Method for detecting the virulent or non-virulent nature of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis

Abstract

The present invention relates to the field of monitoring acute hepatopancreatic necrosis disease in shrimp. More specifically, the present invention discloses the detection of the presence of the alkaline phosphatase Phox enzyme, or the presence of the PiRAvp and / or PirBvp toxins of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease, respectively associated with the avirulence or toxicity of said bacteria. Therefore, an assay capable of detecting said Phox enzyme and / or said toxin may be very useful for monitoring said disease in shrimp.

Description

Technical Field

[0001] The present invention relates to the field of monitoring acute hepatopancreatic necrosis in shrimp. More specifically, the present invention discloses a method for detecting the presence of alkaline phosphatase Phox enzyme, or PiRA of Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis. VP and / or PirB VP The presence of toxins, correlates with the avirulence or virulence of the bacteria, respectively. Therefore, an assay capable of detecting the Phox enzyme and / or the toxins may be very useful for monitoring the disease in shrimp. Background Art

[0002] The Gram-negative marine bacterium Vibrio parahaemolyticus is an important aquatic pathogen, and some strains are capable of causing acute hepatopancreatic necrosis disease (AHPND) and other important diseases in shrimp aquaculture (Jayasree et al., 2006; Li et al., 2017). Shrimp production in areas affected by AHPND sometimes drops dramatically (to ~60%), and the disease causes losses of $1 billion per year to the global shrimp aquaculture industry (Lee et al., 2015; Nunan et al., 2014). Some strains of V. parahaemolyticus do contain the pVA1 plasmid (63-70 kb), which encodes a gene named PirA. VP and PirB VP The binary toxins are homologous to the Photorhabdus luminescens insect-associated (Pir) toxins PirA / PirB (Campa-Córdova et al., 2017; Gomez-gil et al., 2014; Han et al., 2015; Lee et al., 2015). VP and PirB VP Toxins are the main virulence factors of bacteria that mediate AHPND etiology and mortality in shrimp (Campa-Córdova et al., 2017; Dong et al., 2017; Kumar et al., 2018)

[0003] However, it is completely unknown whether the latter toxic phenotype can be converted into an avirulent phenotype and whether the phenotypes can be easily distinguished by an easy-to-use assay measuring the products secreted by each phenotype. Such an assay would be very useful for monitoring AHPND in, for example, shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 Extracellular proteins (VPs) of Vibrio parahaemolyticus from different culture conditions AHPND Coomassie-stained SDS-PAGE gel of ECP, (i) VP AHPNDECP was purified from a culture of Vibrio parahaemolyticus M0904 under constant stirring (120 min -1 ) and no flocs were formed, showing two distinct bands at 13 and 50 kDa (lanes 1-4, showing the results of four repeated cultures). (ii) Under continuous stirring (110 min -1 ) and highly flocculated VP purified from Vibrio parahaemolyticus M0904 culture AHPND ECP showed a single distinct band at 73 kDa (lanes 1-4, showing the results of 4 replicate cultures).

[0005] Figure 2 Expression of toxic AHPND plasmid genes: A-ORF14, B-PirB of Vibrio parahaemolyticus M0904 strain cultured under different culture conditions VP and the gene responsible for floc formation: C-alkaline phosphatase PhoX. Vibrio parahaemolyticus M0904 strain was cultured in 20 ml marine broth for 110 min. -1 (highly flocculated) or 120min -1 (No floc formation) Incubate overnight with continuous stirring. Samples from both culture conditions were collected after 12 and 24 h for gene expression determination. For the toxic AHPND plasmid gene, the expression in the floc formation group was set to 1. However, for the alkaline phosphatase PhoX gene, the expression in the no floc formation group was set to 1. The results are mean ± SE (n = 3) and are expressed relative to V. parahaemolyticus toxR and rpoAmRNA. Asterisks indicate significant differences between V. parahaemolyticus M0904 cultures with flocs (110 rpm) and without flocs (120 rpm) * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001), ***** (P < 0.00001).

[0006] Figure 3 (A) Brine shrimp larvae were cultured in 20 ml of marine bacterial broth for 120 min. -1 or 110 minutes -1 Survival rate (%) of V. parahaemolyticus M0904 culture challenged overnight with continuous stirring 48 h after challenge. 7 Parahaemolyticus challenge with 10 cells / ml of feeding water. Unchallenged larvae were used as negative controls. Error bars represent standard error of five replicates; different letters indicate significant differences (P<0.001).

[0007] (B) Brine shrimp larvae were treated with 1.3, 1.95, 2.6, and 3.9 μg of VP concentrated from a culture of V. parahaemolyticus M0904. AHPNDSurvival rate (%) after 60 h of extracellular protein (ECP) challenge, the culture of V. parahaemolyticus M0904 and PirA VP and PirB VP Toxins in 120min -1 Incubate with constant stirring or with alkaline phosphatase PhoX for 110 min -1 Incubate with constant agitation. Unchallenged larvae were used as negative controls. Error bars represent standard error of three replicates; different letters indicate significant differences (P<0.001).

[0008] (C) Brine shrimp larvae received 1.3, 1.95, 2.6, and 3.9 μg of VP AHPND Purified PirA VP and PirB VP Toxin or VP AHPND Survival rate (%) after 60 h of challenge with purified alkaline phosphatase PhoX. Unchallenged larvae were used as negative control. Error bars represent the standard error of three replicates; different letters indicate significant differences (P<0.001).

[0009] Figure 4 Macrobrachium larvae (8 days old) were cultured in 20 ml marine bacterial broth (MB) for 120 min. -1 or 110 minutes -1 Survival rate (%) after challenge with an overnight culture of Vibrio parahaemolyticus M0904 for 12, 24, 36 and 48 h with continuous agitation. 6 parahaemolyticus challenged with 10 cells / ml of feed water. Unchallenged larvae served as negative controls. Values ​​are expressed as mean ± SE (n = 3). Asterisks indicate larvae that were challenged with 110 rpm or 120 rpm min -1 Significant differences among treatment groups for M0904 culture challenge *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001), *****(P<0.00001), ******(P<0.0000001), *******(P<0.0000001). DETAILED DESCRIPTION

[0010] The present invention discloses that low oscillation speed can trigger phenotypic transformation of Vibrio parahaemolyticus, especially mediating toxic changes in shrimp. The present invention indeed shows that Vibrio parahaemolyticus downregulates PirAB at lower oscillation speeds. VP Toxin expression, simultaneous production and secretion of the alkaline phosphatase PhoX enzyme. Thus, the present invention shows that V. parahaemolyticus exhibits different phenotypes depending on environmental conditions: virulent and avirulent phenotypes.

[0011] Therefore, the present invention firstly relates to the use of the alkaline phosphatase Phox of Vibrio parahaemolyticus as a marker for the avirulent phenotype of said Vibrio parahaemolyticus.

[0012] The term “Vibrio parahaemolyticus” refers to all strains of the Gram-negative marine bacterium that is an important aquatic pathogen and is capable of causing acute hepatopancreatic necrosis disease (AHPND) and several other important diseases in shrimp aquaculture, such as tail necrosis, shell disease, red shrimp disease, loose shell syndrome (LSS) and white gut disease (WGD) (Jayasree et al., 2006; Li et al., 2017). The latter term more specifically refers to strains of V. parahaemolyticus containing the pVA1 plasmid (63-70 kb), which encodes a gene named PirA VP and PirB VP Binary toxins that are homologous to the Photorhabdus luminescens insect-related (Pir) toxins PirA / PirB (Campa-Córdova et al., 2017; Han et al., 2015) (Gomez-Gil et al., 2014; Soto-Rodriguez et al., 2015; Lee et al., 2015). VP and PirB VP Toxins are the main virulence factors of bacteria that mediate the pathogenesis and mortality of shrimp AHPND (Campa-Córdova et al., 2017; Dong et al., 2017; Han et al., 2015). A non-limiting example of such a Vibrio parahaemolyticus strain is strain M0904, deposited at the Belgian Coordinating Collection of Microorganisms (BCCM / LMG strain collection) (Laboratorium voorMicrobiologie, Universiteit Gent (UGent), KLLedeganckstraat 35, B-9000 Gent, Belgium), deposit number (accession number): LMG P-31518.

[0013] The term "avirulent" V. parahaemolyticus indicates that the bacteria do not mediate AHPND etiology and mortality in shrimp, and thus are less toxic to shrimp, compared to bacteria that mediate AHPND etiology and mortality in shrimp.

[0014] The term "alkaline phosphatase Phox" is an enzyme that releases free inorganic phosphate from many phosphate-containing compounds and provides inorganic phosphate as a nutrient for bacteria, as described by Torriani (1990). More specifically, the term refers to an enzyme with accession number-Q87JR9 (UniProt) and having the following amino acid sequence:

[0015] MSKETFDATRYNQSDNKPFEEVLEASLSRRSILKGGLGISAMTAFGAFG

[0016] LAGCNSSSSGTSASNGSGVSK

[0017] AVLNFDSIPGSLTDAVSIPQGYTAQVLVPWGTPLNAQGSAWKNDGSNTS

[0018] SDQLNALGMHHDGMHFFPLND

[0019] STTDGLLCINHEYIDTSALHPNGPTVANGVRTIVDEVRKEINAHGVSVV

[0020] RIQLEDNMWKLVDTDPLNRRY

[0021] TGATVMDLSGPVAHTALTVTRFSPDGSQARGTLNNCGNGYTPWGTYLT

[0022] CEENWPGYFVNAGTRTEEQDRI

[0023] GVDDKSTRYLWETLAGNSEERLDEFTRFNVAPTGTSSADDYRNEANGH

[0024] GYIVEIDPYTQNSRAKKRTALG

[0025] RFRHEGCAFGKLEAGKPVVFYSGHDSRFEYLYKFESAAAWDPADANPA

[0026] NRLATGDKYMDEGTLYVARFNE

[0027] DSTGTWLPLTLDSVTTSGGTLADHFNSLAEIIINTAGAADLVGATPMDR

[0028] PEWCSVDPFTGSVYLTLTNNT

[0029] RRTDETNPANPRLNNKFGHVIRWDEGTSATDFIWDIFVFGSPENGDADT

[0030] NRSGLNELNQFASPDGLAFDG

[0031] RGILWIQTDNGADEVTSYTNDQMLAVVPSKLTNENGDQAVIGADNQAE

[0032] LKRFFVGPNGCEVTGFTISPDY

[0033] KSLFVNIQHPGNWPYSDDAAQETPTGTTVRPRAATVVIRREDGGEIAV

[0034] (SEQ ID N°1)

[0035] The term "phenotypic marker" refers to the use of the presence of alkaline phosphatase PhoX in a substrate (eg shrimp culture water) to indicate the avirulence of V. parahaemolyticus.

[0036] In addition, the present invention discloses the use of the above-mentioned method for monitoring acute hepatopancreatic necrosis of shrimp.

[0037] The term "shrimp" refers to any shrimp known in the art, and more particularly to freshwater shrimp, such as Macrobrachium rosenbergii and Penaeus.

[0038] The term “acute hepatopancreatic necrosis disease (AHPND)” refers to a disease originally called early mortality syndrome (EMS), which is characterized by acute massive shedding of hepatopancreatic tubular epithelial cells and is caused by virulent strains of the Gram-negative marine bacterium Vibrio parahaemolyticus that is ubiquitous in estuarine and coastal waters ( Li et al., 2017 ; Tran et al., 2013 ).

[0039] Furthermore, the present invention relates to the use of a method for detecting the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, or - in other words, to the use of a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus (e.g. in an ELISA / antibody-based assay) for characterizing said Vibrio parahaemolyticus as an avirulent phenotype.

[0040] The term "molecule that binds to alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus" relates to any molecule (e.g., peptide, antibody or fragment thereof, etc.) known in the art that can be used to specifically detect the presence of a specific protein (e.g., alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus) in a matrix (e.g., water).

[0041] More specifically, the present invention relates to the use of the above method, wherein the molecule is an antibody or a fragment thereof that specifically binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus.

[0042] The term "antibody or fragment thereof" relates to an antibody that is capable of specifically binding to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus or any part thereof, or an antigen-binding fragment thereof, in particular an antibody of the F(ab')2, F(ab) or single-chain Fv (scFv) type, or any other type of recombinant antibody known in the art.

[0043] The phrase "specifically (or selectively) detects (or binds to) "alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus", when referring to an antibody, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological preparations. Thus, under specified immunoassay conditions, a specific antibody binds to a particular protein at least two times above background and does not substantially bind to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, a polyclonal antibody raised against a marker "X" of a specific species may be selected to obtain only those antibodies that bind to marker "X" In some embodiments, the present invention relates to a polyclonal antibody that produces a specific immune response but does not produce a specific immune response with other proteins, except for polymorphic variants and alleles of marker "X". This selection can be achieved by subtracting antibodies that cross-react with marker "X" molecules from other species. Various immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are commonly used to select antibodies that specifically immunoreact with proteins (for descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, Antibodies, A Laboratory Manual (1988)). Typically, the specific or selective reaction will be at least twice the background signal or noise, more typically 10 to 100 times higher than the background.

[0044] Furthermore, the present invention relates to an assay (e.g., a lateral flow immunochromatographic assay as described by Delmulle et al. (2005)) comprising a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus and a molecule that binds to the PirA of Vibrio parahaemolyticus. VP and / or PirB VP Toxin-binding molecules.

[0045] The term "PirA of Vibrio parahaemolyticus VP and / or PirB VP “Toxins” refer to extracellular secreted toxins produced by Vibrio parahaemolyticus, which causes AHPND, and are homologous to the insecticidal PirA and PirB toxins (delta-endotoxins) of Photorhabdus and Xenorhabdus (Han et al., 2015). PirA VP PirB binds to certain ligands (e.g., monosaccharides such as N-acetylgalactosamine (GalNAC) and oligosaccharides) on the cell membrane / receptors. VP (containing the N-terminal domain PirB VP N- and C-terminal domains of PirB VPC), is primarily responsible for cell death through pore formation and is involved in protein-protein and protein-ligand interactions ( Kumar et al., 2019 ; Lin et al., 2017 ; Sirikharin et al., 2015 ).

[0046] The term "PirA VP and / or PirB VP The term "molecule that binds to toxin" has a similar meaning to the above-mentioned "molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus".

[0047] The present invention also relates to an assay as described above for classifying said Vibrio parahaemolyticus into an avirulent or virulent phenotype.

[0048] The present invention more particularly relates to an assay as described above, wherein the molecule is a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus and / or to the PirA of Vibrio parahaemolyticus. VP and / or PirB VP Toxin-binding antibodies or fragments thereof.

[0049] The term "PirA VP and / or PirB VP The term "antibody or fragment thereof that specifically binds to toxin" has a meaning similar to that of the antibody or fragment thereof that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus described above.

[0050] The present invention also relates to assays as described above, wherein the assay is an antibody-based assay, such as a lateral flow immunochromatographic assay or an enzyme-linked immunosorbent assay (ELISA). For example, a similar lateral flow test paper (dipstick) based on an immunoassay has been developed for rapid detection of aflatoxin B1 in pig feed (Delmulle et al., 2005).

[0051] Furthermore, the present invention relates to a method for determining said Vibrio parahaemolyticus as an avirulent phenotype, the method comprising:

[0052] - Provide samples, such as shrimp culture water samples,

[0053] - contacting the water sample with a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, and

[0054] - determining the presence or absence of said alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus in said sample, wherein said Vibrio parahaemolyticus is of an avirulent phenotype when said enzyme is present in said sample.

[0055] The present invention also relates to a method as described above, wherein the sample is further contacted with a PiRA that binds Vibrio parahaemolyticus VP and / or PirB VPtoxin molecules, and wherein the presence of PiRA of Vibrio parahaemolyticus in the sample is also determined VP and / or PirB VP toxins, wherein when the enzyme is present in the sample, the Vibrio parahaemolyticus is of an avirulent phenotype, and wherein when at least one of the toxins is present in the sample, the Vibrio parahaemolyticus is of a virulent phenotype.

[0056] Example

[0057] Materials and methods

[0058] Strains and growth conditions

[0059] A total of two strains were used in the study: Vibrio parahaemolyticus M0904 (AHPND strain) and Aeromonas hydrophila LVS3 (Defoirdt et al., 2006; Kumar et al., 2018). LVS3 (autoclaved) was used as feed for Artemia larvae, and M0904 was used to produce toxins and enzymes for challenge assays. The M0904 strain used in experiment M0904 was deposited in the Belgian Coordinated Collection of Microorganisms (BCCM / LMG strain collection) (Laboratorium voorMicrobiologie, Universiteit Gent (UGent), KLLedeganckstraat 35, B-9000 Gent, Belgium) by the Animal Production Department / Laboratory of the Aquaculture and Artemia Reference Center (Campus Coupure F, Coupure Links 653, 9000 Gent, Belgium), with a deposit number (accession number): LMG P-31518. A stock culture of M0904 was streaked onto Marine Bacteria Agar plates (Carl Roth, Belgium) (MA) to obtain pure colonies. A single colony was inoculated into Marine Bacteria Broth (MB) (Carl Roth, Belgium) and incubated at 28°C under constant stirring (120 min -1 ) were incubated overnight, and stock cultures were prepared in 40% glycerol and stored at -80°C. LVS3 cultures (LMG 22148) were collected from the Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Belgium. The strain was streaked onto Marine Bacteria agar plates (Carl Roth, Belgium), and single colonies were then inoculated into Marine Bacteria broth (Carl Roth, Belgium), and stock cultures were prepared in a similar manner.

[0060] Analysis of Vibrio parahaemolyticus cells cultured under different conditions

[0061] (a) Optical and fluorescence microscopy

[0062] Vibrio parahaemolyticus M0904 strain was observed by optical and fluorescence microscopy at 110 min. -1 or 120 minutes -1 Culture under constant stirring to observe changes at the cellular level. First, 20 μl was cultured for 110 min. -1 or 120 minutes -1 The cultured bacterial suspension was placed on a clean glass slide. A cover glass was then placed over the sample and excess liquid was removed by gently pressing the cover glass. The samples were observed at 10×, 40×, 36×, and 100× magnifications in an optical microscope (Zeiss Axioskop 2 plus, Carl Zeiss, Germany) to detect the flocs formed by the strains. Subsequently, the bacterial samples were stained with Calcofluor white dye (Sigma-Aldrich, USA) and the extracellular polysaccharides in the flocs produced by the Vibrio parahaemolyticus M0904 strain were detected by fluorescence microscopy (Zeiss Axioskop 2 plus, Carl Zeiss, Germany). In brief, 20 μl of bacterial suspension was placed on a clean glass slide. Then, 1 drop of Calcofluor white dye and 1 drop of 10% potassium hydroxide (according to the manufacturer's instructions) were added to the sample. The sample was covered with a cover glass and incubated at room temperature for 1 minute. The slides were then examined under a fluorescence microscope at 10×, 40×, 36× and 100× magnifications under excitation with bandpass (BP) 365 / 12, beam splitter FT 395 and emission longpass 397.

[0063] (b) Flow cytometry

[0064] A spectrophotometer (Genesys 20, Thermo Spectronic) was used to measure the -1 or 120 minutes -1 The cells of Vibrio parahaemolyticus M0904 strain cultured under constant stirring were diluted to 5×10 9cells / ml. Afterwards, 1 ml of the bacterial suspension was transferred to a 2 ml sterile eppendorf tube and centrifuged at 8000×g for 10 min at room temperature. The bacterial cells were collected and resuspended in 10 ml Tris-buffered saline (0.13 M NaCl and 10 mM Tris hydrochloride, pH 7.4). Subsequently, 250 μl of bacterial cells in Tris-buffered saline were transferred to a 2 ml sterile eppendorf tube and 1 μl of FITC (fluorescein isothiocyanate)-lectin was added (7 different types of lectins were used with different glycan specificities, Table S1). The bacterial cells and lectin mixture were then transferred to a 96-well plate, and in order to promote the binding of FITC-lectin to bacterial cells, the plate was incubated in the dark at room temperature for 30 min.

[0065] Subsequently, use the CytoFLEX flow cytometer system (Beckman Coulter's LifeSciences, France) analysis sample that is equipped with 37-, μm (aperture) filter, 75-, μm (aperture) orifice and 0.3 neutral density filter and photomultiplier tube are arranged at 500V.The calibration and standardization of flow cytometer are carried out according to manufacturer's instructions.Fluorescent microbeads are used as the standard of fluorescence and volume, and green fluorescence is measured at 525nm (FITC) and 488nm (FSC).Flow cytometer is set as follows: gain FSC (forward scatter)-106, gain FITC-113 and speed-4 (representing that event rate never exceeds 1000 events per second).Counting is recorded as logarithmic signal and is triggered on green fluorescence channel.Use CytExpert software (Beckman Coulter's Life sciences, France) to process data, use electronic gain to separate expected events. Data are presented as FITC / count or FSC / count histograms to optimize the distinction between FITC-lectin labeled bacterial cells and instrument noise or sample background (Frossard et al., 2016; Hammes and Egli, 2010).

[0066] (c) Marine Agar Congo Red (MACR) plate method

[0067] The extracellular mucus produced by V. parahaemolyticus M0904 strain was determined using the Marine Bacteria Agar MACR plate method containing Congo red, following the procedure described by Freeman et al. (Freeman et al., 1989) and Phuoc et al. (Phuoc et al., 2009) with slight modifications. Briefly, V. parahaemolyticus M0904 cultures were incubated in 20 ml of Marine Bacteria Broth (MB) at 28°C for 110 min. -1 or 120 minutes -1Continuous stirring incubation for 24h. Subsequently, the broth was discarded, 5ml FASW was added and the cells attached to the bottom of the conical tube were collected after mixing. The bacterial suspension in FASW was streaked onto a marine bacteria agar (MACR) plate (37.4gMB, 50g sucrose, 15g agar and 0.8g Congo red) containing Congo red. Congo red was prepared as a stock solution, autoclaved, and then added to the culture medium when cooled to 55°C. After inoculation, the plate was cultivated at 28°C for 24h. The bacterial strains producing extracellular mucus formed colonies of different colors on the MACR plate (Freeman et al., 1989). This assay was repeated three times and represents at least two independent experiments.

[0068] Vibrio parahaemolyticus extracellular protein (VP AHPND Purification of ECP

[0069] A total of two separate experiments were performed. Briefly, V. parahaemolyticus M0904 strain was cultured in 20 ml Marine Broth (MB) (Carl Roth, Belgium) in 50 ml Erlenmeyer flasks at 28°C for 110 min. -1 or 120 minutes -1 The culture was stirred overnight. The overnight grown bacterial suspension was centrifuged at 3500×g for 15 min. The supernatant was collected and filtered through a 0.2 μm sieve to obtain the cell-free supernatant (CFS) of V. parahaemolyticus. The CFS of V. parahaemolyticus was then concentrated and washed with phosphate buffered saline (PBS) and 10 kDa The concentrated extracellular protein (VP) was collected by dialysis using ultra-15 centrifugal filters (Merck Millipore, USA) (Kumar et al., 2019). AHPND ECP) and immediately stored at -80°C for further analysis.

[0070] NonoLC-MS / MS analysis from VP AHPND PirA for ECP VP and PirB VP toxins and alkaline phosphatase PhoX enzymes

[0071] To identify putative proteins produced by V. parahaemolyticus AHPND strains under different culture conditions, concentrated VP AHPNDECP samples were mixed with loading buffer, vortexed, heated at 95°C for 5 min, and then electrophoresed in 4-20% SDS-PAGE gels (BioRad, Belgium) with equal volumes (10 μl) of protein loaded per lane. The gels were stained with Coomassie Biosafe (BioRad Laboratories), and the signals were detected by the ChemiDoc MP imaging system (BioRad, Belgium). Protein concentrations were determined by the Bradford method (Bradford, 1976) using bovine serum albumin as a standard. PirA VP and PirB VP Possible candidate bands of toxin and alkaline phosphatase PhoX enzyme on Coomassie-stained SDS-PAGE gel were cut out using a clean scalpel (preferably in a clean bench to avoid keratin contamination) and the gel slices (with clean tweezers) were transferred to sterile eppendorf tubes. The gel slices were rinsed twice with 500 μl sterile distilled water and kept at -20°C for LC-MS / MS analysis.

[0072] The gel pieces were then washed successively with water, an aqueous solution of acetonitrile (ACN) (50 / 50, v / v) and ACN, and then completely dried by vacuum drying. The dried gel strips were rehydrated with 15 μl of a trypsin-containing solution (5 ng / μl in 50 mM ammonium bicarbonate), and 50 mM ammonium bicarbonate was added until the strips were completely submerged. Next, the strips were incubated overnight at 37 ° C to digest and elute the resulting peptides from the gel. Formic acid was added to the eluted peptides to inactivate trypsin, and the samples were vacuum dried. After in-gel digestion, the dried peptide samples were dissolved in 20 μl loading buffer (0.1% TFA in water / acetonitrile, 2 / 98 (v / v)), and half of each sample was analyzed on an Ultimate 3000 RSLC nanoLC (Thermo Scientific, Bremen, Germany) in a tandem configuration connected in series to an LTQ-Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) equipped with a pneu-Nimbus dual ion source (Phoenix S&T) and a Butterfly nano-LC column oven (Phoenix S&T), with the column temperature maintained at 50° C. The sample mixture was first loaded onto a capture column (manufactured in-house, 100 μm ID x 20 mm long, 5 μm beads C18 Reprosil-HD, Dr. Maisch). After washing from the capture column, the sample was loaded onto a reverse phase column (made in-house, 75 μm ID x 200 mm long, 1.9 μm beads C18 Reprosil-HD, Dr. Maisch). The peptides were loaded with loading buffer and separated using a non-linear 1.5 hour gradient from 98% solvent A (0.1% formic acid in water) to 56% solvent B (0.1% formic acid in water / ACN 20:80 (v / v)) at a flow rate of 250 nl / min, followed by a 5 min wash to 99% buffer B.

[0073] The mass spectrometer was operated in data-dependent positive ionization mode, automatically switching between MS and MS / MS acquisition to acquire the 20 most abundant peaks in a given MS spectrum. The source voltage was 3 kV and the capillary temperature was 275 °C. In the LTQ-OrbitrapElite, the Orbitrap (m / z 300-2,000, AGC target 3 × 10 6 ions, maximum ion injection time 100 ms) at a resolution of 60 000 (at 400 m / z). The 20 most intense ions that met the predefined selection criteria (AGC target 5 × 10 3Ions, maximum ion injection time 20 ms, spectral data type: centroid, exclusion of unassigned and 1 positively charged precursor, dynamic exclusion time 20 s) were isolated in a linear ion trap and fragmented in the high pressure cell of the ion trap. The CID collision energy was set to 35 V, and a polydimethylcyclosiloxane background ion of 445.120028 Da was used for internal calibration (lock mass).

[0074] Data analysis was performed using MaxQuant (version 1.6.2.6) using the Andromeda search engine with default search settings including a false discovery rate of 1% at both the peptide and protein levels. VP and PirB VP The spectra were searched against proteins of V. parahaemolyticus strain 20130626002S01 in the database of toxin sequences (GenBank, NCBI) containing 5004 protein sequences. During the main search, the mass tolerance for precursor and fragment ions was set to 4.5 and 20 × g, respectively. The enzyme specificity was set to the C-terminus of arginine and lysine, and cleavage at proline bonds was also allowed with a maximum of two missed cleavages. Variable modifications were set to oxidation of methionine residues, propionamidation of cysteine, and acetylation of the N-terminus of the protein. Only proteins with at least one unique or razor peptide were selected. A minimum ratio count of two unique or razor peptides was required for quantification. The resulting protein list was sorted in descending order by iBAQ ratio, revealing PirA VP 、PirB VP and PhoX were the most abundant proteins in their respective protein bands. VP and PirB VP Toxin peptides were mapped to the corresponding protein sequences.

[0075] PCR confirmation of the presence of AHPND plasmid in V. parahaemolyticus culture

[0076] The AP3 primers were used to confirm the -1 or 120 minutes -1 The cultured V. parahaemolyticus M0904 strain carries the AHPND plasmid (Table S2). The AP3-based PCR fragment is not present in non-AHPND V. parahaemolyticus (Sirikharin et al., 2014; Suong et al., 2017).

[0077] RNA extraction and reverse transcription

[0078] Vibrio parahaemolyticus M0904 strain was cultured in 20 ml sterile marine broth (MB) for 110 min. -1 or 120 minutes -1Culture overnight, repeated three times. After 12 and 24 h of incubation, cells were harvested and total RNA was extracted from bacterial samples using Qiagen RNeasy Plus Mini Kit (Cat. No. 74136) according to the manufacturer's instructions, repeated three times. RNA quality and quantity were measured on a NanoDrop spectrophotometer (ThermoFisher Scientific, Belgium), and RNA samples with A260 / A280 ratios>2.0 and A260 / A230 ratios>1.5 were used for analysis. RNA integrity was checked by agarose gel electrophoresis, and RNA samples were stored at -80°C for subsequent use.

[0079] Use RevertAid according to the manufacturer's instructions. TM Reverse transcription was performed using the H Minus First-Strand cDNA Synthesis Kit (ThermoFisher Scientific, Belgium). Briefly, 1 μg of total RNA and 1 μl of random hexamer primer solution were first mixed. Then, 8 μl of the reaction mixture containing 4 μl of 5× reaction buffer (0.25 mol -1 Tris-HCl pH 8.3, 0.25 mol -1 MgCl2, 0.05 mol -1 DTT), 2 μl of 0.01 mol -1 dNTP mix, 20 units of RNase inhibitor, 200 units of RevertAid TM H minus M-MuLV reverse transcriptase. The reaction mixture was incubated at 25°C for 5 min and then at 42°C for 60 min. The reaction was terminated by heating at 70°C for 5 min and then cooled to 4°C. Complementary deoxyribonucleic acid (cDNA) samples were checked by PCR and stored at -20°C for further use.

[0080] Quantitative real-time PCR (RT-qPCR) analysis

[0081] Two VPs were measured by RT-qPCR with pairs of specific primers using the StepOnePlus Real-Time PCR System (Applied Biosystems). AHPNDExpression of plasmid-associated toxic genes, including genes encoding toxins and toxic plasmids and an alkaline phosphatase PhoX gene associated with floc formation (Table S3). Ct values ​​from two reference genes, rpoA (RNA polymerase A subunit (submit)) and toxR mRNA, used as internal controls, were geometrically averaged and gene expression was calculated relative to rpoA and toxR mRNA levels. Amplification was performed in a total volume of 20 μl, containing 10 μl of 2X Maxima SYBR Green / ROX qPCR Master Mix (Thermo Fisher Scientific), 1 μl of cDNA (50 ng), 8 μl of nuclease-free water, and 0.5 μl of each specific primer. Master mixes for each biological replicate of the sample were prepared in triplicate and RT-qPCR was performed for the target and reference genes using a four-step amplification protocol: initial denaturation (10 min at 95°C); 40 cycles of amplification and quantification (15 s at 95°C, 30 s at 60°C, and 30 s at 72°C); melting curve (55-95°C) with a heating rate of 0.10°C / s, continuous fluorescence measurement, and cooling (4°C). Negative control reactions were included for each primer set by omitting the template cDNA. The expression levels of the target genes were analyzed using the comparative CT method (2-ΔΔCt method) according to Livak and Schmittgen (Livak and Schmittgen, 2001) and validated by the Pfaffl relative standard curve method (Pfaffl, 2002). Log-transformed 2^ΔΔCT values ​​were subjected to t-tests, and p values ​​less than 0.05 were considered statistically significant.

[0082] Sterile Brine Shrimp Hatching

[0083] Sterile brine shrimp larvae were produced by aseptically hatching high-quality cysts (without bacteria) according to the decapping and hatching procedures described by Baruah et al. (Baruah et al., 2014). Briefly, 200 mg of brine shrimp (A. franciscana) cysts ( 21452, INVE Aquaculture, Dendermonde, Belgium) was hydrated in 18 ml of distilled water for 1 h. Sterile cysts and larvae were obtained by dehulling using 660 μl of NaOH (32%) and 10 ml of NaOCl (50%). During the reaction, 0.2 μm filtered aeration was provided. All operations were performed under a clean bench and all tools were sterilized. Dehulling was terminated after 2 min by adding 10 ml of 10 g / l Na2S2O3. The reaction was continued with 35 g / l of instant lysate. The deshelled cysts were washed with filtered autoclaved seawater (FASW) of synthetic sea salt (Aquarium Systems, Sarrebourg, France). The cysts were resuspended in a 50 ml tube containing 30 ml FASW and stirred at 28 °C for 6 min. -1 ) for 24 h, with a constant light intensity of approximately 27 μE / m 2 s. After 28 h of incubation, larvae that hatched at developmental stage II (open mouth to ingest particles) were collected and verified for sterility by plating on marine bacteria agar and marine bacteria broth (100 ml) and adding (500 μl) hatching water, respectively, and then incubated at 28°C for 5 days (Baruah et al., 2012). Experiments that started with non-sterile larvae were discarded.

[0084] Brine shrimp challenge assay

[0085] A total of 3 separate experiments were conducted to determine the presence of V. parahaemolyticus M0904 strains in brine shrimp larvae (under different culture conditions), V. AHPND ECP and purified alkaline phosphatase PhoX enzyme and PirA VP and PirB VP Toxicity of the toxins. Toxicity assays were performed according to the method developed by Kumar et al. (Kumar et al., 2018). In the first experiment, hatched brine shrimp larvae (at developmental stage II) were collected and groups of 20 larvae were transferred to sterile 40 ml glass tubes containing 10 ml FASW. Subsequently, the pathogenic V. parahaemolyticus M0904 strain cultured in 20 ml sterile marine broth (MB) was used at 10 7 cells / ml at 110 min -1 or 120 minutes -1 The culture was cultured under constant agitation at 4°C and the brine shrimp larvae were fed with autoclaved LVS3. Afterwards, the glass tubes were placed back on the stirrer and kept at 28°C. The survival of the Artemia larvae was scored manually 48 h after the addition of the pathogen. Unchallenged larvae were kept as negative controls.

[0086] In the second experiment, groups of 10 brine shrimp larvae were collected and transferred to 2 ml sterile eppendorf tubes containing 1 ml FASW. The brine shrimp larvae were then treated with 1.3, 1.95, 2.6, and 3.9 μg / 100 μl of VP concentrated from a culture of V. parahaemolyticus M0904. AHPND The cultures were challenged with extracellular protein (ECP) and PirA VP and PirB VP Toxin in 120min -1 Incubate with constant agitation or with alkaline phosphatase PhoX enzyme for 110 min -1The larvae were incubated at 4 °C for 24 h. The survival of the larvae was scored 60 h after the addition of toxin. Unchallenged larvae were kept as negative controls.

[0087] In the third experiment, groups of 10 brine shrimp larvae were counted and distributed into sterile 2 ml eppendorf tubes and then treated with 1.3, 1.95, 2.6, and 3.9 μg / 100 μl of VP AHPND Purified PirA VP and PirB VP Toxin or VP AHPND Purified alkaline phosphatase PhoX enzymes were challenged as described above for lethality testing. Unchallenged larvae were kept as negative controls. Assays were performed in five replicates and are representative of at least two independent experiments.

[0088] Macrobrachium rosenbergii culture system

[0089] The experiments were conducted in a thermostatic chamber at the laboratory of the Aquaculture and Artemia Reference Center, Ghent University, Belgium. Acclimated adult freshwater shrimp (Macrobrachium rosenbergii) obtained from the laboratory and kept in four separate freshwater recirculation units were used as broodstock animals. For each experiment, larvae from a single spawning female breeding animal were used. Mature females with fully mature fertilized eggs (indicated by dark grey eggs) were transferred to a hatchery tank (30 l) containing brackish water (6 g / l salinity). 24 h after hatching, the larvae were collected and stocked in two separate brackish water (12 g / l salinity) recirculation units and fed with newly hatched sterile brine shrimp larvae. Broodstock management techniques were as described previously (Baruah et al., 2009; Nhan et al., 2010).

[0090] Macrobrachium challenge assay

[0091] Experiments were conducted to determine the toxicity of V. parahaemolyticus M0904 strain in Macrobrachium prawn larvae (8 days old) cultured under different culture conditions. Groups of 10 Macrobrachium prawn larvae (8 days old) were collected and transferred into 150 ml glass tubes containing 100 ml sterile brackish water (12 g / l salinity) (Rahman et al., 2004). Subsequently, the 10 g / l slurry was added to 20 ml sterile marine broth (MB) at 10 °C. 6 cells / ml at 110 min -1 or 120 minutes -1 Larvae were challenged with pathogenic V. parahaemolyticus M0904 strain cultured under RT (Kumar et al., 2018). Larval survival was scored at 12, 24, 36 and 48 h after pathogen addition. Macrobrachium larvae not challenged with V. parahaemolyticus served as negative controls. The assay was repeated five times and represents at least two independent experiments.

[0092] Statistical analysis

[0093] Survival data of brine shrimp larvae were arcsine transformed when necessary to meet normality and homoscedasticity requirements. The data were then subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test using the statistical software Statistical Package for Social Sciences version 24.0. P values ​​≤ 0.001 were considered significant. Gene expression results are expressed as multiple expression relative to the geometric mean of two internal control genes (toxR and rpoA). The expression level in the floc-forming group (control) was considered to be 1.0, so the expression ratio of the non-floc group (treatment) was expressed relative to the control group. The significant differences in expression levels between the control and treatment were statistically analyzed using the logarithmic transformed data by a one-tailed Student's t test. Logistic regression analysis of the survival data of Macrobrachium rosenbergii was performed using GenStat 16 (VSN international, Hemel Hempstead, UK) to determine the significant differences between the control and treatment.

[0094] result

[0095] Vibrio parahaemolyticus (strain M0904) grows as flocs under low shaking speed conditions (as an example of environmental conditions)

[0096] In the first experiment, the effect of oscillation speed on Vibrio parahaemolyticus M0904 strain was studied, considering that altered environmental conditions may modulate bacteria and trigger a set of adaptive regulatory mechanisms (Balaban et al., 2004; Rossignol et al., 2009; Woude et al., 2006).

[0097] The current results show that within 110 min -1 The Vibrio parahaemolyticus cells (hereinafter referred to as M0904 / 110) cultured under constant stirring flocculated, and at 120 min -1 Cells grown at 100 °C (hereinafter referred to as M0904 / 120) did not produce flocs. This indicates that the shaking speed of M0904 / 110 induces flocculation in the V. parahaemolyticus culture.

[0098] Flocculated cells no longer produce PirA VP and PirB VP toxins, but produces and secretes alkaline phosphatase PhoX

[0099] Since M0904 / 110 and M0904 / 120 cells show different phenotypic characteristics in terms of EPS production levels, we tested whether protein secretion was also affected (Jayaraman, 2011; Sousa et al., 2011; Thomen et al., 2017). To investigate potential changes in the secretome, M0904 / 110 and M0904 / 120 cells (VP AHPND The proteins present in the culture medium of ECP were separated by SDS-PAGE and stained by Coomassie. Notably, this analysis showed that M0904 / 120 cells secreted two major proteins of 13 and 50 kDa, while M0904 / 110 cells secreted a major protein of 73 kDa, as well as some other proteins that were differentially secreted by the two cell types ( Figure 1 i). Thus, V. parahaemolyticus strain M0904 does alter secretory protein production when the shaking speed conditions are slightly changed.

[0100] Next, the major secreted proteins were identified and the corresponding protein bands of 13, 50 and 73 kDa were cut from the gel and subjected to mass spectrometry analysis. This analysis identified the two major proteins produced by M0904 / 120 cells as PirA VP (13 kDa) and PirB VP (50 kDa), which are bacterial toxins encoded by the pVPA1 plasmid of the AHPND V. parahaemolyticus strain ( Han et al., 2015 ).

[0101]

[0102] Table 1: From PirA VP 、PirB VP and alkaline phosphatase PhoX proteins. The corresponding protein bands of 13, 50 and 73 kDa were cut from the gel and analyzed by mass spectrometry. This analysis identified the two major proteins produced by M0904 / 120 cells as PirA VP (13 kDa) and PirB VP (50 kDa). The major protein secreted by M0904 / 110 cells was identified as PhoX, an alkaline phosphatase with a molecular weight of 73 kDa.

[0103] The major protein secreted by M0904 / 110 cells was identified as PhoX, an alkaline phosphatase with a molecular weight of 73 kDa. These results suggest that M0904 / 110 cells cease to produce PirA / B toxins and instead produce another secreted protein with undefined alkaline phosphatase activity.

[0104] Floc formation attenuates toxin gene expression in Vibrio parahaemolyticus

[0105] To further demonstrate that M0904 / 110 reduces the expression of virulence genes (as shown above), transcription of the pVA1 plasmid-bound gene, PirB, which mediates virulence of V. parahaemolyticus in shrimp species, was also investigated. VP (PirB VP Toxin) and ORF-14 (plasmid-encoded gene) were tested (Han et al., 2015; Kumar et al., 2019; Sirikharin et al., 2015). Interestingly, the expression of toxic plasmid genes was significantly downregulated in M0904 / 110 compared with M0904 / 120 at 12 and 24 h ( Figure 2 A, B). This observation is consistent with the results for secreted proteins (described above) and emphasizes that M0904 / 110 cultures significantly reduced transcription of toxic genes.

[0106] Considering that V. parahaemolyticus produces flocs and secretes alkaline phosphatase PhoX enzyme under altered environmental conditions, the temporal expression of alkaline phosphatase PhoX gene in V. parahaemolyticus (M0904 / 110 or M0904 / 120) was investigated. Compared with M0904 / 120, the expression of alkaline phosphatase PhoX gene in M0904 / 110 was significantly increased at 12 and 24 h ( Figure 2 C). Taken together, these results suggest that V. parahaemolyticus changes its phenotype under low oscillation conditions, resulting in decreased transcription of virulence-related genes and increased expression of the flocculant-associated alkaline phosphatase PhoX gene.

[0107] Growing AHPND Vibrio parahaemolyticus under low oscillation conditions reduces the toxicity of AHPND strains to brine shrimp larvae

[0108] Because the environmental conditions in the culture medium of V. parahaemolyticus play an important role in the expression of toxicity-related genes (as shown above). Next, the in vivo toxicity of V. parahaemolyticus was studied using axenic brine shrimp larvae, which is a good model for studying host-pathogen interactions (Baruah et al., 2017, 2015). First, the toxicity of V. parahaemolyticus grown in M0904 / 110 or M0904 / 120 on the survival of brine shrimp larvae was tested. The results showed that larvae challenged with M0904 / 110 showed significantly increased survival compared to larvae challenged with M0904 / 120 ( Figure 3 A) However, it is noteworthy that the virulence of V. parahaemolyticus AHPND strains is mainly regulated by PirA VP and PirB VP The toxin is composed of extracellular proteins (ECPs) mediated by Figure 1Starting earlier in the culture, it was shown that the culture of M0904 / 110 resulted in the secretion of the alkaline phosphatase PhoX; whereas the culture of M0904 / 120 produced PirA VP and PirB VP We next investigated the expression of V. parahaemolyticus extracellular proteins (VPs) purified from M0904 cultures grown in either M0904 / 110 or M0904 / 120. AHPND Interestingly, the results showed that VP purified from M0904 / 120 cultures AHPND Compared with ECP, VP purified from M0904 / 110 AHPND The toxicity of ECP challenged fruit was significantly reduced at 1.3, 2.6, 3.9 and 5.2 μg concentrations ( Figure 3 B) Although VP purified from V. parahaemolyticus grown in M0904 / 110 or M0904 / 120 AHPND The toxicity of ECP was significantly different from that of pure PirA VP and PirB VP It is important to assess the toxicity of pure alkaline phosphatase PhoX compared to toxins. VP and PirB VP In addition to toxins, V. parahaemolyticus may also secrete some other proteins or toxins that may cause the toxicity of ECP in brine shrimp larvae (Sirikharin et al., 2015). To this end, purified alkaline phosphatase PhoX or PirA from M0904 / 110 or M0904 / 120 was added to VP and PirB VP Brine shrimp larvae were challenged with toxin at concentrations of 1.3, 2.6, 3.9, and 5.2 μg / 100 μl. AHPND ECP survival results are consistent with the results that PirA VP and PirB VP The survival rate of brine shrimp larvae exposed to different concentrations of pure alkaline phosphatase PhoX enzyme was significantly increased compared to the toxin ( Figure 3 C). Taken together these results, our in vivo experiments confirmed that oscillation speed does play an important role in mediating the toxicity of V. parahaemolyticus in brine shrimp larvae.

[0109] Floc formation reduces the toxicity of AHPND Vibrio parahaemolyticus M0904 strain to Macrobrachium rosenbergii larvae

[0110] Earlier studies have shown that oscillation speed regulates the toxicity of V. parahaemolyticus in brine shrimp larvae. This led to the suggestion that changing the oscillation speed may reduce the toxicity of V. parahaemolyticus in other shrimp species (e.g., freshwater shrimp, Macrobrachium rosenbergii). To test this hypothesis, additional in vivo experiments were conducted to investigate the toxicity of V. parahaemolyticus grown in M0904 / 110 or M0904 / 120 on the survival of 8-day-old Macrobrachium larvae. The results showed that the survival rate of Macrobrachium larvae exposed to M0904 / 110 was significantly higher at 12, 24, 36, and 48 h after challenge compared to larvae challenged with M0904 / 120 ( Figure 4 ). The results were consistent with the survival results of brine shrimp larvae, indicating that the toxicity of V. parahaemolyticus to shrimp decreased under lower oscillation conditions.

[0111] In summary, the above results indicate that the alkaline phosphatase PhoX and / or PirA secreted by Vibrio parahaemolyticus VP and PirB VP Toxins can be used to determine whether the bacteria are virulent.

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Claims

1. Use of alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus as a marker for determining that the Vibrio parahaemolyticus is of avirulent phenotype.

2. Use of a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus in the preparation of a reagent or composition for monitoring acute hepatopancreatic necrosis in shrimp.

3. Use of a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus for determining that the Vibrio parahaemolyticus is avirulent phenotype.

4. Use of a molecule according to claim 3, wherein the molecule is an antibody or a fragment thereof that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus.

5. Antibodies or fragments thereof that bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus and PiRA that bind to Vibrio parahaemolyticus VP and / or PirB VP Use of toxin-binding antibodies or fragments thereof in an assay to determine whether the Vibrio parahaemolyticus is avirulent or has a virulent phenotype.

6. Use according to claim 5, wherein the assay is a lateral flow immunochromatographic assay or an enzyme-linked immunosorbent assay.

7. A method for determining that Vibrio parahaemolyticus is avirulent phenotype, the method comprising: - Provide samples, - contacting the sample with a molecule that binds to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, and - determining the presence or absence of said alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus in said sample, wherein said Vibrio parahaemolyticus is of an avirulent phenotype when said enzyme is present in said sample; Wherein, the method is used for non-disease diagnosis purposes.

8. The method according to claim 7, wherein the sample is further treated with PiRA that binds Vibrio parahaemolyticus VP and / or PirB VP toxin molecules, and wherein the presence of PiRA of Vibrio parahaemolyticus in the sample is also determined VP and / or PirB VP toxins, wherein when the enzyme is present in the sample, the Vibrio parahaemolyticus is of an avirulent phenotype, and wherein when at least one of the toxins is present in the sample, the Vibrio parahaemolyticus is of a virulent phenotype.

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