Brown Ring Disease of Manila Clams
Category 1 (Not Reported in Canada)
Common, generally accepted names of the organism or disease agent
Brown ring disease of Manila clams, BRD.
Scientific name or taxonomic affiliation
Vibrio tapetis, initially called Vibrio Predominant 1 (Vibrio P1 group or strain) (Paillard and Maes 1990a and b), is characterised as a fermentative, Gram-negative, motile, nonsporulating curved rod that is oxidase-positive and growth inhibited by vibriostat 0/129. It can be differentiated from other species of Vibrio by growth at 4 °C; no growth at temperatures greater than 22°C and at salinities greater than 5% NaCl; no production of arginine dehydrolase, lysine decarboxylase and ornithine decarboxylase; and Voges-Proskauer reaction (Borrego et al. 1996a and b). The virulence of isolates varied depending on bacterial strain and clam species assayed.
Entire European Atlantic coast to North Africa including coasts of France, Portugal, Spain, Italy, England, Ireland and Norway. Occasionally detected in the Mediterranean and Adriatic seas (Paillard 2004, Paillard et al. 2008). In 2006, BRD caused by V. tapetis was reported from Venerupis (=Ruditapes) philippinarum on the west coast of Korea (Park et al. 2006).
Brown ring disease has been detected in wild populations of Venerupis (=Tapes, =Ruditapes) philippinarum, Venerupis aurea, Tapes(=Ruditapes) decussatus and Dosinia exoleta. Of these clams, V. philippinarum is generally most sensitive to the disease and is the only species known to experience associated mortalities. However, V. tapetis has also been isolated from Cerastoderma edule (which shows no signs of disease) in Quiberon, France (Maes and Paillard 1992) and experimentally transmitted to Tapes rhomboïdes. Vibrio tapetis-like strains have also been detected in cultured fish during mortality outbreaks: corkwing wrasse, Crenilabrus (=Symphodus) melops, in Norway (Jensen et al. 2003, Paillard et al. 2008) and halibut, Hippoglossus hippoglossus, in Scotland (Reid et al. 2003a).
Impact on the host
Vibrio tapetis adheres to and progressively colonizes the surface of the periostracal lamina at the mantle edge of the shell. The resulting disruption to the periostracal lamina causes the anomalous deposition of periostracum around the inner shell surface resulting in an accumulation of brown organic material which is symptomatic of the disease. Infection also disturbs the normal calcification process involved in shell deposition. Haemolymph parameters of haemocyte abundance and peptidase activity were affected by experimental challenge (inoculation). Allam et al. (2002) concluded that clam mortality associated with BRD likely resulted from the penetration of V. tapetis into the extrapallial space through disruption of the periostracal lamina. From the extrapallial space, the bacteria eventually penetrate through the mantle epithelium and into the soft tissues where bacterial proliferation can cause severe damage and subsequent death. Also, infected clams had a significant decrease in glycogen suggesting that mass mortalities could be exacerbated by the degeneration of metabolic activity (Plana et al. 1996). Tissue lesions were not observed systemically until advanced stages of the disease when alterations in the digestive gland and mantle were detected (Plana and Le Pennec 1991, Paillard 1992). A negative correlation between the extent (level) of BRD and the condition index of clams (ration of soft tissue wet weight to shell dry weight) and energy budget (maintenance costs) was noted (Park et al. 2006 and Sante Marie et al. 2008, respectively). Consequently, infected clams often show significant weight loss, depressed defence-associated activities and ultimately mass mortality which usually occur during the winter. Clams can recover from the disease by covering the organic brown deposit by shell material by a process called nacrezation (Paillard 1992).
Since BRD was first detected in 1987, it has been associated with mass mortalities on various cultured clam beds along the west coast of France (Paillard et al. 1989) and then spread along the European Atlantic coast (Flassch et al. 1992). In Brittany, France, clam culture was completely decimated and intense venerid culture was not reinitiated in this area (Paillard 2004). In Korea, BRD and isolation of V. tapetis was reported from V. philippinarum collected from an area that experienced mass mortalities and dramatic declines in clam harvests since 1993 (Park et al. 2006). Although these losses in clam production were linked with unusually high levels of Perkinsus olseni, the possible involvement of other pathogens such as V. tapetis must be assessed (Park et al. 2006). A positive relation between a higher parasite load and signs of BRD was also reported in clams from areas of southern Europe (Figueras et al. 1996).
Allam et al. (2001) demonstrated that V. philippinarum from southern Puget Sound, Washington, USA were more resistant to BRD than the same species of clam from the Bay of Brest, Brittany, France and that T. decussatus also collected from the Bay of Brest was more resistant than both stocks of V. philippinarum. Their results suggested that resistance to BRD may be related to the concentration of granular haemocytes and the phagocytic activity of the haemocytes. Several additional studies have examined defence factors of V. philippinarum and other bivalves to V. tapetis and other Vibrio spp. (Oubella et al. 1993, 1994, 1996; Allam and Paillard 1998; Allam and Ford 2006; Allam et al. 2000a, 2000b, 2006; Choquet et al. 2003; Reid et al. 2003b). Castro et al. (2002) reported that diffusible extracellular products from some isolates of Vibrio tubiashii inhibited the growth of 27 strains of V. tapetis.
Gross Observations: Stunted growth and a brownish coloured conchiolin deposit along the leading edge of the mantle adhering to the inner surface of the shell between the pallial line and the outer edge of the shell in juvenile and adult clams. The extent of BRD can be assessed against a classification system described by Paillard and Maes (1994). Note that conchiolin deposits are a defence response of bivalves and not exclusively due to V. tapetis. Thus, gross signs can be diagnosed as BRD only if V. tapetis has been detected in the diseased bivalve (Mortensen et al. 2007).
Histology: Vibrio tapetis can be observed within the periostracal lamina in decalcified sections of shell and disease mantle edge prepared for histological examination and stained with haematoxylin and eosin stain (Paillard and Maes 1995b). Visibility of the bacteria can be enhanced by an indirect immuno-fluorescence technique using polycolnal antibodies against V. tapetis (Paillard 1992; Paillard and Maes 1995a, 1995b; Allam et al. 1996, 2000b).
Electron Microscopy: Alterations of the periostracal lamina with an additional layer, rich in vacuoles and cell debris, between the periostracum and fibrous matrix layer. From 1 to 4 weeks after challenge with V. tapetis, the deposit becomes progressively wider, thicker, and invaded by many bacteria (Paillard and Maes 1995b).
Immunological Assay: Polyclonal antibodies against a V. tapetis reference strain were successfully used in Spain, France and England to detect and identify this bacterium using the slide agglutination test, indirect immuno-fluorescence technique, indirect dot-blot immuno-enzymatic assay and enzyme-linked immunoassay (ELISA) (Paillard 2004). The ELISA can be used to assay extrapallial fluids and haemolymph from clams with gross signs of disease (Noel et al. 1996, Allam et al. 2002). The ELISA detection limit is about 5 x 104 colony forming units per milliliter of extrapallial fluids (Paillard 2004). Only the indirect dot-blot immuno-enzymatic assay showed a weak cross reactivity with a few other species of Vibrio isolated from diseased clams (Castro et al. 1995). Two isolates from Tapes decussatus did not show agglutination with V. tapetis antiserum although they were biochemically similar to V. tapetis (Novoa et al. 1998). Antigenic characterisations indicated that V. tapetis constitute a homogenous group and differ in protein and lipopolysaccharide patterns from other Vibrio species isolated from diseased clams (Castro et al. 1996, Paillard 2004).
Culture: Vibrio bacteria can be isolated on a classical marine agar medium (2216 E, Difco). Selection of potential V. tapetis can be achieved by subculturing isolates on selective media based on four key characteristics: non-utilisation of sucrose and mannitol, growth on TCBS (thiosulfate citrate bile sucrose) medium, and failure to grow above 27 °C. Colonies meeting these four criteria should be isolated and purified and then subjected to phenotypic, serological and molecular characterisation (Paillard 2004). Note that various strains/species of Vibrio have been isolated from V. philippinarum with signs of BRD in Spain (Castro et al. 1992, 2002). The antagonistic relationship established between V. tapetis and the Vibrio spp. microbiota of clams may explain the failure of isolation in plating medium of V. tapetis from BRD-affected clams on the south Atlantic coast of Spain (Castro et al. 2002). Also, isolation of V. tapetis using bacteriological methods is time consuming and may not provide a clear diagnosis because the bacterium grows slowly and is generally not predominant within the total heterotrophic microflora (Mortensen et al. 2007).
Cultures are infective to V. philippinarum when injected into the pallial cavity resulting in the development of clinical signs by 4 weeks after injection. Vibrio tapetis is characterised as a fermentative, Gram-negative, motile, non-sporulating curved rod that is oxidase-positive, with growth inhibited by vibriostatic 0/129. It can be differentiated from other species of Vibrio by growth at 4°C; no growth at temperatures greater than 27 °C and salinities greater than 5% NaCl; no production of arginine dehydrolase, lysine decarboxylase and ornithine decarboxylase; and a positive Voges-Proskauer reaction (Paillard 2004).
DNA Probes: The sequence of at least 4 individual genes (16S rDNA, gyrase, rpoD, recA) can be used to confirm the identity of V. tapetis. A polymerase chain reaction (PCR) detection method was developed for V. tapetis using dot blot hybridization and a species-specific primer that targets the 16S rDNA (Paillard et al. 2006, Park et al. 2006, Rodríguez et al. 2006). The detection limit is about 102 colony forming units per milliliter of clam extrapallial fluids or tissue homogenates (Paillard 2004, Paillard et al. 2006). The DNA sequence of the PCR product should be compared with genomic DNA sequences for V. tapetis available in GenBank and possibly using the Vibrionaceae Multi Locus Sequence Analysis website developed by Marcos Renato R. Araujo and Fabiano L. Thompson, hosted at Campinas State University (Thompson et al. 2005).
Restriction fragment length polymorphism (RFLP) patterns of plasmid DNA from several strains of V. tapetis indicated that the strain from fish (corkwing wrasse) in Norway differed from strains isolated from bivalves in France and Britain (Le Chevalier et al. 2003). Also, different molecular assays including ribotyping, pulsed-field gel electrophoresis and PCR-based typing methods (enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR) revealed genetic groups of V. tapetis that strongly correlated to host origin (Romalde et al. 2002, Rodríguez et al. 2006).
Note: After assessing four techniques for the diagnosis of BRD (shell valve analysis, microbial isolation and characterisation, the PCR assay of Rodríguez et al. (2006), and the PCR assay of Paillard et al. (2006)), Drummond et al. (2006) determined that none of the four techniques was sufficient on its own for effective BRD diagnosis. The combination of shell valve analysis with the PCR assay of Paillard et al. (2006) proved to be the most sensitive and rapid of those tested (Drummond et al. 2006).
Methods of control
Assessing the health of clam seed (juveniles) prior to planting and reducing planting density may be beneficial (Flassch et al. 1992). Laboratory based studies demonstrated that stress caused by the environment was related to disease development. Specifically, Reid et al. (2003b) found that disease prevalence was higher at low salinities (20‰) than at high salinities (40‰). Also, the type of substrate in clam beds significantly affects the disease. Vibrio tapetis has been detected in the marine environment (water and sediment) and in various tissues of clams (extrapallial and pallial fluids, and haemolymph as well as clam faeces and pseudofaeses (Mortensen et al. 2007). Laboratory studies indicated that the most probable transmission route is by means of direct contact with infected clams (Martinez-Manzanares et al. 1998) and by faeces from diseased clams (Mortensen et al. 2007). The observation that the disease is absent in enzootic areas with high summer temperatures was supported by experimental evidence that showed low temperatures (14°C or less) strongly affected BRD development (Paillard et al. 2004) and a recovery process occurring at temperatures of 21°C or higher. Allam et al. (1999) and Paillard et al. (1999, 2004) indicated that warm temperatures increased potential cellular defence mechanism, especially phagocytosis of V. tapetis by haemocytes in the extrapallial fluid of clams as well as reduced the growth of V. tapetis. Other environmental factors may limit the development of BRD. For example, V. tapetis has been detected in V. philippinarum from a number of coastal bays in the north and west of Ireland with no evidence of BRD but V. philippinarum from Ireland were shown to be susceptible to BRD in the laboratory (Drummond et al. 2007).
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Bower, S.M. (2010): Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Brown Ring Disease of Manila Clams.
Date last revised: April 2010
Comments to Susan Bower
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