DISEASES OF AQUATIC ORGANISMS Vol. 22: 135-141,1995 Published June 15 Dis aqua1 Org l

Pathogenicity studies on a anguillarum- related (VAR) strain causing an epizootic in Argopecten purpuratus larvae cultured in Chile

'Departamento de Acuicultura. Facultad de Recursos del Mar, Universidad de Antofagasta, PO Box 170, Antofagasta, Chile 'Departamento de Microbiologia y Parasitologia, Facultad de Biologia, Universidad de Santiago de Compostela, E-15706Santiago de Compostela, Spain

ABSTRACT: A Vibrio anguillarum-related (VAR) strain, isolated in pure culture from an epizootic in a commercial hatchery producing Argopecten purpuratus, was characterized, and its potential patho- genicity to veliger larvae of A. purpuratus determined. Experimental challenges indicated that the bac- terium affects larval survival at concentrations of 104 to 108 cells ml-l The effect of water quality and temperature on pathogenicity was also evaluated. Larval survival in seawater filtered through 5 pm pore-size membranes was 45.6%, whereas using seawater passed through 1 and 0.2pm filters, larval survival increased to 66.4 and 80.4% respectively. Temperature also affected pathogenicity as larval survival at 15OC for 24 h was 69.3% but decreased to 30 and 26.9% at 20 and 25°C respectively. Toxic activity was found in cell-free supernatant of bacterial culture. Larval survival was reduced to 68.9and 36.4% after 20 and 40% (v/v) of supernatant, respectively, was added to the rearing water. These results suggest that exotoxins produced by the VAR strain play an in~portantrole in its pathogenicity for scallop larvae.

KEYWORDS: Argopecten purpuratus - Larvae . Vibrio anguillarum-related (VAR) . Pathogenicity . Exotoxins

INTRODUCTION ture exceeds 18°C (Sinderman 1990). In view of this, parameters such as the concentration of organic matter Great importance is attributed to as patho- and temperature should be monitored during larval gens of bivalves because of their association with high culture since they could be important contributing fac- mortalities of larval cultures with consequent eco- tors in the proliferation of potential pathogenic vibrios nomic losses to the hatcheries (DiSalvo et al. 1978, in a culture system. Elston & Leibovitz 1980, Jeffries 1982, Brown 1983, Although vibriosis caused by Vibrio anguillarum and Nottage & Birkbeck 1986, 1990, Lodeiros et al. 1987, V, tubiashii has been considered the most important Bourne et al. 1989, Navarro et al. 1991). Thus, there is bacterial infection limiting the production of marine an urgent need to develop methods for controlling sud- fish and shellfish throughout the world (see review by den outbreaks of vibriosis in commercial hatcheries. Toranzo & Barja 1990), other vibrios exist in the envi- The occurrence of epizootics caused by vibrios in ronment that are taxonomically and serologically bulk bivalve cultures may be related to their high con- related to these 2 species (Bryant et al. 1986, Fouz et al. centration in the environment, since low concentra- 1990). These other variants have been traditionally tions of vibrios do not necessarily imply a risk for larval considered to be of no pathogenic significance, but culture (Jeffries 1982). Sudden proliferation of mem- have been also associated with disease in larval and bers of the associated with severe larval adult stages of marine fish (Masumura et al. 1989, Fouz mortalities generally occurs in summer when organic et al. 1990, Toranzo et al. 1990, 1993, Myhr et al. 1991, matter increases in the sea and the seawater tempera- Toranzo & Barja 1993) and shellfish (Baticados et al.

0 Inter-Research 1995 136 Dis aquat Org 22: 135-141, 1995

1990, Lavilla-Pitogo et al. 1990, Paillard & Maes 1990, 20E system (Analytab) was employed using half Castro et al. 1992, Fujiwara et al. 1993). These vibrios strength seawater as diluent, and the results were correspond mainly to different biotypes of V splen- scored after 48 h at 22OC. dldus and V pelagius species and can be grouped The taxonomic position of the Vibrio strain recov- under the designation 'V anguillarum-like' or V ered in pure culture from all diseased larvae was anguillarum-related (VAR) organisms (Larsen 1985, determined following the criteria of West & Colwell Fouz et al. 1990, Toranzo & Barja 2990, Myhr et al. (1984), Bergey's Manual of Systematic Bacteriology 1991, Pazos et al. 1993). (1984, 1986), Bryant et al. (1986),Myhr et al. (1991) and The pathogenicity of some vibrios isolated from Austin & Lee (1992). molluscs (oysters) has been assumed to be due to their Drug resistance patterns were determined by the invasive capacity and/or their ability to produce exo- disc diffusion method (Barry & Thornsberry 1991) on toxin(s) (DiSalvo et al. 1978, Elston & Leibovitz 1980, Mueller-Hinton agar (bioMerieux) supplemented with Elston et al. 1981, Birkbeck et al. 1987). 1.5 % NaCl, using the following antimicrobial agents The aim of the current study was to evaluate the (pg disc-'): ampicillin (10),streptomycin (10),chloram- effect of temperature and water quality on the patho- phenicol (30), tetracycline (30), oxytetracycline (30), ------gcnic cspsc:ty of s VAR s:i;in isolated ir, pure ~~ilt~ii~eryihro ril.y. ciii [IS), naualllyclll1 --.- {30),oxuii~lic ctcici (21, from an epizootic in a commercial hatchery for Argo- Furazolidone (300), novobiocin (5) and trimethoprim- pecten purpuratus (Lamarck, 1819). In addition, the sulphamethoxazole (23.75-1.25). The vibriostatic agent role of exotoxins produced by this Vibrio strain on lar- 0/129 (150) was employed only for taxonomic pur- val survival was investigated. poses. Serological analysis was conducted by the slide- agglutination test as described by Serrensen & Larsen MATERIALS AND METHODS (1986) and Toranzo et al. (1987), using the thermo- stable bacterial '0' antigens and rabbit antisera raised Bacterial isolation. In a hatchery located in northern against the 10 '0' serotypes (from 01to 010) of Vibrio Chile (27" 5' 42" S, 69" 51' 48" W) massive larval mor- anguillarum (Ssrensen & Larsen 1986), V tubiashii talities occur in spring-summer. On one of these occa- EX1 (Lode~roset al. 1987) and V splendldus biovar sions, culture tanks with more than 90% of larvae on I ATCC 25914. the bottom were sampled. Swimming and bottom lar- Pathogenicity studies. Virulence assays were per- vae were netted and washed with sterile seawater, formed to determine the pathogenicity of the Vibrio then homogenized using a tissue grinder and spread strain isolated as pure culture from moribund larvae. on TCBS (thiosulfate-citrate-bile sucrose agar) (Oxoid) Healthy larvae of Argopecten purpuratus obtained and marine general medlum STlO (Ishida et al. 1986). from the hatchery were used for these assays. In addition, the bacterial flora of the hatchery water Pathogenicity of the isolated strain was determined supply was determined: unfiltered, seawater in sedi- applying the following variables: (1) bacterial concen- mentation tanks, filtered and UV treated seawater, and tration, (2) water quality and (3) temperature. The water from larval culture ponds. The bacterial flora of assays were performed in triplicate according to a microalgal cultures (stock and production batches) modification of methodology described by Brown used as larval food was also determined. All samples (1983). Argopecten purpuratus larvae were added (at were spread in triplicate on TCBS and STlO media and 2 larvae ml-') to sterile seawater filtered through incubated at 20°C for 48 h on TCBS and for 7 d on 0.2 pm membranes (Millipore) contained in cell culture ST10. Ad.ditionally, broodstock gonads were extracted chambers of 15 m1 capacity. and washed externally with 1% benzalkonium chlo- (1) Effect of bacterial concentration: cul- ride. A small incision was made through the surface of tured in STlO broth were washed by centrifugation the gonads with a heat sterilized scalpel. Gonad con- (3840 X g for 15 min) and suspended in Marine Saline tents were removed with sterile Pasteur pipettes, then Solution (MSS) (Austin 1988). Bacterial cells in this homogenized and spread on TCBS medium. suspension were stained with 1 pg ml-' of DAPI (4',6- Biochemical and serological characterization. Pure diamidino-2-phenylindole) (Porter & Feig 1980) and cultures of the bacteria isolated from moribund larvae counted under epifluorescence microscope (Austin were subjected to standard morphological, physiologi- 1988). Dilutions of the cell suspension were then added cal and biochemical tube and plate tests according to to the larvae. Final concentrations of Vibrio used were the procedures of West & Colwell (1984), Bryant et al. ca 104, 105, 106, 107 and 10"ells ml-l. Chambers with- (1986), Fouz et al. (1.990) and Hansen & Sorheim out addition of bacte.ria were used as controls. These (1991). Plates and tubes were incubated at 22OC for up bioassays were carried out at 20°C, and larval survival to 7 d. In addition, the commercial miniaturized API- at 19 and 24 h was recorded. Riquelme et al.:Vibriosls in Argopecten larvae In Chlle

(2) Effect of water quality: Argopecten purpuratus RESULTS larvae were suspended in seawater filtered through different pore-size membranes: (a) 5 pm (Nytal), Results of bacteriological analysis of samples in the (b)1.2 pm (Millipore)and (c)0.2 pm (Millipore).A final hatchery are presented in Table 1 The filtered and concentration of bacteria 10h cells m1 ' was added to UV-treated water entering larval culture tanks con- each chamber Controls without bacteria were used for tained a low bacterial concentration and undetectable each treatment. Larval survival was recorded at 10, 19 number of Vibrio spp. However, after the filtration and and 24 h. UV treatment, the Vibrio populations increased in the (3) Effect of temperature: To evaluate the effect of cultul-e tanks, reaching values of 92.3% of the total cul- temperature on the pathogenicity of this strain, a bac- turable bacteria. This increase is very high compared terial concentration of 10Qells ml-l was added to each to the 2 2 % composition of vibrios present in the intake chamber containing larvae in filtered (0.2 pm) sea- seawater. In microalgal cultures used as food for the water. The temperatures used for bioassays were 15, larvae, Vlbrio strains were also not detected. 20 and 25°C. Controls without bacteria were used for Bacteriological analysis of moribund larvae revealed each treatment. Larval survival was recorded at 10, 19 the presence of a Vibrio strain in pure culture, which was and 24 h. identified as V: anguillarum related (VAR) (Table 2). Effect of cell-free supernatant of bacterial culture This isolate shared characteristics with V anguillarum, on larval survival. Bacterial cultures in STlO were V: tubiashij and V. splendidus biovar I. However, sero- centrifuged for 15 min at 3840 X g. The supernatant logical tests indicated that this isolate did not share anti- was filtered through 0.2 pm membranes (Millipore), gens with the reference strains of these 3 species. The and added at 20 and 40% v/v to each chamber con- bacterium was resistant to several antimicrobial agents taining larvae in filtered (0.2 pm) seawater. Chambers (Table 2). without filtered supernatant and with 105 and 10' The bioassays on the pathogenicity of the VAR to viable cells ml-" of bacteria were used as controls. Lar- larvae sho~vedthat a concentration of 106 cells n~l-' vae were maintained in these conditions at 20°C a.nd was necessary to produce a decrease in larval survival survival recorded after 24 h. (Table 3) The highest mortality (0% larval survival) Record of larval survival. In all the experimental was recorded at 24 h with a concentration of 10' cells trials, larval survival was quantified by means of obser- ml-' (p < 0.05). However, with a bacterial concentra- vation of the organisms under a microscope. Larvae on tion of 103cells ml-' survival was 94 O/oat 24 h, not sig- the bottom of chambers that showed no apparent nificantly different from the control (p < 0.05). The movement, closed valves and no velar activity were number of colony-forming units (CFU) in the initial considered dead. bacterial inoculum was determined in selective The presence of bacteria on moribund larvae was medium (TCBS) and the number of culturable Vibrio verified by epifluorescence microscopy using DAPI strain corresponded to ca 0.1 5% of the total cells. staining. The results of different treatments of water filtration All assays were repeated at least twice. Results were indicated that in seawater filtered through 5 pm pore- analyzed using the stat~sticalG test (Zar 1984). size membranes, larval survival decreased to 67.3 % in 10 h and 45.6% in 24 h (Fig. 1A).However, using water filtered through 1 pm and 0.2 pm pore-sizes, larval sur-

Table 1 Vibrios and total culturable bacteria (CFU ml-') In vival of 66.4 and 80.4 0/;,respectively ~7asreached at different samples analyzed in the hatchery 24 h. Larval survival in these 3 treatments of seawater to which the VAR was added was significantly lower Samples Vibrio (TCBS) Total bacteria (ST10) (p 0.05) than in the respective controls (Fig. 1B). The pathogenicity of the VAR to larvae of Argo- Entrance seawater 2.0 X 102 9.3 X lo3 pecten purpuratus was affected by culture tempera- Sedimentation tanks 1.0 X 10' 4.9 X lo3 ture (Fig. 2). At 25°C the pathogenic effect was consid- Filtered water treated with UV erable, because larval survival was reduced to 36.8 Water from larval and 26.9% at 10 and 24 h respectively (Fig. 2A). In con- tanks trast, in the controls (without bacteria added) survival Microalgae of larvae greater than 70 % was observed at both times Breeders (Fig. 2B). At 20°C, larval survival of 70.4 and 30% at 10 and 24 h respectively was observed, both of which aBelolv detection limit of the spread-platemethod were lower (p < 0.05) than controls (Fig.2B). bCFU g-' of gonad tissues In the presence of the VAR strain, best larval survival (p < 0.05) was found at 15"C, reaching 81.9 and 69.3 Dis aquat Org 22: 135-141, 1995

Table 2.Identification of Vibrio ang~~illarum(VAR) strain Table 3. Argopecten purpuratus. Effect of different concen- trations of Vibrio anguillarum (VAR) on larval survival Characteristic Response Bacterial concentration Larval survival (%) at Gram stain added (cells ml-') 19 h 24 h Motility Oxidase 1o4 98 95 Catalase 1o5 98 87 Voges-Proskauer 1 O6 76 70 Indole production 1o7 11 3 Citrate utilization 1 o8 0 0 H2S production Control (no Vibrio added) 100 100 O/F (glucose) Gas from glucose Growth at 5'C 15°C 25°C v.17OC 42°C Growth in 0 % NaCl 3 "b NaCl 5 % NaCl 8 % NaCl 10% NaCl Growth on TCBS Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Lipase (Tween 80) P-Galactosidase (ONPG) Urease Gelat~nase Amylase Haemolysls (sheep blood) Acld production from: Glucose Mannose Galactose Fructose Sucrose Rhamnose Arabinose TIME (hours) Amygdaline Fig. 1. Argopecten purpuratus. Effect of differential filtration Melibiose of seawater on larval survival. (A) Larvae with addition of Mannitol VAR strain; (B) control Inositol Sorbitol Sens~tivity/resistanceto: 0/129 at 10 and 24 h, respectively (Fig. 2A). However, at this Novobiocin temperature larval survival in the control was signifi- Ampiclll~n Chloramphenicol cantly lower than in the tanks containing the Vibrio Tetracycline strain (Fig. 2B). Oxytetracycline The addition of cell-free supernatant of Vibrio cul- Streptomycin tures to tanks of larvae showed a clear lethal effect Erythromycin causing a decrease of larval survival to 68.9 and 36.4 % Kanamycin Oxolinic acid after 24 h following addition of 20 and 40% v/v, Furazolidone respectively (Fig. 3). These values were significantly Trimethoprim-sulphamethoxazole lower than those obtained in controls as well as in tanks containing 10' viable cells ml-' Colonization of F: fermentative strain; R:resistant; S: sensitive; I: intermediate bacteria was observed on moribund larvae exposed to the VAR strain for 24 h (Fig. 4). Riquelme et a1 . V~brloslsin Argopecten larvae In Chile 139

for Ostrea edulis (Lodeiros et al. 1987) and Argopecten purpuratus (Riquelme et al. 1994). The microbiological analysis allowed us to identify a VAR strain as responsible for the larval mortalities. In agreement with previous reports (Lodeiros et al. 1987, Myhr et al. 1991, Castro et al. 1992, Pazos et al. 1993) this straln is taxonomically related to Vibrio anguil- larun~,V tublash;, and V. splendidus. Until now, only 2 occurrences of pathogenic V. anguillarum and related strains (VAR) have been reported as responsible for mortalities of mollusc larvae in Chile. V anguillarum has been recovered from Mytilus chilensis (Vial et al. 1988), and VAR organisms were isolated from Argo- pecten purpuratus and Concholepas concholepes cul- ture (Pazos et al. 1993). The multiresistance to antimi- crobial agents of V. anguillarum isolated is remarkable (Table 2),and may be attributed to the frequent use of drugs in larval culture in the hatchery. The assays of pathogenicity conducted with this V. anguillarum indi- cated that a concentration of 106cells ml-' is necessary

20 to cause a pathogenic effect in A. purpuratus larvae. 0 5 10 15 20 25 The lower larval survival observed in bioassays with TIME (hours) seawater filtered through 5 pm membranes can be Fig. 2. Argopecten purpuratus. Effect of temperature on larval attributed to the increase of this bacterium because of survival. (A) Larvae w~thaddition of VAR strain; (B)control the copiotrophic characteristic of vibrios (West & Col- well 1984) and by the highest availability of particles and nutrients in this filtered seawater. The increase of temperature affected the pathogenic- ity of this Vibrio strain, with 69.3 % larval survival at Supernatant 20% 15°C in 24 h, but 30 and 26.9 % respectively at 20 and - I I 25°C. These findings may be attributed to the higher Supernatant 40% temperatures favouring bacterial proliferation. In fact, a F typical feature of the majority of Vibrio species is their improved growth at temperatures above 15°C (West & Colwell 1984).Moreover, at 25OC the heat stress could & adversely affect the larvae and predispose them to CT + V~brloWAR) 106 Vibrio attack. In rearing scallop larvae, temperatures between 18 and 24°C are used (Navarro et al. 1991). CONTROL In control cultures larval survival was lower at 15°C (Fig. 2B). This could be explained by the fact that 15°C is below the optimal temperature for the development SURVIVAL(%) of Argopecten purpuratus larvae. However in 15°C Fig. 3. Argopecten purpuratus. Survival of larvae exposed to: cultures inoculated with the VAR strain, larval survival 105and 106 cells ml-' of vibrios (VAR) and 20 % and 40% (vlv) was higher than in controls. Apparently the VAR strain of culture supernatant of thls strain is less virulent at 15OC, and also partially satisfies some nutritional requirements for larval survival. This point requires further investigation. DISCUSSION The overall results for the effects of water quality and temperature on the pathogenicity of the VAR The results of the survey in the hatchery suggest that strain in Argopecten purpuratus larvae indicate that the probable incorporation route of vibrios to the sys- both parameters are of great importance for controls tem could be the broodstock which contained a sig- against the increase of Vibrionaceae in larval cultures. nificant load of vibrios (2.4 X lo3 CFU g-l) in their The result that the cell-free supernatant of Vibrio reproductive organs. Similar findings of vertical trans- cultures decreased the larval survival suggest that a mission of Vibrio strains have been reported previously probable exotoxin(s) produced by this strain play a role Dis aquat Org 22: 135-141, 1995

Fig. 4. Argopecten purpuratus. Moribund larvae colonized by the VAR strain (a) Epifluorescence microscopy and phase contrast; bar = 30 pm;(b) epifluorescence, arrow shows bac- teria; bar = 8 pm;(c) light rnicroscopy; bar = 8 pm

However it remains necessary to clarify the rnecha- nism(s) of action that the present VAR strain possesses and to discover the nature and pathogenic effect of extracellular products that makes this bacterium a sig- nificant pathogen of Argopecten purpuratus larvae.

Acknowledgements. This investigation was supported by I FONDECYT Grant 92-0997 (Chile) to CR. AE.T Ulanks the ) EEC for financial support th;ough Grant AQ-1-263 UK-F-E.

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Responsible Subject Editor- A. K Sparks, Srattle, Manuscript hrst received: Adarch 9, 1994 IVashington, USA Revised verslon accepted: December 7, 1994