The susceptibility of Irish-grown and Galician-grown Manila clams, Ruditapes philippinarum, to Vibrio tapetis and Brown Ring Disease.

Brown Ring Disease (BRD), which affects the Manila clam in Europe, is caused by the bacterium, Vibrio tapetis. BRD has been diagnosed in Ireland on only one occasion (1997) although the aetiological agent has recently been detected in apparently healthy Manila clams from a number of sites around the Irish coast. The present work investigated the susceptibilities to BRD of two stocks of Manila clams, one from Ireland and the second from Galicia, north-western Spain, where BRD has been reported on a number of occasions. Exposure of the clams was by addition of V. tapetis to the holding waters. Development of BRD was assessed by the appearance of brown ring signs on the host shells, by bacterial isolation and characterization, and by detection of the bacterium by PCR. The pathogen was recovered from infected individuals and confirmed as V. tapetis by biochemical tests and a slide agglutination test. Galician clams experienced significantly higher mortalities, BRD prevalences and V. tapetis levels than Irish clams. Background infection with V. tapetis in the control stocks prevented conclusions being drawn on comparative susceptibility of the two stocks. Irish clams were significantly affected by the experimental challenge, as demonstrated by the development of BRD and an increase in V. tapetis levels. Results illustrate the vulnerability of Irish clams to BRD and have implications for the movement and transfer of clam seed in Ireland.

Evidence of retroviral etiology for disseminated neoplasia in cockles (Cerastoderma edule).

Epizootiologic outbreaks of disseminated neoplasia have been reported in association with massive mortalities of various bivalve species. In cockles, Cerastoderma edule, this pathological condition was described in Ireland and France. Since 1997, different populations affected by this pathology have been detected in Galicia (NW Spain). Transmission electron microscopy allowed the visualization of virus-like particles in neoplastic cells, resembling a retrovirus-like agent. To confirm this hypothesis, we used a commercial kit for detection and quantification of reverse transcriptase (RT) activity, based on the use of bromo-deoxyuridine triphosphate (BrdUTP) and a BrdU binding antibody conjugated to alkaline phosphatase. In addition, we developed a product-enhanced RT assay using RNA of hepatitis A virus as a template. These two assays showed positive RT activity in 90.9 and 81.8% of samples, respectively, from cockles displaying disseminated neoplasia as determined by light microscopy. These results strongly support the hypothesis of retroviral etiology for this pathological condition.

Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD.

Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction- (PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future.

Species-specific polymerase chain reaction primer sets for the diagnosis of Tenacibaculum maritimum infection.

In this study the specificity and sensitivity of 2 primer pairs, MAR1-MAR2 and Mar1-Mar2, for the detection of Tenacibaculum maritimum were evaluated in parallel using 79 T. maritimum strains isolated from different fish species, as well as 53 representatives of related and unrelated bacterial species. Both primer pairs were species-specific for T. maritimum, since no amplification products were obtained from chromosomal DNA of the non-T. maritimum bacteria tested. However, whereas MAR1-MAR2 identified all the T. maritimum strains studied, producing a unique and clear PCR band of the expected 1088 bp length, the Marl-Mar2 primer pair failed to amplify the 400 bp specific band in 3 sole isolates. To verify if these strains belonged to T. maritimum species, 2 endonucleases (PvuI and SacII) were selected as the most adequate enzymes to confirm the specificity of the MAR1-MAR2 amplified fragment. The digestion patterns obtained with both endonucleases supported the assignation of all the strains to T. maritimum. The sensitivity of both PCR detection methods was also different, showing a reduction of sensitivity in at least one order of magnitude of the Marl-Mar2 primer pair in comparison with MAR1-MAR2. When the MAR-MAR2 PCR protocol was applied to different seeded turbot tissues, the detection limit was 10(2) to 10(4) T. maritimum cells per reaction. In addition, a nested PCR protocol for detection of this pathogens based on MAR1-MAR2 was developed, which increased the sensitivity by approximately 2 orders of magnitude, ranging from 1 to 250 T. maritimum cells per reaction depending on the tissue employed. The tissues that allowed the most easy detection of T. maritimum were the skin and mucus. Based on the findings reported here, we propose the nested PCR protocol as the most adequate for an accurate detection of T. maritimum in diagnostic pathology as well as in epidemiological studies of gliding bacterial disease of marine fish.