe useful in epidemiological investigations as V. para-haemolyticus continues to emerge as a foodbornepathogen. Nevertheless, as with other foodborne patho-gens, detection and subtyping analysis will not substitutefor implementation of effective control measures to

decrease contamination of seafood with V. parahaemo-lyticus.

Acknowledgments

This work was supported by a grant from theNational Oceanic and Atmospheric Administration(NA07OAR170428) and by Louisiana Sea Grant CollegeProgram.





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References

Alam, M.J., Tomochika, K., Miyoshi, S. & Shinoda, S. (2001).Analysis of seawaters for the recovery of culturable Vibrio parahae-molyticusand some other vibrios.Microbiology and Immunology,45,393397.

Ansaruzzaman, M., Lucas, M., Deen, J.L. et al. (2005). Pandemicserovars (O3:K6 and O4:K68) ofVibrio parahaemolyticus associated

with diarrhea in Mozambique: spread of the pandemic into theAfrican continent. Journal of Clinical Microbiology, 43, 25592562.

Arakawa, E., Murase, T., Shimada, T., Okitsu, T., Yamai, S. &Watanabe, H. (1999). Emergence and prevalence of a novel Vibrioparahaemolyticus O3:K6 clone in Japan. Japanese Journal ofInfectious Diseases, 52, 246247.

Azanza, P.V., Buckle, K.A. & Fleet, G.H. (1996). Effect of diluents onthe enumeration ofVibrio vulnificus. International Journal of FoodMicrobiology, 30, 385390.

Bag, P.K., Nandi, S., Bhadra, R.K. et al. (1999). Clonal diversityamong recently emerged strains of Vibrio parahaemolyticus O3:K6associated with pandemic spread. Journal of Clinical Microbiology,37, 23542357.

Bej, A.K., Patterson, D.P., Brasher, C.W., Vickery, M.C., Jones, D.D.& Kaysner, C.A. (1999). Detection of total and hemolysin-produc-ing Vibrio parahaemolyticus in shellfish using multiplex PCR

amplification oftl

,tdh

, andtrh

. Journal of Microbiological Methods

,36, 215225.Bhowmick, P.P., Khushiramani, R., Raghunath, P., Karunasagar, I. &

Karunasagar, I. (2008). Molecular typing ofVibrio parahaemolyticusisolated from seafood harvested along the south-west coast of India.Letters in Applied Microbiology, 46, 198204.

Bhuiyan, N.A., Ansaruzzaman, M., Kamruzzaman, M. et al. (2002).Prevalence of the pandemic genotype of Vibrio parahaemolyticus inDhaka, Bangladesh, and significance of its distribution acrossdifferent serotypes. Journal of Clinical Microbiology, 40, 284286.

Bilung, L.M., Radu, S., Bahaman, A.R. et al. (2005). Randomamplified polymorphic DNA-PCR typing ofVibrio parahaemolyt-icusisolated from local cockles (Anadara granosa).American Journalof Immunology, 1, 3136.

Blanco-Abad, V., Ansede-Bermejo, J., Rodriguez-Castro, A. & Mar-tinez-Urtaza, J. (2009). Evaluation of different procedures for theoptimized detection of Vibrio parahaemolyticus in mussels and

environmental samples.International Journal of Food Microbiology,129, 229236.

Broberg, C.A., Calder, T.J. & Orth, K. (2011). Vibrio parahaemolyticuscell biology and pathogenicity determinants. Microbes and Infection,13, 9921001.

Canizalez-Roman, A., Flores-Villasen or, H., Zazueta-Beltran, J.,Muro-Amador, S. & Leo n-Sicairos, N. (2011). Comparative eval-uation of a chromogenic agar medium PCR protocol with aconventional method for isolation ofVibrio parahaemolyticus strainsfrom environmental and clinical samples. Canadian Journal ofMicrobiology, 57, 136142.

Centers for Disease Control and Prevention (CDC) (1998). Outbreakof Vibrio parahaemolyticus infections associated with eating rawoysters Pacific Northwest, 1997. The Journal of the AmericanMedical Association, 280, 126127.

Centers for Disease Control and Prevention (CDC) (1999). Outbreakof Vibrio parahaemolyticus infection associated with eating rawoysters and clams harvested from Long Island SoundConnecticut,New Jersey, and New York, 1998. The Journal of the AmericanMedical Association, 281, 603604.

Cerda -Cue llar, M., Jofre, J. & Blanch, A.R. (2000). A selectivemedium and a specific probe for detection of Vibrio vulnificus.Applied and Environmental Microbiology, 66, 855859.

Chakraborty, R.D. & Surendran, P.K. (2009). Incidence and molec-ular typing ofVibrio parahaemolyticus from Tiger shrimp cultureenvironments along the southwest coast of India. Food Biotechnol-ogy, 23, 284311.

Chang, T.C., Chen, C.H. & Chen, H.C. (1994). Development of a latexagglutination test for the rapid identification ofVibrio parahaemo-lyticus. Journal of Food Protection, 57, 3136.

Chao, G., Jiao, X., Zhou, X. et al. (2009). Serodiversity, pandemicO3:K6 clone, molecular typing, and antibiotic susceptibility offoodborne and clinical Vibrio parahaemolyticus isolates in Jiangsu,China. Foodborne Pathogens and Disease, 6, 10211028.

Chiou, C.S., Hsu, S.Y., Chiu, S.I., Wang, T.K. & Chao, C.S.(2000). Vibrio parahaemolyticus serovar O3:K6 as cause ofunusually high incidence of food-borne disease outbreaks inTaiwan from 1996 to 1999. Journal of Clinical Microbiology, 38,46214625.

Chowdhury, N.R., Chakraborty, S., Eampokalap, B. et al. (2000a).Clonal dissemination of Vibrio parahaemolyticus displaying similarDNA fingerprint but belonging to two different serovars (O3:K6 andO4:K68) in Thailand and India. Epidemiology and Infection, 125,1725.

Chowdhury, N.R., Chakraborty, S., Ramamurthy, T. et al. (2000b).Molecular evidence of clonal Vibrio parahaemolyticus pandemicstrains. Emerging Infectious Diseases, 6, 631636.

Chun, D., Chung, J.K., Tak, R. & Seol, S.Y. (1975). Nature of theKanagawa phenomenon ofVibrio parahaemolyticus. Infection andImmunity, 12, 8187.

Cook, D.W., Bowers, J.C. & DePaola, A. (2002). Density of total and

pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and GulfCoast molluscan shellfish at harvest. Journal of Food Protection, 65,18731880.

Curtis, S.K., Kothary, M.H., Blodgett, R.J., Raybourne, R.B., Ziobro,G.C. & Tall, B.D. (2007). Rugosity in Grimontia hollisae. Appliedand Environmental Microbiology, 73, 12151224.

Daniels, N.A., MacKinnon, L., Bishop, R. et al. (2000a). Vibrioparahaemolyticus infections in the United States, 19731998. TheJournal of Infectious Diseases, 181, 16611666.

Daniels, N.A., Ray, B., Easton, A. et al.(2000b). Emergence of a newVibrio parahaemolyticus serotype in raw oysters: a preventionquandary. The Journal of the American Medical Association, 284,15411545.

Datta, S., Janes, M.E. & Simonson, J.G. (2008). Immunomagneticseparation and coagglutination ofVibrio parahaemolyticuswith anti-flagellar protein monoclonal antibody. Clinical and Vaccine Immu-nology, 15, 15411546.

DePaola, A., Hopkins, L.H., Peeler, J.T., Wentz, B. & McPhearson,R.M. (1990). Incidence of Vibrio parahaemolyticus in U.S. coastalwaters and oysters. Applied and Environmental Microbiology, 56,22992302.

DePaola, A., Kaysner, C.A., Bowers, J. & Cook, D.W. (2000).Environmental investigations of Vibrio parahaemolyticus in oys-ters after outbreaks in Washington, Texas, and New York (1997and 1998). Applied and Environmental Microbiology, 66, 46494654.

DePaola, A., Ulaszek, J., Kaysner, C.A. et al. (2003). Molecular,serological, and virulence characteristics of Vibrio parahaemolyticusisolated from environmental, food, and clinical sources in NorthAmerica and Asia. Applied and Environmental Microbiology, 69,39994005.

Di Pinto, A., Ciccarese, G., Tantillo, G., Catalano, D. & Forte, V.T.(2005). A collagenase targeted multiplex PCR assay for identifi-

cation ofVibrio alginolyticus, Vibrio cholerae, and Vibrio parahae-molyticus. Journal of Food Protection, 68, 150153.Dileep, V., Kumar, H.S., Kumar, Y., Nishibuchi, M., Karunasagar, I.

& Karunasagar, I. (2003). Application of polymerase chain reactionfor detection ofVibrio parahaemolyticus associated with tropicalseafoods and coastal environment. Letters in Applied Microbiology,36, 423427.

Donovan, T.J. & van Netten, P. (1995). Culture media for the isolationand enumeration of pathogenic Vibrio species in foods andenvironmental samples.International Journal of Food Microbiology,26, 7791.





13/15

Drake, S.L., DePaola, A. & Jaykus, L.A. (2007). An overview ofVibrio vulnificus and Vibrio parahaemolyticus. Comprehensive Re-views in Food Science and Food Safety, 6, 120144.

Dziejman, M. & Yildiz, F.H. (2011). Genomics of pathogenic Vibriospecies. In: Genomics of Foodborne Pathogens (edited by W.Wiedman & W. Zhang). Pp. 267310. New York, NY: SpringerScience+Business Media.

Earle, P.M. & Crisley, F.D. (1975). Isolation and characterization ofVibrio parahaemolyticus from Cape Cod soft-shell clams (Myaarenaria). Applied and Environmental Microbiology, 29, 635640.

Ellison, R.K., Malnati, E., DePaola, A., Bowers, J. & Rodrick, G.E.(2001). Populations of Vibrio parahaemolyticus in retail oystersfrom Florida using two methods. Journal of Food Protection, 64,682686.

Fujino, T., Okuno, Y., Nakoda, D. et al. (1953). On the bacteriolog-ical examination of shirasu food poisoning. Medical Journal ofOsaka University, 4 , 299304.

Gonza lez-Escalona, N., Cachicas, V., Acevedo, C. et al. (2005).Vibrioparahaemolyticus diarrhea, Chile, 1998 and 2004. Emerging Infec-tious Diseases, 11, 125131.

Gonza lez-Escalona, N., Blackstone, G.M. & DePaola, A. (2006).Characterization of a Vibrio alginolyticus strain, isolated fromAlaskan oysters, carrying a hemolysin gene similar to the thermo-stable direct hemolysin-related hemolysin gene (trh) of Vibrio

parahaemolyticus. Applied and Environmental Microbiology, 72,79257929.

Gooch, J.A., DePaola, A., Kaysner, C.A. & Marshall, D.L. (2001).Evaluation of two direct plating methods using nonradioactiveprobes for enumeration of Vibrio parahaemolyticus in oysters.Applied and Environmental Microbiology, 67, 721724.

Griffitt, K.J., Noriea, N.F. III, Johnson, C.N. & Grimes, D.J. (2011).Enumeration of Vibrio parahaemolyticus in the viable but noncul-turable state using direct plate counts and recognition of individualgene fluorescence in situ hybridization. Journal of MicrobiologicalMethods, 85 , 114118.

Hagen, C.J., Sloan, E.M., Lancette, G.A., Peeler, J.T. & Sofos, J.N.(1994). Enumeration of Vibrio parahaemolyticus and Vibrio vulnif-icusin various seafoods with two enrichment broths. Journal of FoodProtection, 57, 403409.

Hara-Kudo, Y., Nishina, T., Nakagawa, H., Konuma, H., Hasegawa,J. & Kumagai, S. (2001). Improved method for detection of Vibrio

parahaemolyticus in seafood. Applied and Environmental Microbiol-ogy, 67, 58195823.

Hara-Kudo, Y., Sugiyama, K., Nishibuchi, M. et al. (2003). Preva-lence of pandemic thermostable direct hemolysin-producing Vibrioparahaemolyticus O3:K6 in seafood and the coastal environment inJapan. Applied and Environmental Microbiology, 69, 38833891.

Hashii, N., Kondo, S., Iguchi, T., Nishibuchi, M. & Hisatsune, K.(2000). Chemical and serological properties of lipopolysaccharidesfrom Vibrio parahaemolyticus O-untypeable strains isolated frompatients. Microbiology and Immunology, 44, 229234.

Hisatsune, K., Kiuye, A. & Kondo, S. (1980). Sugar composition ofO-antigenic lipopolysaccharides isolated from Vibrio parahaemolyt-icus. Microbiology and Immunology, 24, 691701.

Hisatsune, K., Iguchi, T., Haishima, Y., Tamura, N. & Kondo, S.(1993). Lipopolysaccharide isolated from a new O-antigenic form(O13) ofVibrio parahaemolyticus.Microbiology and Immunology, 37,

143147.Hi, L., Larsen, J.L., Dalsgaard, I. & Dalsgaard, A. (1998a).Occurrence ofVibrio vulnificus biotypes in Danish marine environ-ments. Applied and Environmental Microbiology, 64, 713.

Hi, L., Dalsgaard, I. & Dalsgaard, A. (1998b). Improved isolation ofVibrio vulnificusfrom seawater and sediment with cellobiose-colistinagar. Applied and Environmental Microbiology, 64 , 17211724.

Honda, T., Chearskul, S., Takeda, Y. & Miwatani, T. (1980).Immunological methods for detection of Kanagawa phenomenonof Vibrio parahaemolyticus. Journal of Clinical Microbiology, 11,600603.

Honda, T., Yoh, M., Kongmuang, U. & Miwatani, T. (1985). Enzyme-linked immunosorbent assays for detection of thermostable directhemolysin ofVibrio parahaemolyticus. Journal of Clinical Micro-biology, 22, 383386.

Honda, T., Nishibuchi, M., Miwatani, T. & Kaper, J.B. (1986).Demonstration of a plasmid-borne gene encoding a thermostabledirect hemolysin in Vibrio cholerae non-O1 strains. Applied andEnvironmental Microbiology, 52, 12181220.

Honda, T., Miwatani, T., Yabushita, Y., Koike, N. & Okada, K.(1995). A novel method to chemically immobilize antibody on nylonand its application to the rapid and differential detection of twoVibrio parahaemolyticus toxins in a modified enzyme-linked immu-nosorbent assay. Clinical and Diagnostic Laboratory Immunology, 2,177181.

Iguchi, T., Kondo, S. & Hisatsune, K. (1995). Vibrio parahaemolyticusO serotypes from O1 to O13 all produce R-type lipopolysaccharide:SDS-PAGE and compositional sugar analysis. FEMS MicrobiologyLetters, 130, 287292.

Janda, J.M., Powers, C., Bryant, R.G. & Abbot, S.L. (1988).Current perspectives on the epidemiology and pathogenesis ofclinically significant Vibrio spp. Clinical Microbiology Reviews, 1,245267.

Kaneko, T. & Colwell, R.R. (1975). Incidence ofVibrio parahaemo-lyticus in Chesapeake Bay. Applied Microbiology, 30, 251257.

Karunasagar, I., Sugumar, G., Karunasagar, I. & Reilley, P.J.A.(1995). Rapid detection ofVibrio cholerae contamination of seafoodby polymerase chain reaction. Molecular Marine Biology andBiotechnology, 4, 365368.

Karunasagar, I., Sugumar, G., Karunasagar, I. & Reilly, P.J.A. (1996).Rapid polymerase chain reaction method for detection of KanagawapositiveVibrio parahaemolyticusin seafoods.International Journal ofFood Microbiology, 31, 317323.

Kaufman, G.E., Blackstone, G.M., Vickery, M.C. et al. (2004). Real-time PCR quantification of Vibrio parahaemolyticus in oysters usingan alternative matrix. Journal of Food Protection, 67, 24242429.

Kaysner, C.A. & DePaola, A. (2004).Vibrio.Bacteriological AnalyticalManual, Chapter 9. Arlington, VA: U.S. Food and Drug Admin-istration.

Kaysner, C.A., Abeyta, C. Jr, Stott, R.F., Lilja, J.L. & Wekell, M.M.(1990). Incidence of urea-hydrolyzing Vibrio parahaemolyticus inWillapa Bay, Washington. Applied and Environmental Microbiology,

56, 904907.Khan, A.A., McCarthy, S., Wang, R.F. & Cerniglia, C.E. (2002).

Characterization of United States outbreak isolates of Vibrioparahaemolyticus using enterobacterial repetitive intergenic consen-sus (ERIC) PCR and development of a rapid PCR method fordetection of O3:K6 isolates. FEMS Microbiology Letters, 206, 209213.

Kourany, M. (1983). Medium for isolation and differentiation ofVibrio parahaemolyticus and Vibrio alginolyticus. Applied andEnvironmental Microbiology, 45, 310312.

Kumar, B.K., Raghunath, P., Devegowda, D. et al. (2011). Develop-ment of monoclonal antibody based sandwich ELISA for the rapiddetection of pathogenic Vibrio parahaemolyticus in seafood. Inter-national Journal of Food Microbiology, 145, 244249.

Lee, C. & Pan, S.F. (1993). Rapid and specific detection of thethermostable direct haemolysin gene in Vibrio parahaemolyticus by

the polymerase chain reaction.Journal of General Microbiology,139

,32253231.Lee, C., Chen, L.H., Liu, M.L. & Su, Y.C. (1992). Use of an

oligonucleotide probe to detect Vibrio parahaemolyticus in artifi-cially contaminated oysters. Applied and Environmental Microbiol-ogy, 58, 34193422.

Levin, R.E. (2010). Vibrio parahaemolyticus. In: Rapid Detection andCharacterization of Foodborne Pathogens by Molecular Techniques(edited by ????. ????). Pp. 167192. Boca Raton, FL: CRC Press.

Lu, P.L., Chang, S.C., Pan, H.J., Chen, M.L. & Luh, K.T. (2000).Application of pulsed-field gel electrophoresis to the investigation of





14/15

a nosocomial outbreak of Vibrio parahaemolyticus. Journal ofMicrobiology, Immunology, and Infection, 33 , 2933.

Marshall, S., Clark, C.G., Wang, G., Mulvey, M., Kelly, M.T. &Johnson, W.M. (1999). Comparison of molecular methods fortypingVibrio parahaemolyticus.Journal of Clinical Microbiology,37,24732478.

Martinez-Urtaza, J., Simental, L., Velasco, D. et al. (2005). PandemicVibrio parahaemolyticus O3:K6, Europe. Emerging Infectious Dis-eases, 11, 13191320.

Martinez-Urtaza, J., Lozano-Leon, A., Vina-Feas, A., de Novoa, J. &Garcia-Martin, O. (2006). Differences in the API20E biochemicalpatterns of clinical and environmental Vibrio parahaemolyticusisolates. FEMS Microbiology Letters, 255, 7581.

Matsumoto, C., Okuda, J., Ishibashi, M. et al. (2000). Pandemicspread of an O3:K6 clone of Vibrio parahaemolyticus andemergence of related strains evidenced by arbitrarily primed PCRand toxRS sequence analyses. Journal of Clinical Microbiology, 38,578585.

McLaughlin, J.C. (1995). Vibrio. In: Manual of Clinical Microbiology,Chapter 35 (edited by E. JoBaron, M.A. Pfaller, F.C. Tenover, R.H.Yolken & P.R. Murray). Pp. 465476. Washington, DC: AmericanSociety for Microbiology (ASM) Press.

McLaughlin, J.B., DePaola, A., Bopp, C.A. et al. (2005). Outbreak ofVibrio parahaemolyticus gastroenteritis associated with Alaskan

oysters. The New England Journal of Medicine, 353, 14631470.Miyamoto, Y., Kato, T., Obara, Y., Akiyama, S., Takizawa, K. &

Yamai, S. (1969). In vitro hemolytic characteristic of Vibrioparahaemolyticus: its close correlation with human pathogenicity.The Journal of Bacteriology, 100, 11471149.

Miyasaka, J., Yahiro, S., Arahira, Y., Tokunaga, H., Katsuki, K. &Hara-Kudo, Y. (2006). Isolation of Vibrio parahaemolyticus andVibrio vulnificusfrom wild aquatic birds in Japan. Epidemiology andInfection, 134, 780785.

Myers, M.L., Panicker, G. & Bej, A.K. (2003). PCR detection of anewly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogenin pure cultures and seeded waters from the Gulf of Mexico.Appliedand Environmental Microbiology, 69, 21942200.

Nair, G.B., Ramamurthy, T., Bhattacharya, S.K., Dutta, B., Takeda,Y. & Sack, D.A. (2007). Global dissemination of Vibrio parahae-molyticusserotype O3:K6 and its serovariants.Clinical MicrobiologyReviews, 20, 3848.

Nasu, H., Iida, T., Sugahara, T. et al. (2000). A filamentous phageassociated with recent pandemic Vibrio parahaemolyticus O3:K6strains.Journal of Clinical Microbiology, 38, 21562161.

Nemoto, J., Sugawara, C., Akahane, K. et al. (2009). Rapid andspecific detection of the thermostable direct hemolysin gene in Vibrioparahaemolyticus by loop-mediated isothermal amplification. Jour-nal of Food Protection, 72 , 748754.

Nemoto, J., Ikedo, M., Kojima, T., Momoda, T., Konuma, H. &Hara-Kudo, Y. (2011). Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitivedetection ofVibrio parahaemolyticus.Journal of Food Protection, 74,14621467.

Nishibuchi, M. & Kaper, J.B. (1995). Thermostable direct hemolysingene ofVibrio parahaemolyticus: a virulence gene acquired by amarine bacterium. Infection and Immunity, 63, 20932099.

Nishibuchi, M., Ishibashi, M., Takeda, Y. & Kaper, J.B. (1985).

Detection of the thermostable direct hemolysin gene and relatedDNA sequences in DNA colony hybridization test. Infection andImmunity, 49, 481486.

Nishibuchi, M., Doke, S., Toizumi, S., Umeda, T., Yoh, M. &Miwatani, T. (1988). Isolation from a coastal fish ofVibrio hollisaecapable of producing a hemolysin similar to the thermostable directhemolysin ofVibrio parahaemolyticus. Applied and EnvironmentalMicrobiology, 54, 21442146.

Nishibuchi, M., Khaeomanee-iam, V., Honda, T., Kaper, J.B. &Miwatani, T. (1990). Comparative analysis of the hemolysin genes ofVibrio cholerae non-O1, V. mimicus, andV. hollisae that are similar

to thetdh gene ofV. parahaemolyticus.FEMS Microbiology Letters,55, 251256.

Nishibuchi, M., Janda, J.M. & Ezaki, T. (1996). The thermostabledirect hemolysin gene (tdh) of Vibrio hollisae is dissimilar inprevalence to and phylogenetically from the tdh genes of othervibrios: implication in the horizontal transfer of the tdh gene.Microbiology and Immunology, 40, 5965.

Nishibuchi, M., Doke, S., Toizumi, S., Umeda, T., Yoh, M. &Miwatani, T. (1998). Isolation from a coastal fish ofVibrio hollisaecapable of producing a hemolysin similar to the thermostable directhemolysin ofVibrio parahaemolyticus. Applied and EnvironmentalMicrobiology, 54, 21442146.

Nordstrom, J.L. & DePaola, A. (2003). Improved recovery ofpathogenic Vibrio parahaemolyticus from oysters using colonyhybridization following enrichment. Journal of MicrobiologicalMethods, 52, 273277.

Nordstrom, J.L., Vickery, M.C., Blackstone, G.M., Murray, S.L. &DePaola, A. (2007). Development of a multiplex real-time PCRassay with an internal amplification control for the detection of totaland pathogenic Vibrio parahaemolyticus bacteria in oysters. Appliedand Environmental Microbiology, 73, 58405847.

Notomi, T., Okayama, H., Masubuchi, H. et al. (2000). Loop-mediated isothermal amplification of DNA.Nucleic Acids Research,28, e63.

Okuda, J., Ishibashi, M., Abbott, S.L., Janda, J.M. & Nishibuchi, M.(1997a). Analysis of the thermostable direct hemolysin (tdh) geneand the tdh- related hemolysin (trh) genes in urease-positive strainsofVibrio parahaemolyticus isolated on the West Coast of the UnitedStates.Journal of Clinical Microbiology, 35, 19651971.

Okuda, J., Ishibashi, M., Hayakawa, E. et al. (1997b). Emergence of aunique O3:K3 clone of Vibrio parahaemolyticus in Calcutta, Indiaand isolation of strains from same clonal group from South EastAsian travelers arriving in Japan. Journal of Clinical Microbiology,35, 31503155.

Okura, M., Osawa, R., Arakawa, E., Terajima, J. & Watanabe, H.(2005). Identification of Vibrio parahaemolyticus pandemic group-specific DNA sequence by genomic subtraction. Journal of ClinicalMicrobiology, 43, 35333536.

Oliver, J.D. (1981). Lethal cold stress ofVibrio vulnificus in oysters.Applied and Environmental Microbiology, 41, 710717.

Oliver, J.D., Guthrie, K., Preyer, J. et al. (1992). Use of colistin-

polymyxin B-cellobiose agar for isolation ofVibrio vulnificus fromthe environment. Applied and Environmental Microbiology, 58, 737739.

Pace, J.L., Chai, T.J., Rossi, H.A. & Jiang, X. (1997). Effect of bile onVibrio parahaemolyticus. Applied and Environmental Microbiology,63, 23722377.

Quilici, M.L., Robert-Pillot, A., Picart, J. & Fournier, J.M. (2005).Pandemic Vibrio parahaemolyticus O3:K6 spread, France. EmergingInfectious Diseases, 11, 11481149.

Raghunath, P., Karunasagar, I. & Karunasagar, I. (2009). Improvedisolation and detection of pathogenic Vibrio parahaemolyticus fromseafood using a new enrichment broth.International Journal of FoodMicrobiology, 129, 200203.

Raghunath, P., Maiti, B., Shekar, M., Karunasagar, I. & Karunasa-gar, I. (2010). Clinical isolates ofAeromonas veronii biovar veroniiharbor a nonfunctional gene similar to the thermostable direct

hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.FEMS Microbiology Letters, 307, 151157.Rahman, M., Bhuiyan, N.A., Kuhn, I. et al. (2006). Biochemical

fingerprinting of Vibrio parahaemolyticus by the PhenePlate system:comparison between pandemic and non-pandemic serotypes. Epi-demiology and Infection, 134, 905989.

Ray, B., Hawkins, S.M. & Hackney, C.R. (1978). Method for thedetection of injured Vibrio parahaemolyticus in seafoods. Appliedand Environmental Microbiology, 35, 11211127.

Sakazaki, R., Tamura, K., Kato, T., Obara, Y., Yamai, S. & Hobo, K.(1968). Studies of the enteropathogenic, facultatively halophilic





15/15

bacteria, Vibrio parahaemolyticus. III. Enteropathogenicity. Japa-nese Journal of Medical Science & Biology, 21, 325331.

Sarkar, B., Chowdhury, N.R., Nair, G.B. et al. (2003). Molecularcharacterization of Vibrio parahaemolyticus of similar serovarsisolated from sewage and clinical cases of diarrhoea in Calcutta,India. World Journal of Microbiology & Biotechnology, 19, 771776.

Sawabe, T., Yoshizawa, A., Kawanishi, Y. et al. (2009). Multi-probe-fluorescence in situ hybridization for the rapid enumeration of viableVibrio parahaemolyticus. Microbes and Environments, 24, 259264.

Simonson, J. & Siebeling, R.J. (1986). Rapid serological identificationofVibrio vulnificus by anti-H coagglutination. Applied and Environ-mental Microbiology, 52, 12991304.

Su, Y.C. & Liu, C. (2007). Vibrio parahaemolyticus: a concern ofseafood safety. Food Microbiology, 24, 549558.

Tada, J., Ohashi, T., Nishimura, N. et al. (1992). Detection of thethermostable direct hemolysin gene (tdh) and the thermostable directhemolysin-related hemolysingene (trh) ofVibrio parahaemolyticus bypolymerase chain reaction.Molecular and CellularProbes, 6, 477487.

Tassin, M.G., Siebeling, R.J., Roberts, N.C. & Larson, A.D. (1983).Presumptive identification of Vibrio species with H anti-serum.Journal of Clinical Microbiology, 18, 400407.

Terada, Y. (1968). Serological studies of Vibrio parahaemolyticus. II.Flagellar antigens. Japanese Journal of Bacteriology, 23, 767771.

Thompson, F.L., Hoste, B., Vandemeulebroecke, K. & Swings, J.

(2003). Reclassification ofVibrio hollisae as Grimontia hollisae gen.nov., comb. nov. International Journal of Systemetic and Evolution-ary Microbiology, 53, 16151617.

Tyagi, A., Saravanan, V., Karunasagar, I. & Karunasagar, I. (2009).Detection ofVibrio parahaemolyticus in shellfish by SYBR greenreal-time PCR and evaluation of three enrichment media. Interna-tional Journal of Food Microbiology, 129, 124130.

U.S. Food and Drug Administration (1997). NSSP: National ShellfishSanitation Program, Sanitation of the harvesting, processing and thedistribution of shellfish.Manual of Operations, Part II. Washington,DC: U.S. Department of Health and Human Services, Food andDrug Administration.

Wagatsuma, S. (1968). On a medium for hemolytic reaction. MediaCircle, 13, 159162 (In Japanese).

Ward, L.N. & Bej, A.K. (2006). Detection of Vibrio parahaemolyticusin shellfish by use of multiplexed real-time PCR with TaqManfluorescent probes. Applied and Environmental Microbiology, 72,

20312042.Watkins, W.D. & Cabelli, V.J. (1985). Effect of fecal pollution on

Vibrio parahaemolyticus densities in an estuarine environment.Applied and Environmental Microbiology, 49, 13071313.

Wong, H.C. (2003). Detecting and molecular typing of Vibrioparahaemolyticus. Journal of Food and Drug Analysis, 11, 100107.

Wong, H.C. & Lin, C.H. (2001). Evaluation of typing of Vibrioparahaemolyticus by three PCR methods using specific primers.Journal of Clinical Microbiology, 39, 42334240.

Wong, H.C., Lu, K.T., Pan, T.M., Lee, C.L. & Shih, D.Y. (1996).Subspecies typing of Vibrio parahaemolyticus by pulsed-fieldgel electrophoresis. Journal of Clinical Microbiology, 34, 15351539.

Wong, H.C., Chen, M.C., Liu, S.H. & Liu, D.P. (1999a). Incidence ofhighly genetically diversified Vibrio parahaemolyticus in seafoodimported from Asian countries. International Journal of FoodMicrobiology, 52, 181188.

Wong, H.C., Ho, C.Y., Kuo, L.P., Wang, T.K., Lee, C.L. & Shih,D.Y. (1999b). Ribotyping of Vibrio parahaemolyticus isolatesobtained from food poisoning outbreaks in Taiwan. Microbiologyand Immunology, 43, 631636.

Wong, H.C., Liu, C.C., Pan, T.M., Wang, T.K., Lee, C.L. & Shih,D.Y. (1999c). Molecular typing of Vibrio parahaemolyticus isolates,obtained from patients involved in food poisoning outbreaks inTaiwan, by random amplified polymorphic DNA analysis. Journalof Clinical Microbiology, 37 , 18091812.

Wong, H.C., Liu, S.H., Wang, T.K. et al. (2000). Characteristics ofVibrio parahaemolyticusO3:K6 from Asia. Applied and Environmen-

tal Microbiology, 66, 39813986.Wright, A.C. & Schneider, K.R. (2009). Pathogenic vibrios in seafood.

In: Pathogens and Toxins in Food (edited by V.K. Juneja & J.N.Sofos). Pp. 146163. Washington, DC: ASM Press.

Yamazaki, W., Ishibashi, M., Kawahara, R. & Inoue, K. (2008).Development of a loop mediated isothermal amplification assay forsensitive and rapid detection of Vibrio parahaemolyticus. BMCMicrobiology, 30, 163.

Yamazaki, W., Kumeda, Y., Misawa, N., Nakaguchi, Y. & Nishibu-chi, M. (2010). Development of a loop mediated isothermalamplification assay for sensitive and rapid detection of the tdh andtrh genes of Vibrio parahaemolyticus and related Vibrio species.Applied and Environmental Microbiology, 76, 820828.

Yeung, P.S. & Boor, K.J. (2004). Epidemiology, pathogenesis, andprevention of foodborne Vibrio parahaemolyticus infections. Food-borne Pathogens and Disease, 1, 7488.

Zhao, G., Xing, F. & Deng, S. (2007). A disposable amperometric

enzyme immunosensor for rapid detection of Vibrio parahaemolyt-icus in food based on agaroseNano-Au membrane andscreen-printed electrode. Electrochemistry communications, 9,12631268.




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