Ž .Aquaculture 196 2001 37–46www.elsevier.nlrlocateraqua-online

Pathogenicity of Vibrio parahaemolyticus in tigerprawn Penaeus monodon Fabricius: possible role of

extracellular proteases

P.S. Sudheesh a,), Huai-Shu Xu b

a Central Institute of Brackishwater Aquaculture, 160-Mahalingapuram Main Road, Chennai-34,Chennai, India

b Ocean UniÕersity of Qingdao, Qingdao, People’s Republic of China

Received 19 July 2000; received in revised form 11 August 2000; accepted 16 November 2000

Abstract

Tiger prawn, Penaeus monodon, were experimentally challenged with a virulent strain ofVibrio parahaemolyticus 25C, originally isolated from Penaeus orientalis in an outbreak of redleg disease at a shrimp farm in the People’s Republic of China. The strain was found to be highly

5 Ž .virulent to P. monodon with an LD value of 1=10 colony forming units CFU rprawn. The50Ž .extracellular products ECP of the bacteria were also found to be toxic with an LD value of 850

mg proteinrprawn. The strain was found to be a poor producer of hemolysins. Extracellularproteases present in the ECP were purified by Sephadex G-150 gel filtration and further analyzedby SDS-polyacrylamide gel electrophoresis. Two fractions viz., F I and F II, were obtained andboth were found toxic to tiger prawns, but heat-inactivated fractions were not toxic. q 2001Elsevier Science B.V. All rights reserved.

Keywords: Vibrio parahaemolyticus; Penaeus monodon; Extracellular protease; Pathogenicity

1. Introduction

Numerous Vibrio species have been reported as pathogenic to various penaeidshrimps. Among them, Vibrio parahaemolyticus has emerged as an important shrimppathogen. Though there are many reports on the involvement of this bacterium in shrimp

) Corresponding author. Tel.: q91-44-8218178, q91-44-8218179; fax: q91-44-8218126, q91-44-8218176.Ž .E-mail address: sudheeshps@yahoo.com P.S. Sudheesh .

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 00 00575-5

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–4638

Žvibriosis Ruangpan and Kitao, 1991; Xu et al., 1994; Chanratchakool et al., 1995;.Alapide-Tendencia and Dureza, 1997 , the pathogenicity mechanism of this bacteria in

Ž .shrimp is poorly understood. Extracellular products ECP of bacteria and their virulencemechanism in fish have been studied in detail in the diseases caused by Aeromonas

Žsalmonicida and V. anguillarum Ellis et al., 1981; Inamura et al., 1984; Kanemori et al.,. Ž .1987 . Extracellular toxins produced by V. parahaemolyticus Xu et al., 1994 , V.

Ž . Ž . ŽÕulnificus Biosca and Amaro, 1996 , V. alginolyticus Lee et al., 1997 , V. harÕeyi Liu. Ž .et al., 1997 , V. splendidus and V. damsela Perez et al., 1998 have also been studied.

Recently, a few reports concerning extracellular proteases produced by pathogenicŽ .vibrios of shrimps have been published Lee et al., 1997; Liu et al., 1997 . This study

was undertaken to investigate the pathogenicity and virulence of V. parahaemolyticus tothe tiger prawn, Penaeus monodon, and also to assess the role of ECP, particularly thesecreted proteases produced by the bacteria involved in the disease of P. monodon.

2. Materials and methods

2.1. Bacteria

V. parahaemolyticus, strain 25C, was isolated from diseased P. orientalis in DahuaŽ .shrimp hatchery Laizhou, China , during an outbreak of vibriosis, popularly known as

Žred leg disease in China. Stock cultures of bacteria were stored in 2216 E broth Oxoid,. Ž .UK pH 7.4 containing 15% glycerol at y808C. Bacteria were routinely grown on

2216 E agar at 288C for 24 h for use in the experiments. The bacteria were identified byŽ .standard biochemical methods Baumann and Schubert, 1984 and the API-20E rapid

Ž .identification system Biomerieux, France .

2.2. Bacterial challenge

Bacteria were grown on 2216 E slants and harvested to sterile phosphate-bufferedŽsaline PBS, 0.01 M NaH PO P2H O, 0.01 M Na HPO P2H O, 0.15 M NaCl, pH2 4 2 2 4 2

. Ž .7.2 and diluted to a standard concentration equal to an optical density OD of 1.0 at540 nm. This standard suspension of bacteria contained approximately 1=109 CFUrml,as determined by standard dilution and plating methods. Healthy, inter-moult tiger

Ž .prawn, P. monodon mean weights10"1 g without any overt disease symptomsŽ .were obtained from Dahua shrimp hatchery Laizhou, China , and maintained in tanks

supplied with seawater of 35 ppt salinity and continuous aeration. The health status ofexperimental animals was further ascertained by culturing hemolymph and hepatopan-creas from few sampled animals on 2216 E agar for the presence of any bacterialpathogens. The prawns were acclimated in 1000-l tanks for 1 week before commencingthe experiment.

ŽThe LD test was conducted on P. monodon with batches of 10 prawnsrdose in50. Ž . Ž 8duplicate by intramuscular im injection of 0.1 ml of a bacterial suspension 1=10 ,

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–46 39

6 4 2 .1=10 , 1=10 and 1=10 CFUrprawn into the prawn at the site between the thirdŽ .and fourth abdominal segments Vera et al., 1992 . Sterile PBS of the same volume was

injected into control groups. Mortalities were observed for 7 days post-challenge.Bacteria were re-isolated from all moribund prawns by culturing hemolymph andhepatopancreas samples on 2216 E agar. The LD value was calculated by the method50

Ž .of Reed and Muench 1938 .

2.3. Kanagawa reaction

Ž .The Kanagawa reaction was performed on Wagatsuma agar Wagatsuma, 1967 .Polymerase chain reaction for the amplification of the tdh gene was carried out

Ž .according to the method of Karunasagar et al. 1996 using the primer pair 456Ž X X. Ž X X.5 -TTTCATGATTATTCAGTTT-3 and 411 5 -TTTGTTGGATATACACAT-3 .

2.4. Preparation of ECP

Ž .ECP was prepared by the cellophane plate technique Liu, 1957 . Briefly, overnightŽ .cultures of bacteria were washed into 5 ml of 0.01 M PBS pH 7.2 . Sterile cellophane

sheets were placed on 2216 E agar plates and inoculated by 0.2 ml of the bacterialsuspension. After 24 h incubation at 288C, grown cells were washed off the sheets into 3ml of PBS. This suspension was centrifuged at 12 000 rpm for 20 min at 48C and thesupernatant obtained was filter sterilized by means of a 0.2-mm membrane filterŽ .Millipore . The ECP was stored at y208C until use.

2.5. Enzyme actiÕities of bacteria and ECP

Caseinase, gelatinase, amylase, phospholipase, lipase and chitinase activities of ECPŽ .were detected by placing 25 ml of sample in wells cut in agarose 1% in PBS, pH 7.2

that contained either 1% casein, 1% gelatin, 0.2% starch, 2.5% egg yolk, 1% Tween-80or 0.3% chitin, respectively. Hemolytic activity was detected by supplementing themedium with 5% washed rabbit, sheep or human erythrocytes. After incubation at 288Cfor 24 h, the diameter of the lytic halo around each well was measured. Similarly,enzyme activities of bacteria were measured by spot inoculating bacteria on agar platescontaining the respective substrates as described above. The diameter of the lytic haloaround each colony was measured. The hemolytic activity of the ECP was also assayed

Ž .spectrophotometrically by the method of Inamura et al. 1984 .

2.6. Purification of protease

The protease enzyme present in the ECP was purified by Sephadex G-150 columnŽ .chromatography Inamura et al., 1984 . Briefly, 4 ml of an ammonium sulfate precipi-

tated and dialyzed ECP solution was applied to a Sephadex G-150 column and elutedŽ .with 0.01 M PBS pH 7.2 . The flow rate was about 0.4 mlrmin and the collected

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–4640

Žfractions were 3 ml each. The elution profiles were recorded at 280 nm Shanghai.Biochemical Instruments, HD-93 . The fractions were stored at y208C until use.

Ž .Protease was assayed by the method of Inamura et al. 1984 . Total proteins wereŽ .measured by the method of Lowry et al. 1951 using bovine serum albumin as a

standard.

2.7. SDS-polyacrylamide gel electrophoresis

The ECP and purified protease samples were further analysed by SDS-polyacryl-Ž .amide gel electrophoresis using the discontinuous buffer system of Laemmli 1970 on a

12% separating gel at a constant current of 15 mA. The gel was stained with coomassieŽ .brilliant blue R-250. Standard protein markers Sigma, St. Louis, CA, USA were used

to compare the molecular weight of proteases.

2.8. Toxicity of ECP to tiger prawn

Ž .Tiger prawns mean weights10"1 g were acclimated in 1000-l tanks for 1 weekbefore commencing experiments. Toxicity of ECP to tiger prawn was tested by injecting0.1 ml of filter sterilized ECP intramuscularly into 10 prawns. Sterile PBS was injectedinto a control group. Prawns were observed every hour for the first 6 h and then every 6h for 2 days and any mortality recorded. The 24-h LD test for ECP with batches of 1050

Ž .prawnsrdose in duplicate was conducted by intramuscular injection of 0.1 ml ECPŽ .20, 10, 5 and 2.5 mgrprawn into the prawns and any mortality was observed for 24 h.Sterile PBS was injected into a control group. The LD value was calculated by the50

Ž .method of Reed and Muench 1938 .

Fig. 1. Dose–response curve of V. parahaemolyticus 25C after 7 days of challenge with bacteria.

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–46 41

Fig. 2. Protease activity and total proteins of ECP after different incubation periods.

In another experiment, 0.1 ml of each Sephadex G-150 purified protease fractions Iand II were intramuscularly injected into 10 tiger prawns. Additionally, each heat-in-

Table 1Enzymatic and hemolytic activities of V. parahaemolyticus 25C and its ECP

Properties Bacteria ECP

Agar plate methodaCaseinase qqqq qqqq

Gelatinase qqqq qqqqAmylase qq qqqqPhospholipase q qLipase q qChitinase qq qqq

Hemolytic actiÕityHuman RBC y y

bSheep RBC q yRabbit RBC y y

Spectrophotometric methodŽ .Protease activity unitsrml ND 1220

Ž .Heat inactivated protease activity 708C for 10 min ND 0Ž .Hemolytic activity unitsrml ND 0

Ž .Heat inactivated Hemolytic activity 1008C for 10 min ND 0cPCR for tdh gene Negative ND

ND—not determined.aŽ . Ž . Žq and y Signs indicate ratio of hydrolysis halo to colony or well diameter in millimeters: qqqq,

.8–10; qqq, 5–8; qq, 2–5; q, 1–2; y, 0 .b No b-hemolysis was observed. Hemolysis was observed only below the colony.cAmplification of a 623-bp fragment of the tdh gene.

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–4642

Fig. 3. Sephadex G-150 chromatography of proteases present in the ECP of V. parahaemolyticus 25C. F I:fraction I; F II: fraction II.

Fig. 4. SDS-polyacrylamide gel electrophoresis of ECP, Protease Fractions I and II of V. parahaemolyticus25C. Lane A—molecular weight marker; lane B—ECP; lane C—Fraction II; lane D—Fraction I.

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–46 43

Ž .activated protease fraction 708C for 10 min was injected into 10 tiger prawns aspositive controls. Control prawns were injected with sterile PBS. Prawns were observedevery hour for the first 6 h, then every 6 h for 2 days and any mortality recorded.

3. Results

3.1. Bacterial challenge

Tiger prawns showed symptoms such as lethargy, loss of balance, whirling movementand general weakness within 6 h after challenge with bacteria. Hemolymph failed to clotand slight reddening of pleopods was also noticed. No mortality was observed in thecontrol group. After 7 days, 100% mortality was observed when injected with a bacterialdose of 1=108 CFUrprawn whereas mortalities of 80%, 20% and 10% were obtainedwith 106, 104 and 102, respectively. The LD value was calculated as 1=105

50

CFUrprawn. The re-isolated bacteria were identified as V. parahaemolyticus. Thedose–response curve of the bacteria is shown in Fig. 1.

3.2. Enzyme actiÕity

The bacteria and its ECP showed high caseinase and gelatinase activity, moderateamylase and chitinase activity and very low phospho-lipase and lipase activity. Theprotease activities after different incubation periods are shown in Fig. 2. The bacteria orits ECP did not show any b-hemolysis on blood agar. The ECP also did not show anydetectable hemolytic activity when assayed by the spectrophotometric method. The PCR

Fig. 5. Dose–response curve of ECP of V. parahaemolyticus 25C after 24 h of injection with ECP.

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–4644

Table 2Mortality of P. monodon injected with ECP and proteases of V. parahaemolyticus 25C

Ž .Treatment Dose Mortality no. GrossŽ .mg proteinrprawn mortalityŽ .Time h

6 12 24 36 48

ECP 20 6 2 2 – – 10r10Protease F I 20 6 2 2 – – 10r10Protease F II 20 5 4 1 – – 10r10

aProtease F I 20 – – – – – 0r10aProtease F II 20 – – – – – 0r10

Ž .Control PBS – – – – – – 0r10

a Heat-inactivated protease fractions from gel filtration of ECP.

amplification of a 623-bp fragment of the tdh gene did not give a positive reaction. Theenzymatic and hemolytic activities of ECP and bacteria are summarised in Table 1.

3.3. Toxicity of ECP

The protease present in the ECP was eluted as two separate peaks in Sephadex G-150Ž . Ž .gel filtration, designated as fraction I F I and fraction II F II as shown in Fig. 3.

Proteolytic activity was observed in both fractions, but no hemolytic activity wasobserved. SDS-polyacrylamide gel electrophoresis of each fraction produced single band

Ž .indicating that the fractions are sufficiently pure Fig. 4 . All prawns injected with ECPand protease fractions I and II died within 24 h post-treatment. The LD value of ECP50

to tiger prawn was calculated as 8 mg proteinrprawn. The dose–response curve of theECP is shown in Fig. 5. Prawns lost balance and showed whirling movement beforesuccumbing to death. The heat-inactivated protease fractions I and II did not cause anymortality. No mortality was observed in the control group injected with sterile PBS. Theresults of ECP and protease toxicity tests are shown in Table 2.

4. Discussion

V. parahaemolyticus 25C was isolated from the hemolymph of P. orientalis with redleg disease. Shrimp ponds with red leg disease have often encountered mortalities of

Ž .more than 90% Xu et al., 1994 . The disease is characterized by an expansion ofchromatophores on the periopods and pleopods, giving the appendages a reddishcolouration. Yellow pigmentation on the branchial region of cephalothorax is alsoobserved in prawns with red leg disease. In the present study, challenge experimentswere carried out on P. monodon and the strain was found to be highly virulent with an

5 Ž .LD value of 1=10 CFUrprawn. Alapide-Tendencia and Dureza 1997 reported that50

100% mortality occurred in tiger prawns after the injection of 107 CFU of V.parahaemolyticus and the typical symptoms of red leg disease were also reproduced.Our results are comparable and support their observation. LD values ranging from 102

50

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–46 45

6 Žto 10 CFU for Vibrio species have been reported by many workers Takahashi et al.,.1985; Jiravanichpaisal et al., 1994; Lee et al., 1996 .

The role of hemolysins in the virulence of V. parahaemolyticus to humans is wellŽ .documented Joseph et al., 1982 Interestingly, the present strain was found to be a poor

producer of hemolysins. Hemolysins produced by the bacteria or its ECP could not bedetected by the blood agar method or by the spectrophotometric method. The negativereaction in the PCR amplification of the tdh gene showed that the bacteria did notpossess the tdh gene. Proteases present in the ECP of bacteria have been implicated as

Ž .virulence determinants in fish diseases Inamura et al., 1984; Kanemori et al., 1987 andŽ .in shrimp vibriosis Lee et al., 1997 . The bacteria, as well as its ECP, showed high

protease activity. Extracellular products secreted by the bacteria were found to be highlyŽtoxic to tiger prawn. Similar findings have been reported for V. alginolyticus Yu, 1995;

. Ž .Lee et al., 1997 . Also, both protease fractions I and II , but not the heat-inactivatedfractions, were found to be highly toxic to tiger prawns. This is the first report of thetoxicity of the extracellular protease of V. parahaemolyticus to tiger prawn. This findingpoints to the possible role of proteases as virulence determinants of the bacteria. Furtherresearch is required to assess the role of extracellular proteases in relation to othervirulence determinants of the bacteria in the disease of shrimp.

Acknowledgements

Ž .This work was partly supported by grants from UNESCO contract no. 861.359.8 .We thank Dr. Indrani Karunasagar for help in the PCR amplification of the tdh gene.We also thank Dr. K.P. Jithendran for critically going through the manuscript. The firstauthor is grateful to the Ministry of Human Resource Development and the IndianCouncil of Agricultural Research, Government of India and the State Education commis-sion, People’s Republic of China for providing a scholarship.

References

Ž .Alapide-Tendencia, E.V., Dureza, L.A., 1997. Isolation of Vibrio spp. from Penaeus monodon Fabriciuswith red leg disease syndrome. Aquaculture 154, 107–114.

Baumann, P., Schubert, R.H.W., 1984. Family II. Vibrionaceae. Veron 1965. In: Krieg, N.R., Holt, J.G.Ž .Eds. , Bergey’s Manual of Systematic Bacteriology, vol. 1. The Williams and Wilkins, Baltimore, MD,USA, pp. 516–550.

Biosca, E.G., Amaro, C., 1996. Toxic and enzymatic activities of Vibrio Õulnificus biotype 2 with respect toŽ .host specifity. Appl. Environ. Microbiol. 62 7 , 2331–2337.

Chanratchakool, P., Pearson, M., Limsuwan, C., Roberts, R.J., 1995. Oxytetracycline sensitivity of Vibriospecies isolated from diseased black tiger shrimp, Penaeus monodon Fabricius. J. Fish Dis. 18, 79–82.

Ellis, A.E., Hastings, T.S., Munro, A.L.S., 1981. The role of Aeromonas salmonicida extracellular products inthe pathology of furunculosis. J. Fish Dis. 4, 41–51.

Inamura, H., Muroga, K., Nakai, T., 1984. Toxicity of extracellular products of Vibrio anguillarum. FishPathol. 19, 89–96.

Jiravanichpaisal, P., Miyazaki, T., Limsuwan, C., 1994. Histopathology, biochemistry and pathogenicity ofVibrio harÕeyi infecting black tiger prawn, Penaeus monodon. J. Aquat. Anim. Health 6, 27–35.

( )P.S. Sudheesh, H.-S. XurAquaculture 196 2001 37–4646

Joseph, S.W., Colwell, R.R., Kaper, J.B., 1982. Vibrio parahaemolyticus and related Vibrios. CRC Crit. Rev.Microbiol. 10, 77–124.

Kanemori, Y., Nakai, T., Muroga, K., 1987. The role of extracellular protease produced by Vibrio anguil-larum. Fish Pathol. 22, 153–158.

Karunasagar, I., Sugumar, G., Karunasagar, I., Reilly, P.J.A., 1996. Rapid polymerase chain reaction methodfor detection of Kanagawa positive Vibrio parahaemolyticus in seafoods. Int. J. Food Microbiol. 31,317–323.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.Ž .Nature London 227, 680–685.

Lee, K.K., Yu, S.R., Chen, F.R., Yang, T.I., Liu, P.C., 1996. Virulence of Vibrio alginolyticus isolated fromdiseased tiger prawn, Penaeus monodon. Curr. Microbiol. 32, 229–231.

Lee, K.K., Yu, S.R., Liu, P.C., 1997. Alkaline serine protease is an exotoxin of Vibrio alginolyticus inKuruma prawn, Penaeus japonicus. Curr. Microbiol. 34, 110–117.

Liu, P.V., 1957. Survey of hemolysin production among species of pseudomonads. J. Bacteriol. 74, 718–727.Liu, P.C., Lee, K.K., Tu, C.C., Chen, S.N., 1997. Purification and characterization of a cysteine protease

produced by pathogenic luminous Vibrio harÕeyi. Curr. Microbiol. 35, 32–39.Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol

reagent. J. Biol. Chem. 193, 265–275.Perez, M.J., Rodriguez, L.A., Nieto, T.P., 1998. The acetylcholinesterase ichthyotoxin is a common compo-

nent in the extracellular products of Vibrionaceae strains. J. Appl. Microbiol. 84, 47–52.Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27,

493–497.Ruangpan, L., Kitao, T., 1991. Vibrio bacteria isolated from black tiger shrimp, Penaeus monodon Fabricius.

J. Fish Dis. 14, 383–388.Takahashi, Y., Shimoyama, Y., Momoyama, K., 1985. Pathogenicity and characteristics of Vibrio sp. isolated

from cultured kuruma prawn, Penaeus japonicus Bate. Bull. Jpn. Soc. Sci. Fish. 51, 721–730.Vera, P., Navas, J.I., Quintero, M.C., 1992. Experimental study of virulence of three species of Vibrio bacteria

Ž .in Penaeus japonicus Bate 1881 juveniles. Aquaculture 107, 119–123.Wagatsuma, S., 1967. On a medium to test the hemolytic reaction of Vibrio parahaemolyticus. Media Circle

Ž .13, 159–162 in Japanese .Xu, B., Xu, H.S., Ji, W.S., Shi, J., 1994. Pathogens and pathogenicity to Penaeus orientalis Kishinouye. Acta

Oceanol. Sin. 13, 297–304.Yu, S.R., 1995. Studies on the characteristics of Vibrio alginolyticus and the lethal factor of the bacteria in

Penaeus monodon and P. japonicus. Master’s Thesis. National Taiwan Ocean University, Taiwan, China.