Vibrio parahaemolyticus Associated with MassMortality of Postlarval Abalone, Haliotis diversicolor

supertexta (L.), in Sanya, China

LU CHENG

Education and Experiment Center, Sun Yat-Sen University, Guangzhou East Campus, Panyu,Guangzhou 510006 China

JIE HUANG AND CHENGYIN SHI

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Qingdao 266071 China

KIM D. THOMPSON

Institute of Aquaculture, University of Stirling, Stirling FK9 4LA UK

BERNARD MACKEY

School of Food Biosciences, University of Reading, Reading RG6 6AT UK

JUNPENG CAI1

College of Light Industry and Food Sciences, South China University of Technology,Guangzhou 510640 China

Abstract

Outbreaks of mass mortality in postlarval abalone, Haliotis diversicolor supertexta (L.), have swept

across south China since 2002 and in turn have resulted in many abalone farms closing. Twenty-five

representative bacterial isolates were isolated from a sample of five diseased postlarval abalone, taken

15 d postfertilization during an outbreak of postlarval disease in Sanya, Hainan Province, China in

October 2004. A dominant isolate, referred to as Strain 6, was found to be highly virulent to postlarvae

in an experimental challenge test, with a 50% lethal dose (LD50) value of 3.2 3 104 colony forming

units (CFU)/mL, while six of the other isolates were weakly virulent with LD50 values ranging from

1 3 106 to 1 3 107 CFU/mL, and the remaining 18 isolates were classified as avirulent with LD50

values greater than 1 3 108 CFU/mL. Using both an API 20E kit and 16S recombinant DNA sequence

analysis, Strain 6 was shown to be Vibrio parahaemolyticus. It was sensitive to 4 and intermediately

sensitive to 5 of the 16 antibiotics used when screening the antibiotic sensitivities of the bacterium.

Extracellular products (ECPs) prepared from the bacterium were lethal to postlarvae when used in

a toxicity test at a concentration of 3.77 mg protein/mL, and complete liquefaction of postlarvae

tissues occurred within 24 h postexposure. Results from this study implicate V. parahaemolyticus as the

pathogen involved in the disease outbreaks in postlarval abalone in Sanya and show that the ECPs may

be involved in the pathogenesis of the disease.

Introduction

Small abalone, Haliotis diversicolor super-texta Lischke, is a commercially importantaquaculture species, widely farmed on the southcoast of China, with total production estimatedto be worth 10 billion Yuan annually. In theyears between 1999 and 2001, however, farmers

experienced mass mortalities in adult abalonebecause of disease (Lee et al. 2001). Since late2002 onward, mass mortalities have also beenexperienced in postlarvae within 30 d postferti-lization, reducing total abalone production byhalf. A number of causative agents have been re-ported to be responsible for causing these mor-talities, including a spheroid virus reported byWang et al. (2004). Vibrios have also been re-ported to be a primary pathogen for the small1 Corresponding author.

JOURNAL OF THE

WORLD AQUACULTURE SOCIETY

Vol. 39, No. 6

December, 2008

� Copyright by the World Aquaculture Society 2008

746

abalone (Liu et al. 2000, 2001; Lee et al. 2001),and bacteria such as Vibrio parahaemolyticusand Vibrio alginolyticus have been isolatedfrom the hemolymph of moribund adults,which have then been demonstrated to be ableto cause the symptoms of vibriosis throughexperimental challenge (Liu et al. 2000, 2001;Lee et al. 2001). However, the causes for themass mortalities seen in the postlarvae of thesmall abalone still remain unclear, even thoughV. parahaemolyticus, V. alginolyticus, and She-wanella alga have all been isolated from post-larvae cultured in farms in GuangdongProvince and have all been shown to be ableto reproduce disease in postlarvae (Cai et al.2006a, 2006b, 2006c).

The aim of the present study was to investi-gate the causative agent involved in the diseaseoutbreaks affecting postlarvae of the small aba-lone farmed in Sanya, Hainan Province, Chinaand to investigate possible mechanisms for itspathogenicity. Ultimately, this study will helpestablish the significance of V. parahaemolyti-cus, V. alginolyticus, and/or S. alga in the epi-demics seen in Sanya.

Materials and Methods

Bacterial Isolation

Five whitened postlarvae, with an averageshell length of approximately 0.6 mm, weresampled 15 d postfertilization during a diseaseoutbreak affecting abalone postlarvae on a farmin Sanya, Hainan Province, China in October2004. The postlarvae were collected from thebiofilms in the pond, using a pipette fitted with-a sterile 1-mL tip. They were placed in 0.5mL sterile phosphate-buffered saline (PBS)(0.01 M, pH 7.2) and transferred to the microbi-ological laboratory on site, where they werehomogenized in a sterile glass grinder contain-ing another 0.5 mL PBS, after first rinsing themthoroughly with fresh PBS. A 10-fold serialdilution of the homogenate was prepared inPBS, and 100 mL aliquot from each dilutionwas plated out onto Zobell’s Marine Agar(Difco, Detroit, MI, USA) supplemented with2.5% (w/v) NaCl. The PBS from the last rinseprior to homogenization was also plated out on

the marine agar as a negative control (Li et al.2005). After 4–6 d incubation at 25 C, a numberof colonies were selected based on their mor-phology and their abundance on the agar, andthese were streaked out on fresh agar to obtainpure cultures of each colony selected. Bacteriafrom pure cultures were stored at �70 C inmarine broth supplemented with 10% (v/v)glycerol.

Bacterial Challenge Tests

Apparently healthy abalone postlarvae, withan average shell length of approximately0.9 mm, were collected from an abalone farmin Shenzhen, Guangdong Province 20 d postfer-tilization. There had been no incidence of dis-ease outbreaks at this site during the statedhatching season. The abalone postlarvae wereused in experimental challenges according tothe protocol specified by Cai et al. (2006a).Seawater, filter-sterilized through a 0.22-mmmembrane (Millipore, Billerica, MA, USA),was used as a negative control in the experiment.All 25 isolates, recovered from the diseased aba-lone homogenate prepared above, were subjectedto challenge experiments, which were performedover a 3-d period. The 50% lethal dose (LD50)values were calculated on Day 3 according toReed and Muench (1938): LD50 values of .108

colony forming units (CFU)/mL were consideredavirulent; values between 104 and 105 CFU/mLwere considered virulent; and values betweenthese were considered weakly virulent in accor-dance with Mittal et al. (1980).

At the end of the challenge experiment, purecultures of bacteria were only obtained forthe most virulent isolate, reisolated from homo-genized moribund postlarvae as describedabove.

Characterization of Bacterial Isolates

The 25 isolates recovered from the pool of thefive postlarval abalone, sampled above, werechecked to establish if they could grow on thio-sulfate citrate bile salt sucrose (TCBS) medium(Difco) supplemented with 2.5% (w/v) NaCl.Apart from this, no further analysis was carriedout to characterize these strains except for themost virulent isolate identified from the challenge

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 747

experiments (i.e., Strain 6). Morphological,physiological, and biochemical analysiswas car-ried out on a pure culture of Strain 6, accordingto Alsina and Blanch (1994). Gram staining,oxidase test, morphology, motility, and suscep-tibility to vibriostatic compound 0/129 (2,4-diamino-6,7-diisopropyl pteridine phosphate,150 mg/disk; Sigma, St. Louis, MO, USA) wereperformed according to Huq et al. (1992). Bio-chemical analysis of the isolate was performedusing a commercial analytical profile index(API) 20E kit (ATB system; BIOMERIEUXSA, Marcy-I’Etoile, France) following themanufacturer’s instructions. All tests were per-formed in triplicate at 32 C, incubating bacteriafor 24 h. The reactions obtained were comparedwith the reference strain of V. parahaemolyticusAmerican Type Culture Collection (ATCC)17802T (Rockville, MD, USA).

Sequence analysis of 16S recombinant DNA(rDNA), which is a widely accepted method ofidentifying bacteria to the species level (orabove), was employed here to speciate Strain 6.The bacterium was grown overnight in marinebroth at 28 C with shaking (200 g). It was thenharvested by centrifugation at 10,000 g for2 min, rinsed, and resuspended in 13 Tris-EDTAbuffer (10 mM Tris–HCl, 100 mM ethylenedia-minetetraacetic acid, pH 8.0). Extraction of geno-mic DNA and amplification of 16S rDNA bypolymerase chain reaction (PCR) were per-formed as described by Cai et al. (2006a). Confir-mation of amplification of the 16S rDNA wascarried out by electrophoresis (5 mL of PCRproducts was run on a 1% (w/v) agarose gel),after which the products were purified usinga PCR purification kit (Takara, Guangzhou,China), according to the manufacturer’s instruc-tions. Direct sequencing of the PCR productswas carried out as reported by Thompson et al.(1992). Nucleotide sequence data were thendeposited in GenBank Nucleotide Sequence DataLibraries. Computer program BLASTn was usedto identify the species of bacteria with the closestmatch to the sequence of Strain 6. A phylogenetictree was constructed using the neighbour-joining(NJ) method (Saitou and Nei 1987), and its reli-ability was tested by bootstrap analysis (Felsen-stein 1985) with 500 trees.

Necropsies of Moribund Abalone Postlarvae

Necropsies were carried on moribund postlar-vae challenged with Strain 6 using transmissionelectron microscopy (Chang et al. 2005). Appar-ently healthy postlarvae were included in theexamination as a negative control. Briefly, mor-ibund and healthy postlarvae were prefixed inglutaraldehyde, diluted to 2.5% (v/v) with0.1 M phosphate buffer (pH 7.2), at 4 C for1 h. After washing for 1 h at 4 C with the samebuffer, samples were postfixed in 1% osmiumtetroxide at 4 C for a further hour, then dehy-drated in ascending concentrations of ethanol(50–100%), embedded in Epon 812 resin, andpolymerized at 70 C for 24 h. Ultrathin sections(70 nm) were directly examined without fur-ther staining under a Tecnai 12 (PHILIPS-FEI,Eindhoven, Holland) electron microscope atan accelerating voltage of 80 kV.

Toxicity Tests of Crude ExtracellularProducts from Strain 6

A preliminary trial to investigate the potentialrole of the extracellular products (ECPs) in thedisease mechanisms of Strain 6 was undertakenusing a crude preparation of ECP, preparedaccording to Liu (1957). Briefly, Strain 6 wascultured on a sterile cellophane overlay placedon the marine agar plates. After incubating at30 C for 24 h, the bacteria and ECPs werewashed off the cellophane with 3 mL PBS(0.01 M, pH 7.2). The bacteria were removedfrom the suspension by centrifuging for 20 minat 4950 g. The supernatant containing the ECPswas then passed through a 0.22-mm filter andstored in 3 mL aliquots at �70 C. Total proteinwas assayed by the Bradford (1976) method,using bovine serum albumin (Sigma) as astandard. Hydrolytic activities (proteolytic,gelatinolytic, lipolytic, phospholipolytic, amy-lolytic, and hemolytic) of the ECP were evalu-ated on agarose plates (0.8% w/v agarose inPBS 0.1 M, pH 7.0) according to Amaro et al.(1992), supplementing the agarose with one ofthe following substrates: 1% (w/v) casein, 1%(w/v) gelatin, 1% (v/v) Tween-80, 1% (v/v)egg yolk, 0.4% (w/v) starch, and 5% (w/v) sheeperythrocytes (Sigma). A 5 mL volume of the

748 CHENG ET AL.

ECP preparation was added, in duplicate, to 2-to 3-mm-diameter wells cut in the agarose.After incubating at 28 C for 24 h, the diameterof the hydrolysis halo around the inoculatedwell was measured, and the ratio of the diame-ter of the halo to the diameter of the inoculatedwell served as a relative quantification of thelytic activities, according to the criteria de-scribed by Biosca and Amaro (1996) (i.e., ratioof $3.1, very strong; 2.1–3.0, strong; 1.1–2.0,weak).

Apparently healthy abalone postlarvae, withan average shell length of approximately1.1 mm, were collected 27 d postfertilizationfrom an abalone hatchery with no experienceof mass mortalities in the stated hatching sea-son. The postlarvae were treated with a mixtureof antibiotics prior to the trial as described byCai et al. (2006a) to render them free of bacterialpathogens. The toxicity of the ECP in the post-larvae was tested in triplicate by bathing 10 ofthe antibiotic-treated postlarvae in 3 mL of un-diluted ECP solution (3.77 mg protein/mL), fil-ter-sterilized through a 0.22-mm filter, in sterileplastic petri dishes. These were left uncoveredand static in an aseptic environment at between25 and 28 C for 3 d. Sterile PBS 0.01 M (pH7.2) was used as the negative control.

Determining Antibiotic Sensitivities of Strain 6

The antibiotic sensitivities of Strain 6 weredetermined using the disc diffusion method rec-ommended by the NCCLS (2000) of America.The discs used in the assay included three cellwall synthesis inhibitors (ampicillin, 10 mg;cefamezin, 30 mg; penicillin G, 10 IU), one cellmembrane permeability interferer (polymyxinB, 300 mg), eight protein synthesis inhibitors(amikacin, 30 mg; chloramphenicol, 30 mg; ery-thromycin, 15 mg; gentamicin, 10 mg; kanamy-cin, 30 mg; neomycin, 30 mg; streptomycin,10 mg; tetracycline, 30 mg), and four nucleicacid synthesis inhibitors (ciprofloxacin, 5 mg;norfloxacin, 10 mg; novobiocin, 5 mg; trimetho-prim–sulfamethoxazole, 25 mg). Escherichiacoli ATCC 25922 was also included in the anal-ysis as a control bacterium. Antibiotic sensitiv-ity to a particular antibiotic (i.e., sensitive,intermediately sensitive or resistant) was as-

sessed according to the recommended cutofflevels of zone size (NCCLS 2000).

Results

Bacterial Isolation

Twenty-five representative colonies wereselected from bacteria that were recovered fromthe diseased abalone postlarvae sampled duringthe disease outbreak in Sanya in October 2004.The selection of these was based on differencein morphology and their abundance on themarine agar plates after culture. One particularisolate, designated as Strain 6, was found to bea dominant colony on the marine agar plates.

Bacterial Challenge Tests

Live bacterial challenges were carried out todetermine the virulence of all 25 strains isolatedrecovered from the diseased postlarvae, and thevirulence of these was found to vary consider-ably. Strain 6 was the most virulent isolate, kill-ing 80% of the postlarvae by Day 3, and hada LD50 value of 3.2 3 104 CFU/mL. Six otherisolates (Nos. 3, 8, 12, 14, 21, and 24) wereweakly virulent, with LD50 values between1 3 106 and 1 3 107 CFU/mL, and the remain-ing 18 strains were avirulent, with LD50 valuesgreater than 1 3 108 CFU/mL (Table 1). Grosssymptoms, similar to those observed in mori-bund postlarvae during natural outbreaks, werealso observed in the postlarvae, while no mor-talities were observed in the control group.Cultures obtained from moribund postlarvaechallenged with Strain 6, consisted of homoge-nous colonies, were identified as the same bacte-rial species used to challenge the postlarvae.Reisolation and identification of the other 24isolates used in the challenge experiments werenot carried out, as they were not considered pri-mary pathogens.

Characterization of Bacterial Isolates

Of the 25 isolates sampled from the diseasedpostlarvae from Sanya, 15 isolates were positiveand 10 negative for growth on TCBS agar. Tax-onomic characterization was only carried out onthe most virulent strain, as determined from thechallenge experiments.

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 749

Strain 6 was a Gram-negative, straight rod,motile, swarming on 2216E marine agar andnot luminous on Trypton-soya agar (+1% w/vNaCl). It produced green colonies on TCBSagar and was sensitive to the vibriostatic agentO/129 (150 mg). Other biochemical characteris-tics, determined from the API 20E kit, areshown in Table 2. The characteristics of this iso-late matched the type strain of V. parahaemoly-ticus (ATCC 17802T) (Baumann et al. 1984).

PCR amplification yielded an amplicon ofapproximately 1.8 kb in size, which containedboth 16S rDNA and ITS (16S–23S intergenicspacer) regions. The nucleotide sequence of16S rDNA region (1516 bp) of Strain 6 wasdetermined and submitted to GenBank underthe accession No. DQ354575. From theBLASTn program, the closest relationship ofthe product was to V. parahaemolyticus [16S:99% and 98% similarities with strain S-2(DQ164802)]. Sequencing analysis confirmedStrain 6 to be V. parahaemolyticus, based onthe fact that 16S rDNA sequence similarity of$97% can be considered as the same species(Thompson et al. 2004). An NJ phylogenetictreeing analysis also revealed that Strain 6 was

closely clustered with the reference strain of V.parahaemolyticuswith 100%confidence (Fig. 1).

Necropsies of Moribund Abalone Postlarvae

There were no signs of lesions or degenera-tion in the tissues of control postlarvae used inthe challenge experiments, with their tissues ap-pearing ordered and cells within clearly defined(Fig. 2). The epithelium was also intact, and nolesions were evident. The moribund postlarvae,on the other hand, had degeneration and lesionsin their tissues (Fig. 3). Ordered cell and tissuestructure had disappeared and was replacedby vacuoles and small, highly disorganized,globe-shaped material. Postlarval epitheliumhad broken down, and the cellular structurebeneath the epithelium had been replaced with

TABLE 1. LD50 (50% lethal dose) values on Day 3 post-

infection, calculated based on the challenge tests carried

out on the 25 isolates recovered from diseased abalone

postlarvae from Sanya.

StrainLD50 valuea

(CFU/mL) StrainLD50 valuea

(CFU/mL)

1 1.25 3 109 14 1.50 3 107

2 2.37 3 109 15 8.33 3 109

3 2.11 3 106 16 4.34 3 108

4 1.53 3 109 17 1.96 3 108

5 4.63 3 108 18 2.30 3 108

6 3.16 3 104 19 1.75 3 108

7 5.20 3 108 20 4.35 3 108

8 2.61 3 107 21 2.78 3 107

9 1.55 3 108 22 5.48 3 108

10 1.38 3 108 23 1.23 3 108

11 6.20 3 108 24 1.12 3 106

12 7.43 3 107 25 3.85 3 108

13 1.65 3 108

CFU 5 colony forming units.a The challenge tests, performed using batches of 20

abalone postlarvae per bacterial concentration, were con-

ducted by bathing the postlarvae in 1 L of test bacteria,

resuspended in sea water at 20 C, and observing for them

for 3 d.

TABLE 2. Comparison of biochemical characteristics of

Strains 6 with the Vibrio parahaemolyticus reference

strain ATCC 17802T using the API 20E kit.

Character Strain 6 ATCC 17802T

Presence of:

Acetoin + +Catalase + +Cytochrome oxidase + +Gelatinase + +Indole + +Lysine decarboxylase + +Ornithine decarboxylase + +Oxidase + +Arginine dihydrolase � �b-Galactosidase � �H2S � �Tryptophane deaminase � �Urease (NO2) � +

Utilization of:

Amygdalin + +Citrate + +Glucose + +Mannitol + +Arabinose � �Inositol � �Melibiose � �Rhamnose � �Sorbitol � �Sucrose � �a Other characteristics in common between Strain 6 and

the reference strain include Gram negative; straight rod;

motile; fermentative; swarming on marine agar plates, not

luminous on TSA (+1% NaCl); producing green colonies

on thiosulfate citrate bile salt sucrose agar plates; sensitive

to O/129 (150 mg); and halophilic (0.5–9.5%).

750 CHENG ET AL.

an empty space (Fig. 3A). Rod-shaped bacteriawere visibly both outside and inside the tissues(Fig. 3).

Preliminary Toxicity Tests of CrudeECPs of Strain 6

The protein concentration of the crude ECP ofStrain 6 was 3.77 mg/mL. Hydrolytic activitiesof the preparation were evaluated on agar platessupplemented with substrates according toAmaro et al. (1992). After 24-h incubation, thesize of hydrolysis halos for protease, gelatinase,lipase, phospholipase, amylase, and hemolysisactivities was calculated as 4.0, 3.5, 1.2, 1.6,3.3, and 2.0, respectively, indicating that the

ECP of Strain 6 contained proteolytic (caseinaseand gelatinase) and amylase activities and rela-tively weak lipase, phospholipase, and hemo-lytic activities.

Preliminary studies were carried out to inves-tigate the involvement of ECPs in postlarval dis-ease using a relatively high dose of undilutedECPs from Strain 6 in a toxicity test. Within6 h of administering the ECPs, most of the tis-sue in the postlarvae had liquefied, and in somecases, even the whole body had liquefied, leav-ing only empty shells (not shown), demonstrat-ing that the ECPs of Strain 6 are highlyhydrolytic and lethal for the abalone postlarvae.No mortality was observed in the control group.

L. anguillarum ; X16895

V. diazotrophicus ; X56577

V. navarrensis ; X74715

V. aestuarianus ; X74689

V. scophthalmi ; U46579

V. ichthyoenteri ; AJ437192

V. parahaemolyticus ; X74720

strain 6

V. parahaemolyticus ; DQ164802

V. fortis ; AJ514915

V. chagasii ; AJ316199

V. kanaloae ; AJ316193

V. splendidus ; X74725

V. superstes ; AY546651

V. halioticoli ; AB000390

V. ezurae ; AY426981

V. agarivorans ; AJ310647

V. gallicus ; AY257972

V. fischeri ; X74702

V. wodanis ; AJ132227

V. logei ; AY292934

V. salmonicida ; AY292918

V. logei ; AJ437616

V. rumoiensis ; AB013297

V. albensis ; AB166875

V. litoralis ; DQ097523

V. mimicus ; X74713

V. cholerae ; X74695

V. calviensis ; AF118021

100

100100

9587

96

99

100

7693

75

7377

65100

50

51

35

3024

66

13

23

21

5

2

0.005

FIGURE 1. Unrooted dendrogram depicting the interrelationship among Strain 6, Vibrio (V.) parahaemolyticus, Listonella

(L.) anguillarum (5Vibrio anguillarum), and other Vibrio. The tree is based on 16S rDNA sequence data and was

constructed using neighbour-joining method. Bootstrap confidence was calculated from 500 trees.

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 751

Sensitivity to 16 Antibiotics

Strain 6 was susceptible to 4 and moderatelysusceptible to 5 of the 16 antibiotics tested asshown in Table 3, indicating that Strain 6 ex-hibited 56.2% susceptibility to the antibioticstested.

Discussion

Much of the work published on abalone dis-eases so far has been related to adult or juvenileabalone (Elston and Lockwood 1983; Dixonet al. 1991; Haaker et al. 1992; Nishimoriet al. 1998; Reuter and McOrist 1999; Friedman

et al. 2000; Liu et al. 2000, 2001; Handlingeret al. 2001; Nicolas et al. 2002; Cheng et al.2004a, 2004b; Wang et al. 2004; Chang et al.2005), although there are few reports publishedon diseases affecting larval and/or postlarvalabalone (Anguiano-Beltran et al. 1998; Deveney2002; Cai et al. 2006a, 2006b, 2006c). Usingan experimental challenge model, Anguiano-Beltran et al. (1998) showed that V. alginolyti-cus was able to cause mortalities in larvae andpostlarvae of red abalone, Haliotis rufescens.Deveney (2002) reported the occurrence ofmass mortalities in greenlip abalone, Haliotis

FIGURE 2. Micrographs (A) and (B) showing the ordered tissue structure of an unidentified area from an apparently

healthy postlarva from the control group of the bacterial challenge test. Bar represents 2 mm. MV 5 microvilla;

MT 5 muscle tissue; Ep 5 epithelium; SC 5 secretory cell.

752 CHENG ET AL.

laevigata, postlarvae from South Australia, pre-ceded by shell detachment, with Vibrios gener-ally associated with this infection. However, itis unclear if the symptoms of themassmortalitiesin Deveney’s report are similar to those reportedin the present work because of the limited infor-mation available relating to the former.

In the present study, postlarvae becamelethargic, their body color-whitened, and bodymuscles shrunk prior to falling off the biofilmson which they grew, or their shells would

become empty of tissue. This particular diseasehas affected postlarvae on the south coast ofChina since 2002 and has already caused largenumbers of abalone farms to close. Althoughthe causes of the outbreak are still open to ques-tion, there are strong indications that bacteriaare involved, and even if not a major factor inthe disease, the strategic application of antibio-tics may lessen the severity or even preventdisease outbreaks. More recently, Cai et al.(2006a, 2006b, 2006c) have demonstrated that

FIGURE 3. Micrograph showing bacteria in the tissue of a postlarva exposed to live bacteria in the challenge tests. Note

the severe necrosis of postlarval tissues. (A) Bar represents 5 mm. (B) Bar represents 1 mm. B 5 bacteria;

Ep 5 epithelium/surface line of a postlarva.

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 753

V. parahaemolyticus,V. alginolyticus, and S. algaare associated with postlarval mass mortalitiesin Shanwei, Guangdong Province.

According to Nicolas et al. (2002), the pres-ence of a dominant bacterial type in recentlydead abalone implicates the bacterium to thedisease. In this way, they identified Vibriocarchariae (synonym, Vibrio harveyi) as a path-ogen involved in mass mortalities of both wildand farmed grown abalone, Haliotis tubercu-lata, which have been occurring along theFrench coast since 1997. Using the same princi-ple, we isolated a dominant isolate from the dis-eased small abalone postlarvae from Sanya onmarine agar, which we designated Strain 6,together with 24 less dominant isolates. Fromthe live bacterial challenge experiments, a doseof 1 3 106 CFU/mL of Strain 6 was able tocause 80% mortalities within Day 3 postinfec-tion and had an LD50 value as low as 3.2 3 104

CFU/mL, while the other 24 strains caused lessthan 20% mortalities in the postlarvae. ThisLD50 value is similar to values obtained byAnguiano-Beltran et al. (1998), who reportLD50 values for V. alginolyticus of 104.5 CFU/mL in larvae and 103.9 CFU/mL in postlarvaeof H. rufescens. Cai et al. (2006b, 2006c) also

reported LD50 value of 104 CFU/mL for V. algi-nolyticus and S. alga, although they obtaineda slightly lower value for V. parahaemolyticusof 103 CFU/mL (Cai et al. 2006a). In accor-dance with the degree of virulence stated byMittal et al. (1980), Strain 6 is clearly shownto be highly virulent.

Characterization of a bacterial isolate nor-mally involves two components, traditionalphysiological characterization and modernmolecular phylogenetic analysis, particularly16S rDNA sequencing. Typing using API 20Eanalysis, as a traditional method, is useful forthe identification of Vibrios from aquatic origins(Austin and Austin 1989) and was used here tocharacterize Strain 6 as V. parahaemolyticuswhen compared to the V. parahaemolyticus ref-erence strain (ATCC 17802T). To confirm thatStrain 6 was V. parahaemolyticus, sequenceanalysis of nearly complete 16S rDNAwas alsoperformed. Both sets of analysis showed thatStrain 6 (DQ354575) displayed a close relation-ship to V. parahaemolyticus, with 21 out of 22reactions the same in API 20E analysis, and98–99% 16S rDNA sequence similarity whencompared with V. parahaemolyticus referencestrain (X74720) and Strain S-2 (DQ164802).The close relationship was also confirmed byphylogenetic clustering of Strain 6 with thereference strain, with a bootstrapping value of100% (Fig. 1). As Thompson et al. (2004)pointed out, similarities of $97% for the 16Ssequence can be considered as the same species,so Strain 6 was therefore identified as V. para-haemolyticus by sequence analysis.

Mortality caused by Vibrios is very commonin reared fish and shellfish, especially duringthe early larval stages, and these can occursuddenly, sometimes leading to the death ofthe entire population (Iwamoto et al. 1995;Ishimaru et al. 1995, 1996; Borrego et al. 1996;Lee et al. 1996; Nicolas et al. 1996; Lambertet al. 1998; Novoa et al. 1998; Hansen andOlafsen 1999; Diggles et al. 2000; Olafsen2001; Thompson et al. 2004, 2005). We demon-strated that V. parahaemolyticus Strain 6 is ableto cause mortality in the postlarvae of abaloneusing a live bacterial challenge model. Largeamounts of bacteria, relatively uniform in shape,

TABLE 3. Sensitivity of Strain 6 to 16 different antibiotics.

AntibioticsDisc

content (mg) Sensitivitya

Penicillin G 10 IU R

Cefamezin 30 I

Kanamycin 30 I

Gentamicin 10 S

Chloramphenicol 30 S

Polymyxin B 30 S

Norfloxacin 10 I

Ampicillin 10 R

Streptomycin 10 S

Amikacin 30 R

Trimethoprim–

sulfamethoxazole

25 I

Erythromycin 15 I

Tetracycline 30 R

Ciprofloxacin 5 R

Novobiocin 5 R

Neomycin 30 R

a R 5 resistance; S 5 sensitive; I 5 intermediate. Dif-

ferent sensitivities of Strain 6 to an antibiotic were assessed

according to the NCCLS (2000) guidelines.

754 CHENG ET AL.

were found in the degenerated tissues of postlar-vae exposed to live bacteria of this isolate, andthe tissues appeared severely degenerated tothe point where no proper tissue morphologycould be recognizable (Fig. 3).

The severe degeneration in the tissues impliedthe involvement of ECPs in the pathogenesisof the bacterium (Ellis et al. 1981; Sakai 1985;Biosca and Amaro 1996). Bacterial ECPs havebeen implicated as virulent factors in fish andshellfish bacterial diseases (Biosca and Amaro1996; Lee et al. 1997; Perez et al. 1998), andECPs like exoenzymes (protease, gelatinase,amylase, lipase, and phospholipase) and exotox-ins (hemolysin, cytotoxin, etc.) are lesional fac-tors that are responsible for extensive tissuedamage (Biosca and Amaro 1996). Strain 6was found to produce ECPs with strong protease(caseinase and gelatinase) and amylase activi-ties and relatively weak lipase, phospholipase,and hemolysis activities. The results of the pre-liminary toxicity tests with the ECPs preparedfrom Strain 6 showed that a protein concentra-tion of 3.77 mg/mL could liquefy most partsof the tissues in some postlarvae and the wholebody of others, leaving empty shells in a rela-tively short period of time. To reveal whichcomponents of the ECPs of Strain 6 were themost potent virulence factors, we plan to purifythe different components in the ECP preparationand test their toxicities alone or in combination.

The hydrolytic properties of ECP can contrib-ute to the colonization and invasion of the host,leading to breakdown and degeneration of thetissue when the toxic effects of ECPs cannotbe neutralized by the host immune system. Forexample, Ellis et al. (1981) suggested that rain-bow trout serum contains a serum factor, prob-ably an a-globulin, capable of neutralizing thetoxic effects of ECP. This may be supportedby the fact that inbreeding is widespread in thesmall abalones, grown for nearly a decade insouthern China, and this in turn could weakenthe immune system of the abalone, making themmore susceptible to disease.

It is important to take effective action toreduce the risk of disease outbreaks in the hatch-ery system. Fortunately, Strain 6 appears sus-ceptible to 4 and moderately susceptible to 5

of the 16 screened in the study (Table 2). How-ever, it must be pointed out that some of the anti-biotics tested, including chloramphenicol, areprohibited for use in aquaculture, so for the longterm, environmental friendly strategies arerequired to the prevent disease outbreaks on theabalone farms. A possible alternative to the useof antibiotics may be the application of probiotics,especially since Macey and Coyne (2005) demon-strated improved growth and increased diseaseresistance in Haliotis midae, after treating the cul-ture system with probiotics.

It is interesting that we were able to isolateV. parahaemolyticus from two abalone farms [inShanwei (Cai et al. 2006a) and in Sanya (thisstudy)], 1000 km apart on the coast of SouthChina. These two strains showed identical bio-chemical characteristics (22 out of 22 reactionsmatched in API 20E analysis), antibiotic suscepti-bilities (showing differences in 3 out of the 16 anti-biotics tested, i.e., norfloxacin, ampicillin, andtrimethoprim–sulfamethoxazole), and 16S rDNAsequencewith 99.3% identity, suggesting that theyshare a close or an identical genealogical origin.Another reason to support this is that there is anactive exchange of broodstock and juvenilesamong abalone farms in south China, providingan ideal opportunity for V. parahaemolyticus tobe unintentionally transmitted between farms, thusspreading the pathogen. We plan to use moleculartechniques such as karyotyping and RandomAmplification Polymorphic DNA-PCR (RAPD-PCR) in follow-up studies to examine the genea-logical relationship between isolates from theregion.

Acknowledgment

This work was supported by the Natural Sci-ences Fund of China under the grant NSFC-40776091.

Literature Cited

Alsina, M. and A. Blanch. 1994. Improvement and update

of a set of keys for biochemical identification of

Vibrio species. Journal of Applied Bacteriology 77:

719–721.

Amaro, C., E. G. Biosca, C. Esteve, B. Fouz, and A. E.

Toranzo. 1992. Comparative study of phenotypic and

virulence properties in Vibro vulnificus biotypes 1 and

2 obtained from a European eel farm experiencing

mortalities. Diseases of Aquatic Organisms 13:29–35.

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 755

Anguiano-Beltran C., R. Searcy-Bernal, and M. L.

Lizarraga-Partida. 1998. Pathogenic effects of Vibrio

alginolyticus on larvae and post-larvae of the red

abalone, Haliotis rufescens. Diseases of Aquatic Or-

ganisms 33:119–122.

Austin, B. and D. A. Austin. 1989. Methods for the

microbiological examination of fish and shellfish.

Eillis Horwood Ltd., Chichester, UK.

Baumann, P., A. L. Furniss, and J. V. Lee. 1984. Genus I,

Vibrio Pacini 1854. Pages 518–538 in N.R. Krieg

and J.G. Holt, editors. Bergey’s Manual of System-

atic Bacteriology, volume 1. Williams & Wilkins,

Baltimore, Maryland, USA.

Biosca, E. G. and C. Amaro. 1996. Toxic and enzymatic

activities of Vibrio vulnificus biotype 2 with respect to

host specificity. Applied Environmental Microbiology

62:2331–2337.

Borrego, J. J., D. Castro, A. Luque, C. Paillard, P. Maes,

M. T. Garcia, and A. Ventosa. 1996. Vibrio tapetis sp.

nov., the causative agent of the brown ring disease

affecting cultured clams. International Journal of

Systematics and Bacteriology 46:480–484.

Bradford, M. M. 1976. A rapid and sensitive method for

the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding. Analyt-

ical Biochemistry 72:248–254.

Cai, J., Y. Han, and Z. Wang. 2006a. Isolation of Vibrio

parahaemolyticus from abalone (Haliotis diversicolor

supertexta L.) post-larvae associated with mass mor-

talities. Aquaculture 257:161–166.

Cai, J., H. Chen, K. D. Thompson, and C. Li. 2006b.

Isolation and identification of Shewanella alga and its

pathogenic effects on post-larvae of abalone Haliotis

diversicolor supertexta. Journal of Fish Diseases

29:505–508.

Cai, J., H. Han, Z. Song, C. Li, and J. Zhou. 2006c.

Isolation and characterization of pathogenic Vibrio

alginolyticus from diseased postlarval abalone, Hal-

iotis diversicolor supertexta (Lischke). Aquaculture

Research 37:1222–1226.

Chang, P. H., S.T. Kuo, S. H. Lai, H. S. Yang, Y. Y. Ting,

C. L. Hsu, and H. C. Chen. 2005. Herpes-like virus

infection causing mortality of cultured abalone Hal-

iotis diversicolor supertexta in Taiwan. Diseases of

Aquatic Organisms 65:23–27.

Cheng, W., I. S. Hsiao, C. H. Hsu, and J. C. Chen. 2004a.

Change in water temperature on the immune response

of Taiwan abalone Haliotis diversicolor supertexta and

its susceptibility to Vibrio parahaemolyticus. Fish and

Shellfish Immunology 17:235–243.

Cheng, W., C. H. Li, and J. C. Chen. 2004b. Effect of

dissolved oxygen on the immune response of Haliotis

diversicolor supertexta and its susceptibility to Vibrio

parahaemolyticus. Aquaculture 232:103–115.

Deveney, M. 2002. Summer mortality in abalone. South

Australian Animal Health Quarterly 5:2.

Diggles, B. K., J. Carson, P. M. Hine, R. W. Hickman,

and M. J. Tait. 2000. Vibrio species associated with

mortalities in hatchery-reared turbot (Colistium nudi-

pinnis) and brill (C. guntheri) in New Zealand.

Aquaculture 183:1–12.

Dixon, M. G., T. Hecht, and C. R. Brandt. 1991.

Identification and treatment of a Clostridium and

Vibrio infection in South African abalone, Haliotis

midae L. Journal of Fish Diseases 14:693–695.

Ellis, A. E., T. S. Hastings, and A. L. S. Munro. 1981. The

role of Aeromonas salmonicida extracellular products

in the pathology of furunculosis. Journal of Fish

Diseases 4:41–51.

Elston, R. and G. S. Lockwood. 1983. Pathogenesis of

vibriosis in cultured juvenile red abalone, Haliotis

rufescens Swainson. Journal of Fish Diseases 6:

111–128.

Felsenstein, J. 1985. Confidence limits on phylogenies: an

approach using the bootstrap. Evolution 39:783–791.

Friedman, C. S., K. B. Andree, K. A. Beauchamp, J. D.

Moore, T. T. Robbins, J. D. Shields, and R. P.

Hedrick. 2000. ‘‘Candidatus Xenohaliotis californien-

sis’’ a newly described pathogen of abalone, Haliotis

spp., along the west coast of North America. Interna-

tional Journal of Systematic Evolution and Microbiol-

ogy 50:847–855.

Haaker, P. L., D. O. Parker, H. Togstad, D. V. Richards,

G. E. Davis, and C. S. Friedman. 1992. Mass

mortality and withering syndrome in black abalone

Haliotis cracherodii, in California. Pages 214–224

in S. A. Shepard, M. J. Tegner, and S. A. Guzman,

editors. Abalone of the World. Blackwell Scientific,

Oxford, UK.

Handlinger, J., D. Taylor, and J. Carson. 2001.

Flavobacterium-like infection of abalone. Page 209

in European Association of Fish Pathologists. Tenth

International Conference ‘‘Diseases of fish and shell-

fish’’. Trinity College, Dublin, Ireland.

Hansen, G. H. and J. A. Olafsen. 1999. Bacterial

interactions in early life stages of marine cold water

fish. Microbial Ecology 38:1–26.

Huq, A., M. Alam, S. Parveen, and R. R. Colwell. 1992.

Occurrence of resistance to vibriostatic compound 0/

129 in Vibrio cholerae 01 isolated from clinical and

environmental samples in Bangladesh. Journal of

Clinical Microbiology 30:219–221.

Ishimaru, K., M. Akagawa-Matsushita, and K. Muroga.

1995. Vibrio penaeicida sp. nov., a pathogen of the

kuruma prawns (Penaeus japonicus). International

Journal of Systematics and Bacteriology 45:134–138.

Ishimaru, K., M. Akagawa-Matsushita, and K. Muroga.

1996. Vibrio ichthyoenteri sp. nov., a pathogen of

Japanese flounder (Paralichthys olivaceus). Interna-

tional Journal of Systematics and Bacteriology

46:155–159.

Iwamoto, Y., Y. Suzukki, A. Kurita, Y. Watanabe,

T. Shimizu, H. Ohgami, and Y. Yanagihara. 1995.

Vibrio trachuri sp. nov., a new species isolated from

diseased Japanese horse mackerel. Microbiology and

Immunology 39:831–837.

756 CHENG ET AL.

Lambert, C., J. L. Nicolas, V. Cilia, and S. Corre. 1998.

Vibrio pectenicida sp. nov., a pathogen of scallop

(Pecten maximus) larvae. International Journal of

Systematics and Bacteriology 48:481–487.

Lee, K. K., S. R. Yu, F. R. Chen, T. I. Yang, and P. C.

Liu. 1996. Virulence of Vibrio alginolyticus isolated

from diseased tiger prawn, Penaeus monodon. Current

Microbiology 32:229–231.

Lee, K. K., S. R. Yu, and P. C. Liu. 1997. Alkaline serine

protease is an exotoxin of Vibrio alginolyticus in Kur-

uma prawn, Penaeus japonicus. Current Microbiology

34:110–117.

Lee, K. K., P. C. Liu, Y. C. Chen, and C. Y. Huang. 2001.

The implication of ambient temperature with the

outbreak of vibriosis in cultured small abalone Haliotis

diversicolor supertexta Lischke. Journal of Thermal

Biology 26:585–587.

Li, H., F. Medina, S. B. Vinson, and C. J. Coates. 2005.

Isolation, characterization, and molecular identifica-

tion of bacteria from the red imported fire ant

(Solenopsis invicta) midgut. Journal of Invertebrate

Pathology 89:203–209.

Liu, P. C., Y. C. Chen, C. Y. Huang, and K. K. Lee. 2000.

Virulence of Vibrio parahaemolyticus isolated from

cultured small abalone Haliotis diversicolor super-

texta, with withering syndrome. Letters in Applied

Microbiology 31:433–437.

Liu, P. C., Y. C. Chen, and K. K. Lee. 2001. Pathogenicity

of Vibrio alginolyticus isolated from diseased small

abalone Haliotis diversicolor supertexta. Microbios

104:71–77.

Liu, P. V. 1957. Survey of hemolysin production among

species of pseudomonas. Journal of Bacteriology

74:718–727.

Macey, B. M. and V. E. Coyne. 2005. Improved growth

rate and disease resistance in farmed Haliotis midae

through probiotic treatment. Aquaculture 245:249–261.

Mittal, K. R., G. Lalonde, D. Leblanc, G. Olivier, and

R. Lallier. 1980. Aeromonas hydrophila in rainbow

trout: relation between virulence and surface character-

istics. Canadian Journal of Microbiology 26:1501–1503.

NCCLS (National Committee for Clinical Laboratory

Standards). 2000. Performance standard for antimi-

crobial susceptibility testing. Tenth informational sup-

plement. NCCLS Document M100–S10. National

Committee for Clinical Laboratory Standards, Wayne,

Pennsylvania, USA.

Nicolas, J. L., S. Corre, G. Gauthier, R. Robert, and

D. Ansquer. 1996. Bacterial problems associated with

scallop Pecten maximus larval culture. Diseases of

Aquatic Organisms 27:67–76.

Nicolas, J. L., O. Basuyaux, J. Mazurie, and A.

Thebault. 2002.Vibriocarchariae (syn.Vibrioharveyi),

a pathogen of the abaloneHaliotis tuberculata. Diseases

of Aquatic Organisms 50:35–43.

Nishimori, E., O. Hasegawa, T. Numata, and H.

Wakabayashi. 1998. Vibrio carchariae causes mass

mortalities in Japanese abalone, Sulculus diversicolor

supratexta. Fish Pathology 33:195–502.

Novoa, B., A. Luque, D. Castro, J. J. Borrego, and

A. Figueras. 1998. Characterization and infectivity of

four bacterial strains isolated from brown ring disease-

affected clams. Journal of Invertebrate Pathology

71:34–41.

Olafsen, J. A. 2001. Interactions between fish larvae and

bacteria in marine aquaculture. Aquaculture 200:223–

247.

Perez, M. J., L. A. Rodriguez, and T. P. Nieto. 1998. The

acetylcholinesterase ichthyotoxin is a common com-

ponent in the extracellular products of Vibrionaceae

strains. Journal of Applied Microbiology 84:47–52.

Reed, L. J. and H. Muench. 1938. A simple way of

estimating fifty percent end points. American Journal

of Hygiene 27:493–497.

Reuter, R. E. and S. McOrist. 1999. Mortality due to

Vibrio harveyi in farmed blacklip abalone, Notohalio-

tis ruber. Page 629 in C. L. Browdy and R Fletcher,

editors. The Annual International Conference and

Exposition of the World Aquaculture Society, Sydney,

Australia.

Saitou, N. and M. Nei. 1987. The neighbour-joining

method: a new method for reconstructing phylogenetic

trees. Molecular Biology and Evolution 4:406–425.

Sakai, D. K. 1985. Loss of virulence in a protease-deficient

mutant of Aeromonas salmonicida. Infection and

Immunity 48:146–152.

Thompson, D. E., J. T. Balsdon, J. Cai, and M. D.

Collins. 1992. Studies on the ribosomal RNA operons

of Listeria monocytogenes. FEMS Microbiological

Letters 75:219–234.

Thompson, F. L., T. Iida, and J. Swings. 2004. Bio-

diversity of Vibrios. Microbiology and Molecular

Biology Reviews 68:403–431.

Thompson, R., H. L. Macpherson, A. Riaza, and

T. H. Birbeck. 2005. Vibrio splendidus biotype 1 as

a cause of mortalities in hatchery-reared larval turbot,

Scophthalmus maximus (L.). Journal of Applied Micro-

biology 99:243–250.

Wang, J. Y., Z. X. Guo, J. Feng, G. F. Liu, and L. W.

Xu. 2004. Virus infection in cultured abalone Haliotis

diversicolor reeve in Guangdong province, China.

Journal of Shellfish Research 23:1163–1168.

VIBRIO PARAHAEMOLYTICUS PATHOGENIC TO ABALONE POSTLARVAE 757