Cultured Juvenile Pacific Oysters (Crassostrea gigas) by. Cytophaga-Like ..... in 50% seawater cytophaga broth and differential-inter- ..... center. Bar, 50,um. .... Association for Science fellowship while completing graduate stud- ies at the ...
APPLIED
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ENVIRONMENTAL MICROBIOLOGY, May 1989, p. 1128-1135
Vol. 55, No. 5
0099-2240/89/051128-08$02.00/0 Copyright C) 1989, American Society for Microbiology
Evidence for Colonization and Destruction of Hinge Ligaments in Cultured Juvenile Pacific Oysters (Crassostrea gigas) by Cytophaga-Like Bacteria CHRISTOPHER F. DUNGAN,lt* RALPH A. ELSTON,' AND MICHAEL H. SCHIEWE2 Center for Marine Disease Control, Battelle Marine Research Laboratory, 439 West Sequim Bay Road, Sequim, Washington 98382,1 and Northwest Fisheries Center, National Marine Fisheries Service, Seattle, Washington 981122 Received 1 November 1988/Accepted 14 February 1989
Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20°C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G+C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.
Qualitative and quantitative studies of bacterial populations associated with adult and juvenile Pacific and Eastern oysters, Crassostrea gigas and C. virginica, indicate that the genera Alcaligenes (Achromobacter), Bacillus, Corynebacterium, Cytophaga, Flavobacterium, Micrococcus, Pseudomonas, and Vibrio are routinely represented and the genera Pseudomonas and Vibrio are usually numerically dominant (24). The suggestion of several researchers that bacterial groups associated with oysters represent a characteristic commensal microflora (7, 21, 23) is speculative in the absence of information concerning the nature of interactions between bacteria and oysters. Indeed, members of the genera Pseudomonas and Vibrio have also been implicated as oyster pathogens (6, 10, 33). Moreover, while bacteria could be cultured from homogenates of tissues containing external and digestive epithelia of healthy, feeding oysters, examination of these same epithelia by scanning electron microscopy failed to confirm the presence of a surfaceassociated microflora (13). The external surfaces of oyster valves were the only surfaces observed to be routinely colonized by bacteria. High prevalences of degenerative lesions within the proteinaceous hinge ligaments of juvenile oysters from cultured populations experiencing high mortality rates have been described (8, 9, 11). Because the oyster hinge ligament serves to bind and align the valves and to oppose the adductor muscle in opening the valves, it plays a critical role in physiological functions and host defense essential to growth and survival (12). Of the two structural elements composing the oyster hinge ligament, the resilium is responsible for opening the valves (32) and is also the most common
site of these erosive lesions (8). In a previous study, electron microscopy of eroding ligament surfaces from areas deep within degenerative lesions revealed the presence of a dense and morphologically homogeneous population of bacteria oriented at right angles to intact resilium and surrounded by remnants of degraded resilium (8). These bacteria were characterized by cell lengths greater than 3.0 pum, a gramnegative-type cell wall with apparent flexibility, and the absence of flagella. In an effort to identify the etiological agent(s) of hinge ligament erosion, the present study was initiated to investigate the nature of bacterial populations associated with hinge ligaments of commercially cultured juvenile C. gigas and to test selected isolates for degradative capabilities consistent with an ability to mediate characteristic ligament lesions. MATERIALS AND METHODS Oysters. Live juvenile oysters were obtained from different production groups at four public and commercial culture facilities located along the Pacific coast of the United States. Sixteen samples containing 50 to 500 oysters (2- to 10-mm shell height) were shipped in clean, refrigerated containers and arrived within 48 h of shipment; all contained live animals. Four groups of healthy juvenile oysters obtained from culture facilities in both Washington and California were kept at low densities (40 to 120 oysters per aquarium) under laboratory conditions in separate 38-liter aquaria containing aerated seawater. The aquaria were maintained at 8°C, and the oysters were fed a suspension of the diatom Thalassiosira pseudonana. Aquarium water was replaced weekly, at which time all oysters were counted and dead oysters were removed. These data were used to calculate the mortality rate for each oyster group in the month preceding the date when hinge ligaments were sampled for microbiological analysis.
* Corresponding author. t Present address: Oregon State University Microbiology Depart-
ment, Hatfield Marine Science Center, 2030 Marine Science Drive, Newport, OR 97365. 1128
VOL. 55, 1989
Oysters in 12 other samples were either processed for microbiological analysis immediately upon arrival or kept overnight at 4°C in a sterile petri dish containing filtered seawater (0.45-,um pore size). The mortality profiles of populations from which these 12 oyster samples were drawn were supplied by the managers of the facilities that submitted samples. For this investigation, groups or populations of juvenile oysters with mortality rates below 5% during the month preceding analysis of bacteria associated with their hinge ligaments were classified as having low mortality. Groups or populations with mortality rates above 20% during a similar period were classified as having high mortality. Bacteriological methods. Ten animals were selected at random from each oyster group or sample for microbiological analysis of their hinge ligaments. With the dorsal surface uppermost to prevent contamination of the hinge ligament with dissection products, a pointed scalpel was used to sever the adductor muscle at its articulations with the valves, and the oyster tissues and shell liquor were removed ventrally. The valves were then separated, and the hinge ligament was excised. Hinge ligaments were pooled in a drop of sterile saline (1.5% [wt/vol] NaCl) in an iced petri dish. Pooled ligaments were ground in a chilled glass tissue grinder with 1.0 ml of saline as the diluent. Hinge ligament homogenates were diluted with chilled saline in a series of six 10-fold dilutions, 0.1-ml portions of which were placed on solid media and spread with a glass rod. Initial tests of selected media, including several supplemented with oyster hinge ligament homogenates or extracts, indicated that the greatest total number of colonies, as well as the greatest diversity of colony morphologies, was observed when hinge ligament homogenates were plated on either marine agar (MA; Difco Laboratories, Detroit, Mich.) or cytophaga agar (1) formulated with 50% seawater (SWCA). Because colonies of cytophaga-like bacteria (CLB) plated on MA failed to develop the rhizoidal colony margins which facilitated their identification and because rapid growth by eubacterial colonies on this medium often masked the presence of slower-growing CLB colonies, SWCA was selected for initial plating of hinge ligament homogenates. Plated dilutions of hinge ligament homogenates were incubated for 5 or 7 days at 20 or 17°C, respectively, before colonies were enumerated. For all oyster groups or samples, both the total number of CFU and the total number of CLB colonies were recorded. From dilution plates of hinge ligament homogenates of oysters within the four groups that were kept in the laboratory, 5 to 10 of the most numerous colony types were separately enumerated and isolated. On the basis of differences in colony morphology, at least 90% of the bacteria growing on dilution plates were isolated as pure cultures in this manner. All isolates were broadly characterized as pseudomonads, vibrios, aeromonads, CLB, or enteric bacteria with a small battery of determinative tests based on those of Shewan (14, 30). These tests included the following: motility and morphology by phase-contrast microscopy, Gram stain, Kovac spot oxidase test, oxidation-fermentation of glucose, and sensitivity to the vibriostatic compound 0/129 (2,4-diamino6,7-diisopropyl pteridine phosphate). Glucose utilization and 0/129 sensitivity were assessed by the methods of Schiewe et al. (29). Bacterial strains known to be positive or negative for the properties tested were used as controls with each group of isolates tested and included Vibrio anguillarum 775, Aeromonas salmonicida, Pseudomonas fluorescens, and Escherichia coli K-12. Further determinative testing of se-
OYSTER LIGAMENT GLIDING BACTERIA
1129
TABLE 1. Geographic source of seven CLB strains isolated from hinge ligaments of cultured juvenile C. gigas and their estimated percent contributions to the total ligament-associated microflora based on the ratio of CLB CFU to total CFU %
CLB isolate
Contribution
Geographic source of oysters
C1B-2 85-48-1 C4-1 WB1-A WB1-B1 K17-1 K17-2
30.5
