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Characterization of a nonmotile Vibro sp. pathogenic to larvae of Mercenaria mercenaria and Crassostrea virginica
Aquaculture, 74( 1988) 195-204 Elsevier Science Publishers B.V., Amsterdam -
195 Printed in The Netherlands
Characterization of a Nonmotile Vibro sp. Pathogenic to Larvae of Mercenaria mercenaria and Crussostreu virginicu CAROLYN BROWN’and LISA PETTI TETTELBACH National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Center, Milford Laboratory, Milford, CT 06460 (U.S.A.) Address for correspondence: Dr. C. Brown, U.S. Department of Commerce, NOAA, National Marine Fisheries Service, 1335 East-West Highway, Silver Spring, MD 20910 (U.S.A.) (Accepted 8 June 1988)
ABSTRACT Brown, C. and Tettelbach, L.P., 1988. Characterization of a nonmotile Vibrio sp. pathogenic to larvae of Mercenaria mercenaria and Crassostrea virginica. Aquaculture, 74: 195-204. Bioassay studies showed that a bacterial strain isolated from moribund, hatchery-reared larvae of the hard shell clam Mercenaria mercenaria was pathogenic to developing clam and oyster embryos. Results indicated that concentrations as low as 10’ colony forming units/ml of embryonic culture water caused visible adverse effects within 48 h. Biochemical and physiological characterization of the pathogen revealed that it was a nonmotile Vibrio sp. which produced acid in 16 of the 24 sugars tested, as well as fermenting both lactose and sucrose. The bacterial strain did not grow at 37°C; however, it did survive a 30-min exposure to 65°C. Weekly subculturing of the microbe over a period of 9 months resulted in a loss of virulence concomitant with an appearance of motile bacterial cells. Bioassay and bacteriological studies showed that the nonmotile strain was killed by exposure to ultraviolet light (93 312 PW s-r cmm2).
INTRODUCTION
Unfortunately the optimal conditions for growth and development of clam larvae in hatcheries favor the growth and multiplication of bacteria and the accumulation of their metabolites. Elevated numbers of bacteria associated with larval cultures occasionally lead to disease. Previously reported bacterial diseases of hatchery-reared clam larvae have been caused by Gram-negative, motile rods belonging to the genera Pseudomonas and Vibrio (Guillard, 1959; Tubiash et al., 1965; Brown, 1974). The following study was conducted to describe a nonmotile, pathogenic bacterium which was isolated from moribund clam larvae reared in a commercial hatchery.
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The disease occurred during the summer of 1983 and persisted through the month of November. The symptoms were consistent throughout the disease period: the clam larvae appeared to develop normally during the first 10 days of development; after the 10th day, the hatchery operators noticed that the larvae no longer fed. Microscopic examination of larval samples revealed that many of these larvae were disoriented (i.e., they swam in circles). Some of the larvae had what appeared to be oil globules inside of them. Atypically, neither fungal nor bacterial swarming was associated with the disease; survival 3 days following the first obvious signs of stress was less than 1%. MATERIALS AND METHODS
Isolation of bacteria Four sets of streaked agar plates were obtained from a commercial clam hatchery experiencing a disease problem. Each set had been streaked on site with either ground clam larvae (two sets), line effluent seawater, or algal cultures (Thulassiosira pseudonuna and Isochrysis galbana, Tahitian strain). Each set of plates consisted of three types of isolation media: marine agar (0.1% Trypticase, 0.1% yeast extract, 1.0% agar dissolved in 23.0%0 seawater that previously had been aged for about 6 months and then filtered through a 0.45 pm membrane); Sabouraud Dextrose Agarl (Difco, Detroit, MI, U.S.A.); and TCBS Agar (BBL, Baltimore, MD, U.S.A.). A sample of well water was later plated at the laboratory onto marine and TCBS agar plates at dilutions ranging from 10-l to 10e5. Morphologically distinct colonies were selected after a 7-day incubation period at 26’ C in marine broth (same constituents as marine agar, less the agar ). Broth cultures were incubated overnight and then streaked onto marine agar plates to verify their purity. Each pure culture was maintained on marine agar slants. Bacteriological methods described by Evelyn (1971) and Gerhardt (1981) were employed to determine morphological and physiological characteristics. Bacteria were classified to the genus level, using schemes described by Krieg (1984) and Shewan et al. (1960). Test animals Fertilized eggs of hard clam (Mercenaria mercenaria) and American oyster (Crassostrea uirginica) were used as test animals to determine the ability of the microbial isolates to cause disease and to test the efficacy of various disinfectants in disease control. Embryos were reared in 1.3-1polypropylene beak‘Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.
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ers at a density of about 15 embryos/ml of seawater and maintained in a constant-temperature water bath at 26’ C for 2 days. On the second day, larvae were collected on a 36-pm Nitex screen and resuspended in 250 ml of seawater contained in a 250-ml graduated cylinder. A 4-ml representative aliquot obtained from each beaker was fixed with 5% neutral formalin. Each sample was placed in a Sedgwick-Rafter counting cell and examined with the aid of a compound microscope. Larvae were classified into two groups: normal larvae comprised those which after 48 h had developed the standard “D” shaped shell, and abnormal larvae whose shells differed morphologically from the normal shape. These two groups were subdivided further according to whether they were living or dead prior to fixation. A larva was considered dead if its velum was ball-shaped or its tissues had atrophied. Biomsays To determine which microbial isolate was responsible for the spontaneous disease in the commercial hatchery, preliminary experiments were conducted by adding 1.0 ml of 18-24-h bacterial broth cultures to beakers prior to the addition of fertilized clam eggs. Broth culture filtrates, washed cells, broth cultures, and heated broth culture of the suspected pathogen were examined subsequently using procedures described by Brown and Losee (1978) to pinpoint the component(s) responsible. Disease control studies Disinfection efficacy tests were conducted in an attempt to identify a method of neutralizing the effects of the bacterial pathogen. The antibiotics streptomycin sulfate, 50 mg/l; neomycin sulfate, 100 mg/l; and erythromycin, 100 and 50 mg/l were tested in the presence and absence of the pathogenic bacterium. The efficacy of ultraviolet light radiation was also determined. A 135-l covered fiberglass tank was filled with l-pm filtered seawater. Control beakers were then filled with seawater taken from the tank and exposed to UV light (93 312-155 520 ,uW s-l cmm2) following the procedure described by Brown and Russo (1979). The remaining seawater in the tank was inoculated with a 140-ml suspension of the pathogenic bacterial cells to give a final concentration of lo5 pathogenic colony-forming units (CFU) /ml. Experimental beakers were filled with inoculated seawater either taken directly from the tank or after exposure to UV light. Fertilized oyster eggs were used as test animals. Bacteriological studies Bacteriological studies of the pathogen were conducted concomitant with the bioassays to determine viable bacterial cell numbers. Beakers were pre-
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pared as indicated above; however, no fertilized eggs were added. Samples were taken from each beaker at 0 and 24 h. Dilutions of each sample were prepared in sterile seawater, and O.l-ml aliquots of the original samples and of the 10-l and 10e2 dilutions were spread with an alcohol-flamed glass rod on marine agar plates. The inoculated plates were incubated at 26’ C for 2 weeks and then the bacterial colonies were counted. Statistical analysis Results in the Tables are expressed as mean + standard error. Student’s t test was used to determine differences between means. Significance was determined at the P < 0.05 level. RESULTS
Identification of bacteria Fifteen bacterial isolates were selected for study: five from ground clam larvae; three from line effluent seawater; four from well water; and three from a culture of Isochrysis galbana (Tahitian strain). No bacterial colonies were observed on plates inoculated with the sample of a Thalassiosirupseudonana culture. Data suggest that more bacterial diversity existed on plates inoculated with samples of Isochrysis galbana and ground clam larvae than on plates streaked with water samples. Only motile bacteria were isolated from water samples; plates streaked with line effluent seawater yielded three strains of the genus Pseudomonas, while those inoculated with well water gave rise to one strain of the genus Photobacterium and three strains of Pseudomonas. The latter strains were different from those isolated from line effluent seawater. Plates streaked with Isochrysis galbuna yielded one strain each of the genera Pseudomonas, Photobacterium, and Flavobacterium, while those with ground clam larvae had a respresentative from each of the genera Pseudomonas, FZavobacterium, Acinetobacter, a Mora3ceZZa-likebacterium, and Vibrio. Preliminary bioassays revealed that the Vibrio isolate was pathogenic to clam and oyster larvae. Characterization of pathogen The pathogenic bacterium was an anaerogenic, Gram-negative rod which was sensitive to Vibriostat O/129 (iV, N-dimethyl-p-phenylenediamine HCl), and was capable of fermenting glucose and producing oxidase. Growth was inhibited when sodium chloride wac either absent or in a high concentration (10% ). Unlike other reported Vibrio spp., the pathogen fermented both sucrose and lactose and also produced acid anaerogenically in a wide variety of
199 TABLE 1 Characteristicsof a shellfish-pathogenic CTibrio sp. Gram strain Motility in marine broth Fermentativein: Glucose Lactose Sucrose Gas production from glucose Oxidase (Kovacs) Pigmentation Sensitive to: o/129 Penicillin Growth at: 4°C 27°C 37°C Ureaseproduction Catalaseproduction Nitrate reduction Indole production Growth in: 0% NaCl 0.5% NaCl 2.5% NaCl 7.5% NaCl 10.0% NaCl
+ +
+ + + -
Acid production in: Adonitol Arabinose Cellobiose Dulcitol Fructose Gala&se Glucose Glycerol Inositol Inulin Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sorbose Sucrose Trehalose Xylose Esculin hydrolysis
+ + + + + + + + + + + + + + + + -
Methyl red reaction Voges-Proskauer reaction Hemolysis (sheep rbc) Starch hydrolysis Lipase (Tween 80) Citrate utilization Lecthinase Casein hydrolysis Gelatin hydrolysis Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolaze Phenylalaninedeaminase Hydrogen sulfide production in: Lead acetate Sodium thiosulfate Ammonium production Malonate utilization Gluconate utilization Requirementfor organic growth factors Growth on TCBS Agar digestion Catalaseproduction DNase production
+ + + + + + + + -
+ + -
+ + +
sugars (Table 1). The pathogen was also unique in that initially it was nonmotile and lacked flagellation when grown in marine broth. The bacterial cells were motile in Leifson’s O/F medium supplemented with sucrose and overlayed with sterile petrolatum; in contrast the cells remained nonmotile when either glucose or lactose was substituted for sucrose. About 9 months after the initial isolation, some cells of the pathogen grown in marine broth were observed to be motile. The motile strain was identical to the parental strain except the former was motile, had polar monotrichous flagellation, and its virulence was reduced. Further studies showed that although the pathogen (i.e., the parental strain) did not grow at 37 oC, it could survive exposure to 65 oC for 30 min. Bioassay studies
The mean of live-normal development of fertilized clam eggs was 73.4% in untreated controls, this was reduced to 11.4% and 16.6% when eggs were ex-
TABLE 2 Percentage development of clam embryos exposed to different preparations of the pathogenic bacterium Vibrio sp. Type of development
Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Untreated control
Heated broth culture
Sterile filtrate
Washed cells
73.4k4.0 13.1 k6.0 7.0k6.7 4.9-t 1.7 98.5 k 1.8 10
21.8& 13.9* 41.9 rt 19.7* 7.lf 6.5 15.2f 7.5* 85.9 !I 11.6 10
16.6 !I 11.1* 29.7 f 11.4* 13.9 f 10.7 19.7-t 7.4* 79.9f 7.2* 10
11.4f. k3.2* 33.5 Ik17.3* 8.9& 7.3 15.7-t 4.6* 69.6 If:15.7* 10
“Mean values f standard error. *Significantly different (P< 0.05) from untreated control. TABLE 3 Percentage development of clam embryos in presence of various amounts of washed Vibrio sp. cells Type of development
Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Untreated control
104CFU/ml
lo3 CFU/ml
10’ CFU/ml
lo* CFU/ml
63.3f 9.3 6.11 2.1 8.2f 3.9 3.2 t- 2.0 77.1k10.7 9
9.8f 2.6* 55.3klO.l* 4.4+ 1.8 6.8+ 3.1 76.2f 9.8 9
22.9 f 10.9* 39.42 8.5* 11.2+ 5.8 5.6+ 2.3 79.1* 9.9 9
42.4+ 9.6* 32.7 If:13.7* 9.4f 2.5 4.2+ 3.2 87.4f 13.1 9
46.5f 5.1* 18.5 f 11.5* 10.2f 2.2 4.62 3.9 79.4 f 10.6 9
“Mean values + standard error. *Significantly different (P< 0.05) from untreated control.
posed to washed bacterial cells or to sterile broth culture filtrate of the pathogen, respectively (Table 2). The data also showed that exposure to 65 oC for 30 min reduced the virulence of the pathogen; the mean of live-normal development was 21.8% in the presence of the heated broth culture. After exposure to 65”C, both the bacterial cells and the filtrate of the broth culture of the pathogen continued to affect larval development adversely; there was a significant increase in the percentage of dead-normal and dead-abnormal clam larvae. Live-normal development averaged only 20.9% in the presence of heated pathogenic cells and 51.9% in the presence of the heated supernatant fluid. Untreated controls achieved 64.4% mean live-normal development.
201
The percentage of clam embryos dying after reaching the straight-hinge increased significantly in the presence of the pathogenic Vibrio sp. at concentrations as low as lo1 CFU/ml of embryonic culture water (Table 3). Mean livenormal development of 63.3% in untreated controls dropped to 46.5% in the presence of lo1 CFU/ml. Development continued to drop as the number of CFUs was increased: 42.4%, 22.9%, and 9.8% in the presence of 102, 103, and lo4 CFU/ml of embryonic culture water, respectively. Disinfection efficacy studies Although all three antibiotics tested in vitro were capable of inhibiting growth of the pathogen, only two proved to be effective in neutralizing the effects of TABLE 4 Effects of two antibacterials on percentage development of oyster embryos reared in seawater seeded with pathogen Type of development
Live-normal Live-abnormal Dead-normal Dead-abnormal Replicates
Treatment” Streptomycin (50 mg/l)
Neomycin (100 mg/l)
Treated control ( lo5 CFU/ml)
Untreated control
52.4 + 7.5 13.7 4 6.3* 11.2 f 2.5* 4.4 + 1.6* 8
56.1& 8.2 12.7 + 3.6* 8.4f2.2* 2.520.8 8
oJ3+0.5* 14.7a5.7* 2.6 + 1.3* 17.5 5 6.4* 8
58.1 k 8.2 5.6k2.0 6.6 !I 4.7 2.5 +_1.1 8
“Mean values f standard error. *Significantly different (P< 0.05) from untreated control. TABLE 5 Effects of UV treatment on percentage development of fertilized oyster eggs reared in seawater with pathogenic Vibrio sp. Type of development Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Pathogen + UV
Pathogen
Control
57.0 k 3.9 4.6f0.9 10.12 1.9 2.7 f 0.9 74.4 + 4.8 8
31.8f4.7* 14.9 * 3.9* 15.2 I!I1.7* 5.9 k 1.3* 67.8k4.8 8
60.5 k 4.5 4.4If: 1.3 8.1 t- 1.8 3.2-t 1.1 76.2 f 6.8 8
“Mean values k standard error. *Significantly different (P< 0.05) from control.
202
the microbe upon larval oysters. Both streptomycin sulfate (50 mg/l) and neomycin sulfate (100 mg/l) were able to neutralize the effects of the pathogen (Table 4). Mean live-normal development in the presence of the pathogen was 56.1% and 52.4% when neomycin and streptomycin were added, respectively; untreated controls (i.e., embryonic cultures not inoculated with the pathogen) was 58.1%. At a concentration of 100 mg/l of embryonic culture water, erythromycin was toxic to larvae and at 50 mg/l it was ineffective. Ultraviolet light irradiation of seawater also proved to be effective; mean live-normal development was 57.0% when seawater inoculated with the pathogen was treated with UV light prior to the introduction of fertilized eggs, and 60.5% in control beakers. Embryos in untreated, inoculated seawater had a mean live-normal development of only 31.8% (Table 5). DISCUSSION
The results of the study indicate that a nonmotile, toxin-producing, marine Vibrio sp. was the causative agent of a disease problem which plaqued a commercial hatchery. The disease was quite similar to one which involved a commercial oyster hatchery on Long Island Sound (Brown, 1981). In both incidents, the shellfish larval cultures did not begin to show overt signs of disease until the 10th day of development. At that time neither bacterial or fungal swarming inside nor around larvae was observed, globules, however, were found inside otherwise healthy looking larvae. In the present study the microbe was isolated from moribund clam larvae, but not from line effluent seawater. This does not mean, however, that the pathogen was absent from the seawater. In the earlier study the pathogens were isolated from the seawater sample only after the sample had been held overnight. Apparently the pathogens were present in the flowing seawater at numbers too low to be detected (Brown, 1981). The disease outbreak in the commercial hatchery was not evident until after the 10th day of development; laboratory experiments, however, using lo1 CFU/ml of embryonic culture water resulted in massive mortality within the first 2 days of development. This further supports our contention that the pathogen was present in hatchery effluent seawater in very low numbers. The pathogenic Vibrio sp. under study is quite similar to Vibrio anguilhum. There are, however, some notable differences which suggest that this Vibrio is a different species or, at the very least, a different subspecies. The most notable differences are that the study pathogen was nonmotile and lacked flagellation when grown in marine broth and it was able to ferment both sucrose and lactose. Unlike V. anguillarum, the study pathogen also produced acid in maltose and salicin, and hydrogen sulfide in sodium thiosulfate; it was not able to attack arginine. Data show that the bacterium could survive exposure to 65’ C, suggesting that flushing seawater pipelines with hot tap water is not sufficient to kill this
203
pathogen. Although the exposure to 65°C did not kill the pathogenic bacterial cells, it did reduce its virulence. The experiments reported here do not explain why virulence was reduced, however, the data suggest that the bacterial cells either were producing a lower quantity of the toxin or producing it in an altered state that rendered it less toxic. Several other disease outbreaks in commercial hatcheries caused by Vibrio anguillarum (DiSalvo et al., 1978) or other Vibrio spp. (Leibovitz, 1978, Elston et al., 1981) have been reported. These and other outbreaks have caused hatchery operators to look for methods to eliminate pathogens from their systems; the use of antibacterials has been adopted by some operators. The results of the present study show that the test pathogen could be controlled through the use of either streptomycin sulfate (50 mg/l), neomycin sulfate (100 mg/l), or UV light radiation (93 312 PW s-l cmm2) treatments of seawater prior to the, introduction of fertilized eggs. There is the danger of resistance associated with using streptomycin, neomycin, or other antibiotics; naturally occurring Vibrio anguillarum ( Aoki et al., 1974) as well as other vibrios (Koditschek and Guyre, 1974) possessing R plasmids which confer resistance to a wide spectrum of antibacterials have been reported. Jeffries (1982) noted that the presence of these R plasmids in naturally occurring marine bacteria means that even hatcheries which do not routinely use antibiotics are in danger of infestation by resistant strains. He further stated that there is the possibility of the resistance factor being transferred from vibrios to Enterobacteriaceae; this would have medical consequences. Because of these serious concerns, the authors do not recommend the chronic use of antibiotics. Routine application of UV light is recommended as a preventive measure.
REFERENCES Aoki, T., Egusa, S. and Arai, T., 1974. Detection of R factors in naturahy occurring Vibrio anguillarum strains. Antimicrob. Agents Chemother., 6: 534-538. Brown, C., 1974. A pigment-producing pseudomonad which discolors culture containers of embryos of a bivalve mollusk. Chesapeake Sci., 15: 17-21. Brown, C., 1981. A study of two shellfish-pathogenic Vibrio strains isolated from a Long Island hatchery during a recent outbreak of disease. J. Shellfish. Res., 1: 83-87. Brown, C. and Losee, E., 1978. Observations on natural and induced epizootics of vibriosis in Crossostrea virginica. J. Invert. Pathol., 31: 41-47. Brown, C. and Russo, D.J., 1979. Ultraviolet light disinfection of shellfish hatchery sea water. I. Elimination of five pathogenic bacteria. Aquaculture, 17: 17-23. DiSalvo, L.H., Blecka, J. and Zebal, R., 1978. Vibrio anguillarum and larval mortality in a Cahfornia coastal shellfish hatchery. Appl. Environ. Microbial., 35: 219-221. Elston, R., Leibovitz, L., Relyea, D. and Zatila, J., 1981. Diagnosis of vibriosis in a commercial oyster hatchery epizootic: diagnostic tools and management features. Aquaculture, 24: 53-62. Evelyn, T.P.T., 1971. First records of vibriosis in Pacific salmon cultured in Canada, and taxonomit studies of the responsible bacterium, Vibrio anguillarum. J. Fish. Res. Board Can., 28: 517-525.
204 Gerhardt, P. (Editor-in-Chief), 1981. Manual of Methods for General Bacteriology, 7. American Society for Microbiology, Washington, DC, 524 pp. Guillard, R.R.L., 1959. Further evidence of the destruction of bivalve larvae by bacteria. Biol.. Bull., 117: 258-266. Jeffries, V.E., 1982. Three Vibrio strains pathogenic to larvae of Crassostrea gigas and Ostrea edulis. Aquaculture, 29: 201-226. Koditschek, L.K. and Guyre, P., 1974. Antimicrobial-resistant coliforms in New York Bight. Mar. Pollut. Bull., 5: 71-74. Krieg, N.R., (Editor), 1984. Bergey’s Manual of Determinative Bacteriology, Vol. 1. Williams and Wilkins, Baltimore MD, 964 pp. Leibovitz, L., 1978. A study of vibriosis at a Long Island shellfish hatchery. New York Sea Grant Institute Reprint Series, New York Sea Grant Institute, Albany, NY, NYSG-RR-79-02. Shewan, J.M., Hobbs G. and Hodgkiss, W., 1960. A determinative scheme for the identification of certain genera of Gram-negative bacteria, with special reference to the pseudomonadaceae. J. Appl. Bacterial., 23: 379-390. Tubiash, H.S., Chanley, P.E. and Leifson, E., 1965. Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology. J. Bacterial., 90: 1036-1044.
195 Printed in The Netherlands
Characterization of a Nonmotile Vibro sp. Pathogenic to Larvae of Mercenaria mercenaria and Crussostreu virginicu CAROLYN BROWN’and LISA PETTI TETTELBACH National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Center, Milford Laboratory, Milford, CT 06460 (U.S.A.) Address for correspondence: Dr. C. Brown, U.S. Department of Commerce, NOAA, National Marine Fisheries Service, 1335 East-West Highway, Silver Spring, MD 20910 (U.S.A.) (Accepted 8 June 1988)
ABSTRACT Brown, C. and Tettelbach, L.P., 1988. Characterization of a nonmotile Vibrio sp. pathogenic to larvae of Mercenaria mercenaria and Crassostrea virginica. Aquaculture, 74: 195-204. Bioassay studies showed that a bacterial strain isolated from moribund, hatchery-reared larvae of the hard shell clam Mercenaria mercenaria was pathogenic to developing clam and oyster embryos. Results indicated that concentrations as low as 10’ colony forming units/ml of embryonic culture water caused visible adverse effects within 48 h. Biochemical and physiological characterization of the pathogen revealed that it was a nonmotile Vibrio sp. which produced acid in 16 of the 24 sugars tested, as well as fermenting both lactose and sucrose. The bacterial strain did not grow at 37°C; however, it did survive a 30-min exposure to 65°C. Weekly subculturing of the microbe over a period of 9 months resulted in a loss of virulence concomitant with an appearance of motile bacterial cells. Bioassay and bacteriological studies showed that the nonmotile strain was killed by exposure to ultraviolet light (93 312 PW s-r cmm2).
INTRODUCTION
Unfortunately the optimal conditions for growth and development of clam larvae in hatcheries favor the growth and multiplication of bacteria and the accumulation of their metabolites. Elevated numbers of bacteria associated with larval cultures occasionally lead to disease. Previously reported bacterial diseases of hatchery-reared clam larvae have been caused by Gram-negative, motile rods belonging to the genera Pseudomonas and Vibrio (Guillard, 1959; Tubiash et al., 1965; Brown, 1974). The following study was conducted to describe a nonmotile, pathogenic bacterium which was isolated from moribund clam larvae reared in a commercial hatchery.
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers B.V.
196
The disease occurred during the summer of 1983 and persisted through the month of November. The symptoms were consistent throughout the disease period: the clam larvae appeared to develop normally during the first 10 days of development; after the 10th day, the hatchery operators noticed that the larvae no longer fed. Microscopic examination of larval samples revealed that many of these larvae were disoriented (i.e., they swam in circles). Some of the larvae had what appeared to be oil globules inside of them. Atypically, neither fungal nor bacterial swarming was associated with the disease; survival 3 days following the first obvious signs of stress was less than 1%. MATERIALS AND METHODS
Isolation of bacteria Four sets of streaked agar plates were obtained from a commercial clam hatchery experiencing a disease problem. Each set had been streaked on site with either ground clam larvae (two sets), line effluent seawater, or algal cultures (Thulassiosira pseudonuna and Isochrysis galbana, Tahitian strain). Each set of plates consisted of three types of isolation media: marine agar (0.1% Trypticase, 0.1% yeast extract, 1.0% agar dissolved in 23.0%0 seawater that previously had been aged for about 6 months and then filtered through a 0.45 pm membrane); Sabouraud Dextrose Agarl (Difco, Detroit, MI, U.S.A.); and TCBS Agar (BBL, Baltimore, MD, U.S.A.). A sample of well water was later plated at the laboratory onto marine and TCBS agar plates at dilutions ranging from 10-l to 10e5. Morphologically distinct colonies were selected after a 7-day incubation period at 26’ C in marine broth (same constituents as marine agar, less the agar ). Broth cultures were incubated overnight and then streaked onto marine agar plates to verify their purity. Each pure culture was maintained on marine agar slants. Bacteriological methods described by Evelyn (1971) and Gerhardt (1981) were employed to determine morphological and physiological characteristics. Bacteria were classified to the genus level, using schemes described by Krieg (1984) and Shewan et al. (1960). Test animals Fertilized eggs of hard clam (Mercenaria mercenaria) and American oyster (Crassostrea uirginica) were used as test animals to determine the ability of the microbial isolates to cause disease and to test the efficacy of various disinfectants in disease control. Embryos were reared in 1.3-1polypropylene beak‘Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.
197
ers at a density of about 15 embryos/ml of seawater and maintained in a constant-temperature water bath at 26’ C for 2 days. On the second day, larvae were collected on a 36-pm Nitex screen and resuspended in 250 ml of seawater contained in a 250-ml graduated cylinder. A 4-ml representative aliquot obtained from each beaker was fixed with 5% neutral formalin. Each sample was placed in a Sedgwick-Rafter counting cell and examined with the aid of a compound microscope. Larvae were classified into two groups: normal larvae comprised those which after 48 h had developed the standard “D” shaped shell, and abnormal larvae whose shells differed morphologically from the normal shape. These two groups were subdivided further according to whether they were living or dead prior to fixation. A larva was considered dead if its velum was ball-shaped or its tissues had atrophied. Biomsays To determine which microbial isolate was responsible for the spontaneous disease in the commercial hatchery, preliminary experiments were conducted by adding 1.0 ml of 18-24-h bacterial broth cultures to beakers prior to the addition of fertilized clam eggs. Broth culture filtrates, washed cells, broth cultures, and heated broth culture of the suspected pathogen were examined subsequently using procedures described by Brown and Losee (1978) to pinpoint the component(s) responsible. Disease control studies Disinfection efficacy tests were conducted in an attempt to identify a method of neutralizing the effects of the bacterial pathogen. The antibiotics streptomycin sulfate, 50 mg/l; neomycin sulfate, 100 mg/l; and erythromycin, 100 and 50 mg/l were tested in the presence and absence of the pathogenic bacterium. The efficacy of ultraviolet light radiation was also determined. A 135-l covered fiberglass tank was filled with l-pm filtered seawater. Control beakers were then filled with seawater taken from the tank and exposed to UV light (93 312-155 520 ,uW s-l cmm2) following the procedure described by Brown and Russo (1979). The remaining seawater in the tank was inoculated with a 140-ml suspension of the pathogenic bacterial cells to give a final concentration of lo5 pathogenic colony-forming units (CFU) /ml. Experimental beakers were filled with inoculated seawater either taken directly from the tank or after exposure to UV light. Fertilized oyster eggs were used as test animals. Bacteriological studies Bacteriological studies of the pathogen were conducted concomitant with the bioassays to determine viable bacterial cell numbers. Beakers were pre-
198
pared as indicated above; however, no fertilized eggs were added. Samples were taken from each beaker at 0 and 24 h. Dilutions of each sample were prepared in sterile seawater, and O.l-ml aliquots of the original samples and of the 10-l and 10e2 dilutions were spread with an alcohol-flamed glass rod on marine agar plates. The inoculated plates were incubated at 26’ C for 2 weeks and then the bacterial colonies were counted. Statistical analysis Results in the Tables are expressed as mean + standard error. Student’s t test was used to determine differences between means. Significance was determined at the P < 0.05 level. RESULTS
Identification of bacteria Fifteen bacterial isolates were selected for study: five from ground clam larvae; three from line effluent seawater; four from well water; and three from a culture of Isochrysis galbana (Tahitian strain). No bacterial colonies were observed on plates inoculated with the sample of a Thalassiosirupseudonana culture. Data suggest that more bacterial diversity existed on plates inoculated with samples of Isochrysis galbana and ground clam larvae than on plates streaked with water samples. Only motile bacteria were isolated from water samples; plates streaked with line effluent seawater yielded three strains of the genus Pseudomonas, while those inoculated with well water gave rise to one strain of the genus Photobacterium and three strains of Pseudomonas. The latter strains were different from those isolated from line effluent seawater. Plates streaked with Isochrysis galbuna yielded one strain each of the genera Pseudomonas, Photobacterium, and Flavobacterium, while those with ground clam larvae had a respresentative from each of the genera Pseudomonas, FZavobacterium, Acinetobacter, a Mora3ceZZa-likebacterium, and Vibrio. Preliminary bioassays revealed that the Vibrio isolate was pathogenic to clam and oyster larvae. Characterization of pathogen The pathogenic bacterium was an anaerogenic, Gram-negative rod which was sensitive to Vibriostat O/129 (iV, N-dimethyl-p-phenylenediamine HCl), and was capable of fermenting glucose and producing oxidase. Growth was inhibited when sodium chloride wac either absent or in a high concentration (10% ). Unlike other reported Vibrio spp., the pathogen fermented both sucrose and lactose and also produced acid anaerogenically in a wide variety of
199 TABLE 1 Characteristicsof a shellfish-pathogenic CTibrio sp. Gram strain Motility in marine broth Fermentativein: Glucose Lactose Sucrose Gas production from glucose Oxidase (Kovacs) Pigmentation Sensitive to: o/129 Penicillin Growth at: 4°C 27°C 37°C Ureaseproduction Catalaseproduction Nitrate reduction Indole production Growth in: 0% NaCl 0.5% NaCl 2.5% NaCl 7.5% NaCl 10.0% NaCl
+ +
+ + + -
Acid production in: Adonitol Arabinose Cellobiose Dulcitol Fructose Gala&se Glucose Glycerol Inositol Inulin Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sorbose Sucrose Trehalose Xylose Esculin hydrolysis
+ + + + + + + + + + + + + + + + -
Methyl red reaction Voges-Proskauer reaction Hemolysis (sheep rbc) Starch hydrolysis Lipase (Tween 80) Citrate utilization Lecthinase Casein hydrolysis Gelatin hydrolysis Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolaze Phenylalaninedeaminase Hydrogen sulfide production in: Lead acetate Sodium thiosulfate Ammonium production Malonate utilization Gluconate utilization Requirementfor organic growth factors Growth on TCBS Agar digestion Catalaseproduction DNase production
+ + + + + + + + -
+ + -
+ + +
sugars (Table 1). The pathogen was also unique in that initially it was nonmotile and lacked flagellation when grown in marine broth. The bacterial cells were motile in Leifson’s O/F medium supplemented with sucrose and overlayed with sterile petrolatum; in contrast the cells remained nonmotile when either glucose or lactose was substituted for sucrose. About 9 months after the initial isolation, some cells of the pathogen grown in marine broth were observed to be motile. The motile strain was identical to the parental strain except the former was motile, had polar monotrichous flagellation, and its virulence was reduced. Further studies showed that although the pathogen (i.e., the parental strain) did not grow at 37 oC, it could survive exposure to 65 oC for 30 min. Bioassay studies
The mean of live-normal development of fertilized clam eggs was 73.4% in untreated controls, this was reduced to 11.4% and 16.6% when eggs were ex-
TABLE 2 Percentage development of clam embryos exposed to different preparations of the pathogenic bacterium Vibrio sp. Type of development
Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Untreated control
Heated broth culture
Sterile filtrate
Washed cells
73.4k4.0 13.1 k6.0 7.0k6.7 4.9-t 1.7 98.5 k 1.8 10
21.8& 13.9* 41.9 rt 19.7* 7.lf 6.5 15.2f 7.5* 85.9 !I 11.6 10
16.6 !I 11.1* 29.7 f 11.4* 13.9 f 10.7 19.7-t 7.4* 79.9f 7.2* 10
11.4f. k3.2* 33.5 Ik17.3* 8.9& 7.3 15.7-t 4.6* 69.6 If:15.7* 10
“Mean values f standard error. *Significantly different (P< 0.05) from untreated control. TABLE 3 Percentage development of clam embryos in presence of various amounts of washed Vibrio sp. cells Type of development
Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Untreated control
104CFU/ml
lo3 CFU/ml
10’ CFU/ml
lo* CFU/ml
63.3f 9.3 6.11 2.1 8.2f 3.9 3.2 t- 2.0 77.1k10.7 9
9.8f 2.6* 55.3klO.l* 4.4+ 1.8 6.8+ 3.1 76.2f 9.8 9
22.9 f 10.9* 39.42 8.5* 11.2+ 5.8 5.6+ 2.3 79.1* 9.9 9
42.4+ 9.6* 32.7 If:13.7* 9.4f 2.5 4.2+ 3.2 87.4f 13.1 9
46.5f 5.1* 18.5 f 11.5* 10.2f 2.2 4.62 3.9 79.4 f 10.6 9
“Mean values + standard error. *Significantly different (P< 0.05) from untreated control.
posed to washed bacterial cells or to sterile broth culture filtrate of the pathogen, respectively (Table 2). The data also showed that exposure to 65 oC for 30 min reduced the virulence of the pathogen; the mean of live-normal development was 21.8% in the presence of the heated broth culture. After exposure to 65”C, both the bacterial cells and the filtrate of the broth culture of the pathogen continued to affect larval development adversely; there was a significant increase in the percentage of dead-normal and dead-abnormal clam larvae. Live-normal development averaged only 20.9% in the presence of heated pathogenic cells and 51.9% in the presence of the heated supernatant fluid. Untreated controls achieved 64.4% mean live-normal development.
201
The percentage of clam embryos dying after reaching the straight-hinge increased significantly in the presence of the pathogenic Vibrio sp. at concentrations as low as lo1 CFU/ml of embryonic culture water (Table 3). Mean livenormal development of 63.3% in untreated controls dropped to 46.5% in the presence of lo1 CFU/ml. Development continued to drop as the number of CFUs was increased: 42.4%, 22.9%, and 9.8% in the presence of 102, 103, and lo4 CFU/ml of embryonic culture water, respectively. Disinfection efficacy studies Although all three antibiotics tested in vitro were capable of inhibiting growth of the pathogen, only two proved to be effective in neutralizing the effects of TABLE 4 Effects of two antibacterials on percentage development of oyster embryos reared in seawater seeded with pathogen Type of development
Live-normal Live-abnormal Dead-normal Dead-abnormal Replicates
Treatment” Streptomycin (50 mg/l)
Neomycin (100 mg/l)
Treated control ( lo5 CFU/ml)
Untreated control
52.4 + 7.5 13.7 4 6.3* 11.2 f 2.5* 4.4 + 1.6* 8
56.1& 8.2 12.7 + 3.6* 8.4f2.2* 2.520.8 8
oJ3+0.5* 14.7a5.7* 2.6 + 1.3* 17.5 5 6.4* 8
58.1 k 8.2 5.6k2.0 6.6 !I 4.7 2.5 +_1.1 8
“Mean values f standard error. *Significantly different (P< 0.05) from untreated control. TABLE 5 Effects of UV treatment on percentage development of fertilized oyster eggs reared in seawater with pathogenic Vibrio sp. Type of development Live-normal Dead-normal Live-abnormal Dead-abnormal Total Replicates
Treatment” Pathogen + UV
Pathogen
Control
57.0 k 3.9 4.6f0.9 10.12 1.9 2.7 f 0.9 74.4 + 4.8 8
31.8f4.7* 14.9 * 3.9* 15.2 I!I1.7* 5.9 k 1.3* 67.8k4.8 8
60.5 k 4.5 4.4If: 1.3 8.1 t- 1.8 3.2-t 1.1 76.2 f 6.8 8
“Mean values k standard error. *Significantly different (P< 0.05) from control.
202
the microbe upon larval oysters. Both streptomycin sulfate (50 mg/l) and neomycin sulfate (100 mg/l) were able to neutralize the effects of the pathogen (Table 4). Mean live-normal development in the presence of the pathogen was 56.1% and 52.4% when neomycin and streptomycin were added, respectively; untreated controls (i.e., embryonic cultures not inoculated with the pathogen) was 58.1%. At a concentration of 100 mg/l of embryonic culture water, erythromycin was toxic to larvae and at 50 mg/l it was ineffective. Ultraviolet light irradiation of seawater also proved to be effective; mean live-normal development was 57.0% when seawater inoculated with the pathogen was treated with UV light prior to the introduction of fertilized eggs, and 60.5% in control beakers. Embryos in untreated, inoculated seawater had a mean live-normal development of only 31.8% (Table 5). DISCUSSION
The results of the study indicate that a nonmotile, toxin-producing, marine Vibrio sp. was the causative agent of a disease problem which plaqued a commercial hatchery. The disease was quite similar to one which involved a commercial oyster hatchery on Long Island Sound (Brown, 1981). In both incidents, the shellfish larval cultures did not begin to show overt signs of disease until the 10th day of development. At that time neither bacterial or fungal swarming inside nor around larvae was observed, globules, however, were found inside otherwise healthy looking larvae. In the present study the microbe was isolated from moribund clam larvae, but not from line effluent seawater. This does not mean, however, that the pathogen was absent from the seawater. In the earlier study the pathogens were isolated from the seawater sample only after the sample had been held overnight. Apparently the pathogens were present in the flowing seawater at numbers too low to be detected (Brown, 1981). The disease outbreak in the commercial hatchery was not evident until after the 10th day of development; laboratory experiments, however, using lo1 CFU/ml of embryonic culture water resulted in massive mortality within the first 2 days of development. This further supports our contention that the pathogen was present in hatchery effluent seawater in very low numbers. The pathogenic Vibrio sp. under study is quite similar to Vibrio anguilhum. There are, however, some notable differences which suggest that this Vibrio is a different species or, at the very least, a different subspecies. The most notable differences are that the study pathogen was nonmotile and lacked flagellation when grown in marine broth and it was able to ferment both sucrose and lactose. Unlike V. anguillarum, the study pathogen also produced acid in maltose and salicin, and hydrogen sulfide in sodium thiosulfate; it was not able to attack arginine. Data show that the bacterium could survive exposure to 65’ C, suggesting that flushing seawater pipelines with hot tap water is not sufficient to kill this
203
pathogen. Although the exposure to 65°C did not kill the pathogenic bacterial cells, it did reduce its virulence. The experiments reported here do not explain why virulence was reduced, however, the data suggest that the bacterial cells either were producing a lower quantity of the toxin or producing it in an altered state that rendered it less toxic. Several other disease outbreaks in commercial hatcheries caused by Vibrio anguillarum (DiSalvo et al., 1978) or other Vibrio spp. (Leibovitz, 1978, Elston et al., 1981) have been reported. These and other outbreaks have caused hatchery operators to look for methods to eliminate pathogens from their systems; the use of antibacterials has been adopted by some operators. The results of the present study show that the test pathogen could be controlled through the use of either streptomycin sulfate (50 mg/l), neomycin sulfate (100 mg/l), or UV light radiation (93 312 PW s-l cmm2) treatments of seawater prior to the, introduction of fertilized eggs. There is the danger of resistance associated with using streptomycin, neomycin, or other antibiotics; naturally occurring Vibrio anguillarum ( Aoki et al., 1974) as well as other vibrios (Koditschek and Guyre, 1974) possessing R plasmids which confer resistance to a wide spectrum of antibacterials have been reported. Jeffries (1982) noted that the presence of these R plasmids in naturally occurring marine bacteria means that even hatcheries which do not routinely use antibiotics are in danger of infestation by resistant strains. He further stated that there is the possibility of the resistance factor being transferred from vibrios to Enterobacteriaceae; this would have medical consequences. Because of these serious concerns, the authors do not recommend the chronic use of antibiotics. Routine application of UV light is recommended as a preventive measure.
REFERENCES Aoki, T., Egusa, S. and Arai, T., 1974. Detection of R factors in naturahy occurring Vibrio anguillarum strains. Antimicrob. Agents Chemother., 6: 534-538. Brown, C., 1974. A pigment-producing pseudomonad which discolors culture containers of embryos of a bivalve mollusk. Chesapeake Sci., 15: 17-21. Brown, C., 1981. A study of two shellfish-pathogenic Vibrio strains isolated from a Long Island hatchery during a recent outbreak of disease. J. Shellfish. Res., 1: 83-87. Brown, C. and Losee, E., 1978. Observations on natural and induced epizootics of vibriosis in Crossostrea virginica. J. Invert. Pathol., 31: 41-47. Brown, C. and Russo, D.J., 1979. Ultraviolet light disinfection of shellfish hatchery sea water. I. Elimination of five pathogenic bacteria. Aquaculture, 17: 17-23. DiSalvo, L.H., Blecka, J. and Zebal, R., 1978. Vibrio anguillarum and larval mortality in a Cahfornia coastal shellfish hatchery. Appl. Environ. Microbial., 35: 219-221. Elston, R., Leibovitz, L., Relyea, D. and Zatila, J., 1981. Diagnosis of vibriosis in a commercial oyster hatchery epizootic: diagnostic tools and management features. Aquaculture, 24: 53-62. Evelyn, T.P.T., 1971. First records of vibriosis in Pacific salmon cultured in Canada, and taxonomit studies of the responsible bacterium, Vibrio anguillarum. J. Fish. Res. Board Can., 28: 517-525.
204 Gerhardt, P. (Editor-in-Chief), 1981. Manual of Methods for General Bacteriology, 7. American Society for Microbiology, Washington, DC, 524 pp. Guillard, R.R.L., 1959. Further evidence of the destruction of bivalve larvae by bacteria. Biol.. Bull., 117: 258-266. Jeffries, V.E., 1982. Three Vibrio strains pathogenic to larvae of Crassostrea gigas and Ostrea edulis. Aquaculture, 29: 201-226. Koditschek, L.K. and Guyre, P., 1974. Antimicrobial-resistant coliforms in New York Bight. Mar. Pollut. Bull., 5: 71-74. Krieg, N.R., (Editor), 1984. Bergey’s Manual of Determinative Bacteriology, Vol. 1. Williams and Wilkins, Baltimore MD, 964 pp. Leibovitz, L., 1978. A study of vibriosis at a Long Island shellfish hatchery. New York Sea Grant Institute Reprint Series, New York Sea Grant Institute, Albany, NY, NYSG-RR-79-02. Shewan, J.M., Hobbs G. and Hodgkiss, W., 1960. A determinative scheme for the identification of certain genera of Gram-negative bacteria, with special reference to the pseudomonadaceae. J. Appl. Bacterial., 23: 379-390. Tubiash, H.S., Chanley, P.E. and Leifson, E., 1965. Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology. J. Bacterial., 90: 1036-1044.