Comparison of techniques for diagnosis of brown ring disease and detection of Vibrio tapetis in the Manila clam, Venerupis (Ruditapes) philippinarum.

ABSTRACT The bacterium, Vibrio tapetis, is the aetiological agent of Brown Ring Disease (BRD), which affects the Manila clam, Venerupis (Ruditapes) philippinarum. Two PCR assays for detection of V. tapetis were applied to a sample of 52 Manila clams, and evaluated in comparison with the more traditional V. tapetis detection method of microbiological isolation and characterization, as well as the traditional BRD diagnostic technique of shell valve analysis. The pathogen was detected in 15.4% of the sample using the PCR assay of Rodriguez et al. (2003, 2006) in 50% of the sample using the PCR assay of Paillard et al. (2006) and in 36.5% of the sample by microbiological methods. Whereas shell valve analysis was the least sensitive technique, detecting BRD in 7.7% of the sample, it was an essential diagnostic tool because it was the only technique that identified the disease, rather than the aetiological agent. None of the four techniques was sufficient on its own for effective BRD diagnosis; rather various combinations of two techniques were the minimum required. The combination of shell valve analysis with the assay of Paillard et al. (2006) proved to be the most sensitive and rapid of those tested. Shell valve analysis was the most time-efficient and cost- effective technique, whereas microbiological characterization was the most time-consuming, and PCR detection, using either set of primers, the most expensive. These results may need to be considered in light of diagnosis and management of BRD.

KEY WORDS: brown ring disease, Vibrio tapetis, Venerupis (Ruditapes) philippinarum, diagnostics, PCR, microbiology

INTRODUCTION

The Manila clam, Venerupis (Ruditapes) philippinarum (Adams & Reeve, 1850), is the second most important cultivated mollusc species in the world, contributing 20% of the total world aquaculture production of molluscs (FAO 2002). Globally, production of the Manila clam has increased by 60% from 1997 to 2002 (FAO 2002). In Europe, production takes place predominantly in Italy, France, Spain and the United Kingdom. Ireland's Manila clam production, though small, has been steadily increasing in recent years, with an output of 181 tons achieved in 2004 (Parsons 2005). The increase in production worldwide, with its concentration of large quantities of clams in confined areas, has facilitated the proliferation of pathogens such as Perkinsus sp. (in Asia) (Park & Choi 2001, Park et al. 2006a) and Vibrio tapetis (in Europe and Asia) (Paillard 2004, Park et al. 2006b). Consequently, there is a need for rapid, effective and accurate techniques for the diagnosis of Manila clam diseases.

Brown ring disease (BRD) caused by the bacterial pathogen, V. tapetis, is not regulated for disease control under existing European Union (EU) legislation, nor is it a disease notifiable to the Office International des Epizooties (OIE). Nevertheless, since the early 1990s, it has been responsible for mass mortalities in the clam cultivation industry in Europe, and has been diagnosed from the majority of clam cultivation areas (Paillard & Maes 1990, Castro et al. 1992, Paillard et al. 1994, Robledo et al. 1994, Castro et al. 1995, Figueras et al. 1996, Castro et al. 1997, Novoa et al. 1998, Clarke 1999, Allam et al. 2000b).

Diagnostic methods for BRD have traditionally involved examination of the interior shell valves for analysis of conchiolin deposition, as described by Paillard & Maes (1994). Histopathology is not useful for BRD diagnosis because tissue lesions are not systematically observed in diseased clams (Paillard et al. 1994) and alterations of the digestive gland and mantle are observed only in the more advanced stages of the disease (Plana & Le Pennec 1991, Paillard et al. 1994, Paillard 2004). Notwithstanding the analysis of shell valves for BRD signs, all other diagnostic techniques focus on detection of the aetiological agent, V. tapetis, rather than the disease itself. Bacteriological isolation procedures are in place and have resulted in the isolation of V. tapetis from BRD-affected clams from France, England and Galicia, northwestern Spain (Paillard & Maes 1990, Borrego et al. 1996, Novoa et al. 1998, Allam et al. 2000b). An immunological assay was developed in southwestern Spain, which demonstrated that V. tapetis was the aetiological agent of BRD in this region (Castro et al. 1997). An enzyme-linked immunoassay (ELISA), using monoclonal antibodies, which react specifically with V. tapetis, is also available for identification of the bacterium (Noel et al. 1996).

Molecular techniques are now available for detection of V. tapetis, in the form of two polymerase chain reaction (PCR) assays, each of which amplifies a specific target region of V. tapetis DNA (Rodriguez et al. 2003, 2006, Paillard et al. 2006). Previous studies on other pathogens in other bivalves have indicated that PCR amplification has the ability to detect a higher prevalence of a pathogen than traditional diagnostic methods (Carnegie et al. 2000, Harwood et al. 2004, Lynch et al. 2006).

The aims of this study were to apply V. tapetis-specific PCR assays for V. tapetis detection and to evaluate them against the more traditional detection method of microbiological isolation and characterization, as well as the traditional BRD diagnostic technique of shell valve analysis; and to assess which technique, or combination of techniques, is most suitable for BRD diagnosis and V. tapetis detection in terms of efficiency of detection, time required and cost-effectiveness.

MATERIALS AND METHODS

Clams

A sample of 52 clams, of 25-30 mm in length, was obtained from Mulroy Bay, Donegal, northwest Ireland, in April 2005, from a cultured stock in which BRD was suspected. Clams were placed in a cold room at 4[degrees]C prior to dissection, to encourage relaxation of their adductor muscles, for easier opening of the shell valves. After opening of the valves, over 200 [micro]L of extrapallial fluid (EPF) was extracted from each individual by inserting a needle between the mantle and interior shell valve. The EPF was placed in a labeled eppendorf tube. The soft parts of the animal were discarded. The shell valves were retained, labeled and left to dry for 24 h.

Macroscopic and Microscopic Analysis of BRD on Shell Valves

The stage of progression of BRD on the shell valves was monitored according to the classification system of Paillard & Maes (1994), in which the syndrome is characterized by two stages, which may occur simultaneously in a given individual: a conchiolin deposit stage (CDS), in which layers of an organic conchiolin deposit are laid down between the mantle edge and periostracal lamina, and a shell repair stage (SRS), in which the clam lays down layers of shell on top of the conchiolin deposit, for repair and recovery from the disease. CDS index is based on the use of a scale, which takes into account the extent and thickness of the brown ring deposit. For example, CDS 1 (Stage 1) is not visible to the naked eye and can be seen only with a microscope. CDS 2 (Stage 2) corresponds to a limited deposit and CDS 3 through to CDS 7 to more extensive deposits on the inner shell surfaces. Three stages of shell repair are defined: SRS 1 corresponds to the initial accumulation of calcified material; SRS 2 to the partial covering of the deposit by wide calcified plates and SRS 3 to the completely covering of the deposit by shell layers. When the processes of conchiolin deposition (CDS) and shell repair (SRS) are considered together, three phases of the disease are defined: Phase 1 corresponds to development of the disease and is characterized only by accumulation of conchiolin; Phase 2 is characterized by the simultaneous occurrence of both processes in a given individual; and Phase 3 corresponds to the end of the recovery process, when the individual has completely covered the conchiolin deposit (Paillard & Maes 1994).

Microbiological Isolation and Characterization of Vibrio tapetis

One-hundred micro liters of EPF from each individual was spread, using a "hockey stick" spreader, on a Thiosulfate Citrate Bile Sucrose (TCBS) (Oxoid Ltd.) agar plate and incubated for 24 h at 18[degrees]C. TCBS agar is selective for Vibrio species: V. tapetis is saccharose-negative on TCBS agar and, as such, appears as a green colony. Four green, saccharose-negative colonies from each TCBS plate were selected and isolated on marine agar to obtain pure cultures. They were subsequently stored in vials of 15% glycerol/85% marine broth at -80[degrees]C until further characterization. All the remaining colonies were collected from each TCBS plate, placed in eppendorf tubes containing 1 mL sterile distilled water (in order that each tube contained the colonies of an individual clam) and stored at -20[degrees]C until further analysis by PCR.

Isolated colonies were characterized by a series of standard morphological, physiological and biochemical tests (Cowan 1974, Smibert & Krieg 1981, West & Colwell 1984, Borrego et al. 1996). All biochemical tests were performed using marine agar (DIFCO) unless otherwise stated and the incubation temperature was 18 [+ or -] 1[degrees]C unless otherwise stated. The tests, and the criteria for characterization as V. tapetis, are listed in Table 1. The ingredients and recipes used in each test are described in Appendix 3. The type V. tapetis strain, CECT 4600 was used as a positive control for all tests. Isolates, which did not meet the V. tapetis criteria, were eliminated from further tests as results became available. Colonies that met the requirements for characterization as V. tapetis, had their identities confirmed by PCR detection of V. tapetis DNA (Rodriguez et al. 2003, 2006).

PCR Detection of Vibrio tapetis

Two PCR assays were used for the detection of V. tapetis: the first, developed by Rodriguez et al. (2003, 2006), uses template DNA extracted from bacterial colonies, and the second, developed by Paillard et al. (2006), employs template DNA extracted from the EPF of clams.

Rodriguez Assay (2003, 2006)

Colonies from TCBS plates, in suspension in sterile distilled water, were thawed. DNA from individual clams was extracted using InstaGene Matrix (BIO-RAD) according to manufacturers' instructions and stored at -20[degrees]C until required for PCR reactions. PCR amplification of template DNA (at a concentration of 1-3 [micro]g) was undertaken using one Pure Taq Ready-To-Go PCR Bead (Amersham Biosciences) in every reaction: each bead contained 1.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1 mM Mg[Cl.sub.2] and 200 mN each dNTP. For a 25-[micro]L reaction, the bead was combined with 2 pmol (1 [micro]L) of forward primer, 2 pmol (1 [micro]L) of reverse primer, 22 [micro]L of sterile distilled water and 1 [micro]L of DNA template solution. DNA from V. tapetis CECT 4600 was used as a positive control and distilled deionized water was used as a negative control. Amplification was performed in a Hybaid thermal cycler programmed as follows: an initial denaturation step at 94[degrees]C for 3 min; 35 cycles of 94[degrees]C for 60 sec, 64[degrees]C for 60 sec and 72[degrees]C for 90 sec; and a final cycle of 72[degrees]C for 5 min. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and photographed under UV light. A 50-2,000 bp ladder (Sigma Chemical Company) was used as a molecular marker. In cases where either the positive or negative control was not confirmed by PCR as positive and negative respectively, the results were discarded and the analysis repeated. PCR results were graded as either negative or positive.

Paillard Assay (2006)

DNA was extracted from 100 [micro]L of EPF, of each of the 52 clams, using the chelex method (Saulnier et al. 2000). PCR reactions were carried out in a total volume of 50 [micro]L, including 10 mM Tris-Cl, 50 mM KCl containing 200 [micro]M of each dNTP, 1.5 mM Mg[Cl.sub.2], 400 [micro]M of each primer, VtF and VtR, 0.2U of Taq DNA polymerase (Sigma) and 200 ng of genomic DNA (5 [micro]L of template DNA). DNA from V. tapetis CECT 4600 was used as a positive control and distilled deionized water was used as a negative control. PCR amplification was performed in a Hybaid thermal cycler and the thermal cycle consisted of an initial denaturation step at 94[degrees]C for 3 min, followed by 35 cycles of 94[degrees]C for 60 sec, 63[degrees]C for 60 sec and 72[degrees]C for 45 sec. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and photographed under UV light. A 50-2,000 bp ladder (Sigma Chemical Company) was used as a molecular marker. In cases where either the positive or negative control was not confirmed by PCR as positive and negative respectively, the results were discarded and the analysis repeated. PCR results were graded as either negative or positive.

Comparison of Techniques for Diagnosis of BRD and Detection of Vibrio tapetis

The results of screening for BRD by shell analysis, V. tapetis detection by bacteriological techniques and V. tapetis detection by two PCR assays were subsequently compared. A chi-square test was used to ascertain if there were significant differences in the detection rate between each diagnostic technique.

RESULTS

Macroscopic and Microscopic Analysis of BRD on Shell Valves

Prevalence of BRD in the sample of 52 clams was 7.7% as classified by the system of Paillard & Maes (1994): 48 did not exhibit BRD symptoms. Of the four symptomatic clams, one displayed CDS 2 and no shell repair, and so was in Phase 1 of BRD; two clams exhibited CDS 3 and SRS 1, and thus were in Phase 2 of BRD and the fourth clam showed CDS 5 and SRS 1 and was thus also in Phase 2 of BRD. Two (50%) were BRD/V. tapetis-positive by other diagnostic techniques: Table 2 shows that none of the shell analysis positives were detected by microbiology; one was detected by the Rodriguez assay and two were detected by the Paillard assay. There appeared to be no correlation between stage of disease and detection by other techniques: the clam displaying CDS 5 and one of the clams displaying CDS 3 were not detected as positive by any of the other methods; the second clam exhibiting CDS 3 was positive by both PCR assays; whereas the individual showing CDS 2 was positive by the Paillard assay. Two (50%) of the animals positive by shell analysis were not detected as positive by any other technique. Of 48 animals negative by shell analysis, 31 (64.6%) were deemed positive by one or more of the other techniques.

Microbiological Isolation and Characterization of Vibrio tapetis

From 52 TCBS plates, each plate representing an individual clam, a total of 208 suspected Vibrio colonies were isolated. After salt and temperature tests (Table 1), 162 colonies were eliminated. Following characterization by the remaining tests, a total of 23 isolates from 19 individuals were identified as V. tapetis. Sixteen of the 23 (70%) isolates were derived from individual clams, whereas two isolates originated from each of two clams, and three isolates originated from one individual. Overall, V. tapelis was detected in 19 individuals (36.5%) of the sample, 13 (68%) of which were also detected by at least one other technique. Table 2 shows that none of the microbiology V. tapetis-positive individuals was positive by BRD shell analysis, three were positive by the Rodriguez assay and 13 by the Paillard assay. Six (32%) of the microbiology positives were not detected by any of the other techniques and 17 (52%) microbiology negatives were deemed positive by one or more techniques.

PCR Detection of Vibrio tapetis

Rodriguez Assay (2003; 2006)

V. tapetis was detected in 15.4% (8 individuals) of the sample. Six (75%) of these positives were also positive by other diagnostic methods, of which, one was detected by BRD shell analysis; three by microbiology; and six by the Paillard assay (Table 2). Twenty-six (59%) of the negatives by the Rodriguez assay were not confirmed by any of the other techniques.

Paillard Assay (2006)

V. tapetis was detected in 50% (26 individuals) of the sample. Seventeen (65%) of the 26 positives were confirmed by other diagnostic methods, of which 2 were detected by BRD shell analysis; 13 by microbiology and 6 by the Rodriguez assay (Table 2). Nine (35%) Paillard PCR-positives were not detected by any other technique and 10 (39%), which were negative by the Paillard assay were detected as positive by one or more of the other techniques.

Comparison of Techniques for Diagnosis of BRD and Detection of Vibrio tapetis

The four techniques detected BRD and V. tapetis at different levels within the same sample (Table 3) and there was a significant difference between the numbers of BRD/V. tapetis positives and negatives detected by the four methods ([[chi square].sub.3] = 29.45, P < 0.001). BRD prevalence detected by shell analysis was 7.7%; V. tapetis detection by isolation and characterization was 36.5%; and by the primers of Rodriguez et al. (2003, 2006) and Paillard et al. (2006) was 15.4% and 50% respectively. When two diagnostic techniques were applied, detection levels ranging from 21.2% to 61.5% were revealed: a combination of BRD shell analysis and PCR by Rodriguez et al. (2003; 2006) produced the lowest detection levels at 21.2%, whereas the combination of microbiological characterization and PCR by Paillard et al. (2006) produced the highest detection levels at 61.5%. Applications of three techniques led to detection levels ranging from 51.9% to 65.4%, and finally, a combination of all four techniques detected the highest levels of BRD/ V. tapetis (i.e., 69.2%).

Shell valve analysis was found to be the most cost-efficient technique: only a microscope was required. Microbiological methods entailed the cost of a wide variety of reagents and media; but the most expensive techniques were V. tapetis detection by PCR. With regards to time-efficiency, BRD diagnosis by shell analysis of 52 clams was completed in less than two hours. Only four individuals were BRD-positive and the time taken would be expected to be longer in samples containing a higher percentage of diseased individuals, because of the time required to establish the CDS and SRS of each individual. PCR detection by the Paillard et al. (2006) primer was the next most time-efficient method, with the whole process from DNA extraction through to the visualization of PCR products on an electrophoretic gel taking less than 10 h. The Rodriguez et al. (2003; 2006) assay entailed growth of bacterial colonies on TCBS agar prior to DNA extraction and this added a further 24-48 h to the process. By far the most time-consuming technique was the bacteriological method, which required much preparatory and manipulative work. Additionally, V. tapetis colonies require approximately 24-48 h growth on agar media for isolation and the same time or longer for the characterization tests that follow. In this study, the procedure from colony isolation from the extrapallial fluid through to characterization as V. tapetis took 5-6 days to complete for any one bacterial isolate.

DISCUSSION

Developments in molecular biology have led to new methods for diagnosing shellfish diseases. The advantages of molecular techniques such as PCR are that they are potentially faster and more sensitive than methods such as culture, serology and histology that are traditionally used to identify shellfish diseases (Cunningham 2002). The currently available PCR primers for detection of V. tapetis in clam tissue differed significantly in detection rate with the primers by Rodriguez et al. (2003, 2006) detecting fewer positives (15.4%) than those by Paillard et al. (2006) (50%). Optimization of reaction conditions for the Rodriguez primers was required prior to use, but not for the Paillard primers. However, this optimization may have been necessary because of geographic variation in V. tapetis strain (Romalde et al. 2002, Paillard et al. 2006) and perhaps the strain detected in Mulroy Bay was more amenable to detection by the Paillard assay than by the Rodriguez assay. The two sets of primers also differed with respect to their source DNA material: the Paillard primers were applied to the clam's EPF, whereas the Rodriguez primers were applied to DNA from bacterial colonies. Culture of V. tapetis is often met with variable success, depending on the stability of culture conditions and the composition of the microbiota and could possibly have compounded the lower detection rate of the Rodriguez assay. Overall, V. tapetis detection by PCR was the fastest technique used in the current study, thus making it suitable for situations in which a rapid diagnosis is required. Because PCR is used to amplify certain regions of DNA, it can also achieve significant increases in the sensitivity of detection (McPherson et al. 1991). This was demonstrated by the Paillard assay, which was the single most sensitive assay, capable of detecting V. tapetis levels down to [10.sup.2]cfu [mL.sup.-1] (Paillard et al. 2006).

In this study, nine PCR-positives by the Paillard assay and two by the Rodriguez assay went undetected by any one of the other techniques and could potentially have signified false positives. One of the limitations of PCR is that when used in disease diagnosis, false positives identifying DNA similar to, but not of, the target organism can occur, because of low specificity of the primers. To ensure the greatest possible specificity of PCR primers to the target DNA, DNA sequencing of PCR positives prior to routine use of the primers is recommended (Cunningham 2002). In the current study, PCR products obtained using the Paillard et al. (2006) primers were sequenced and found to be consistent with reference V. tapetis sequences from the European Molecular Biology Laboratory (EMBL) database, thereby suggesting that the nine PCR positives, detected by this assay only, reflected real V. tapetis DNA. In general, however, a PCR positive result alone is not considered enough to confirm the presence of a pathogen, and should be supported by other diagnostic methods before a confirmatory diagnosis is obtained (Walker & Subasinghe 2000).

Two individuals, who displayed brown conchiolin deposits, went undetected for V. tapetis by both PCR assays. It is possible that the conchiolin was deposited in response to an agent other than V. tapetis, because conchiolin deposition unassociated with V. tapetis has been observed in other bivalves (Sindermann 1990, Paillard 2004); this seems unlikely, however, as the pathogen was detected in other individuals within the current sample. Alternatively, the deposits may have persisted in these animals from the conchiolin deposit stage (developmental) of the disease, but the causative agent may have been reduced to nondetectable levels during the recovery stage: the shell repair process is associated with a decrease in V. tapetis burden (Allam et al. 1996). The PCR-negative results could also be explained by the pathogen being localized predominantly in tissues other than those targeted for DNA extraction and PCR. In this case, PCR analysis was carried out on the EPF of individuals, but V. tapetis is also known to heavily colonize the periostracal lamina and the mantle surface (Paillard & Maes 1995a, 1995b; Allam et al. 1996, Paillard 2004). It is possible that in these two individuals, the pathogen was localized predominantly in the periostracal lamina or on the mantle surface, neither of which was analyzed by PCR and therefore went undetected by both assays.

Isolation and characterization of V. tapetis by bacteriological methods produced detection levels of 36.5%, the second highest detection level of the single techniques. However, the lack of sensitivity of the technique is highlighted by the fact that a significant proportion of its negatives (17 of 33) were detected as positive by one or more of the other techniques. Problems associated with the microbiological isolation and characterization of V. tapetis have been described in previous studies. In southwestern Spain, microbiological methods failed to detect V. tapetis in cultured Manila clams displaying disease signs identical to those of BRD (Castro et al. 1992, 1995; 1997). In bacteriological analyses of the clams' conchiolin deposits, none of the isolates were phenotypically typed as V. tapetis (Castro et al. 1997). The pathogen was subsequently detected by an indirect immunofluorescence technique and the authors suggested that lower pathogen levels, detected in V. philippinarum from this region in comparison with BRD-affected clams from other regions, were the reason that standard microbiological methods failed to isolate the pathogen (Castro et al. 1997). Similarly, Paillard (2004) stated that isolation of V. tapetis might not provide a clear diagnosis because the bacterium grows slowly and is generally nonpredominant within the total heterotrophic microflora. Immunological techniques for the detection of bacterial pathogens in other species have previously been found to be more sensitive than classical microbiological tests (Bernoth 1991, Lee & Gordon 1987).

Nevertheless, isolation of the causative agent is often essential in diseases of a bacterial etiology. Microbiological detection proved to be the most time-consuming and labor-intensive of the techniques in the present study. In epidemiological studies, involving large sample sizes of clams, the time taken and labor required for completion of microbiological tests would render this technique unsuitable. There are commercial kits available, such as the API 20E (BioMtrieux, France), for the rapid identification of Vibrios, but whereas previous studies have found them to be effective for V. tapetis identification (Allam et al. 2000b, Jensen et al. 2003), the authors of the current study found them unreliable (unpubl. data).

BRD diagnosis has traditionally been achieved by analysis of the brown conchiolin deposit on the shell valves (Paillard & Maes 1994, Allam et al. 1996, Figueras et al. 1996, Paillard et al. 1997, Novoa et al. 1998, Allam et al. 2000a, 2000b; 2001, Reid et al. 2003b, Paillard et al. 2004, Soudant et al. 2004). Whereas it proved to be the least sensitive technique in the current study, failing to detect many positives detected by the other techniques, it is essential for BRD diagnosis: it is the only technique of those used in this study, which detected the actual disease, as opposed to the aetiological agent, which is detected by PCR detection and microbiological isolation. Shell analysis is also useful for classification of BRD into its stages, and thus its assessment of how chronic the infection is and the state of recovery in an individual (Paillard & Maes 1994).

In the current sample, just four animals were BRD-positive as detected by shell analysis, yet up to 32 animals were positive for V. tapetis (by a combination of the other techniques). This suggests that there was far more V. tapetis than BRD in the sample, and that this may also be true of V. tapetis and BRD in the environment. The bacterium has already been detected in other clam species (e.g., R. decussatus and Venerupis aurea; Maes & Paillard 1992, Novoa et al. 1998) and in other animals such as the cockle, Cerastoderma edule (Maes & Paillard 1992), the wrasse Symphodus melops (Jensen et al. 2003) and the halibut, Hippoglossus hippoglossus (Reid et al. 2003a), suggesting it is widespread in the environment in general. Like most Vibrios, it is likely to be an opportunistic pathogen, which does not always generate disease: V. philippinarum has been shown to harbor V. tapetis without experiencing BRD and associated mortalities (unpubl. data; Castro et al. 1997). Consequently, the detection of V. tapetis alone is not sufficient for a clear diagnosis of BRD, because it is not indicative of disease. The observation of clinical disease signs on the hosts' shells, even if only in small numbers as in the current sample, is necessary. Shell valve analysis is thus an essential tool in BRD diagnosis. An exception perhaps arises in cases of acute disease, in which the clam dies prior to manifestation of brown ring signs: this has occurred after injection of V. tapetis into the clam's extrapallial space or adductor muscle (Allam et al. 2002). There is no known documented evidence of such an occurrence in the wild.

The major drawback of BRD diagnosis by shell analysis alone is that the brown ring symptom is neither exclusive to BRD, nor exclusive to clams. Essentially, the brown conchiolin deposit is a defense mechanism and has been observed in many bivalves undergoing infection such as oysters (Perkins 1996, Cuif & Dauphin 1996) and abalone (Sherperd & Huchette 1997) See review by Paillard (2004). Parasites such as fungi, annelids and trematodes can disturb the extrapallial area by boring into or irritating the mantle epithelium (Sindermann 1990, Paillard et al. 1994). As such, brown rings on shell valves of clams are not exclusively caused by infection by V. tapetis. For this reason, a diagnosis of BRD based on shell analysis alone is insufficient; evidence of the aetiological agent is also required.

A combination of diagnostic techniques is therefore necessary for an accurate BRD diagnosis. The results here demonstrated the effectiveness of various combinations. The more techniques used in a particular combination did not necessarily lead to a higher detection rate, although the use of all four methods produced the highest detection levels. There was significant variability in detection rates when various combinations of two techniques were used, from 21.2% to 61.5% detection in the sample; each combination of two tests involving the Paillard assay produced detection levels above 50%, whereas those not using the assay produced detection levels below 50%. It would appear therefore that for sensitive detection, any combination of two techniques should involve the assay of Paillard et al. (2006). There was less variability in detection levels when combinations of three techniques were used: 51.9% to 65.4%. Diagnosis by a combination of three techniques provided detection levels not dissimilar to those produced from a combination of all techniques (i.e., 69.2%), indicating that it is unnecessary to use all four diagnostic methods in an analysis, unless the objective is to ensure, as far as possible, that clams are BRD/V. tapetis-negative.

In BRD management, the diagnostic methods, or combinations thereof, applied in an analysis of clams for BRD/V. tapetis will depend on a number of factors, specifically, the information required from the analysis; the number of individual clams in the sample to be analyzed; the time available and the financial resources available. A clam sample should first be assessed for overt BRD signs on the shell and then for the presence of the aetiological agent. The testing of clam seed for V. tapetis/BRD in the hatchery prior to purchase would best be accomplished by analyses for detection of the pathogen, as clinical BRD signs cannot be observed in juveniles under 2 mm (and in larvae) (Paillard et al. 2006).

In conclusion, the analyses of diagnostic techniques carried out in the current study demonstrate that none of the four techniques was sufficient on its own for an accurate diagnosis of BRD. BRD shell analysis was an essential technique and is recommended as the first technique in the analysis of a clam sample for BRD, followed by confirmation, either by PCR or microbiology, that the brown deposits are caused by V. tapetis. The combination of BRD shell analysis and PCR detection according to Paillard et al. (2006) was the most sensitive and rapid combination in this study. In instances where isolation of the aetiological agent is required and time is not a limiting factor, BRD shell analysis in conjunction with microbiological techniques is recommended. PCR detection is valuable if sensitivity for detection of lower pathogen levels is required. A comparison of the cost-efficiency of each method favored BRD shell analysis, followed in increasing cost by bacteriological isolation and characterization, and finally, either of the two sets of primers.

The results here were based on a single sample, and the sample size of 52 individuals is less than the 60 recommended by Simon & Schill (1984) for detection of an infection present at 5% prevalence in a population of 1 x [10.sup.6]. Additionally, the sample represents a single month, and therefore does not consider seasonal variation in BRD and V. tapetis prevalence. Further studies, which would encompass seasonal sampling, are required for validation of the detection efficiency of each technique. However, the results here provide a basis for deciding on diagnostic and detection techniques of choice for BRD and V. tapetis in different situations.

ACKNOWLEDGMENTS

The authors thank Dr. Jests L. Romalde and Dr. Christine Paillard for training in the use of PCR for V. tapetis screening and Dr. Jamie Coughlan and Dr. Eileen Dillane for sequencing work. This project was carried out with the support of the Marine Institute and the Marine RTDI Measure, Productive Sector Operational Programme, National Development Plan 2000-2006.

LITERATURE CITED

Allam, B., K. A. Ashton-Alcox & S. E. Ford. 2001. Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp. clams. Dev. Comp. Immunol. 25:365-375.

Allam, B., C. Paillard & M. Auffret. 2000a. Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tupetis. J. Invert. Pathol. 76:63-69.

Allam, B., C. Paillard & S. E. Ford. 2002. Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams. Dis. Aquat. Org. 48:221-231.

Allam, B., C. Paillard & P. Maes. 1996. Localization of the pathogen Vibrio P1 in clams affected by brown ring disease. Dis. Aquat. Org. 27:149-155.

Allam, B., C. Paillard, A. Howard & M. Le Pennec. 2000b. Isolation of the pathogen Vibrio tapetis and defense parameters in brown ring diseased Manila clams Ruditapes philippinarum cultivated in England. Dis. Aquat. Org. 41:105-113.

Bernoth, E. M. 1991. Identification of cultured Aeromonas salmonicida by an indirect dot blot immunoassay. J. Fish Dis. 14:419-422.

Borrego, J. J., D. Castro, A. Luque, C. Paillard, P. Maes, M. T. Garcia & A. Ventosa. 1996. Vibrio tapetis sp. nov., the causative agent of the brown ring disease affecting cultured clams. Int. J. Syst. Bacteriol. 46:480-484.

Carnegie, R. B., B. J. Barber, S. C. Culloty, A. J. Figueras & D. L. Distel. 2000. Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia. Dis. Aquat. Org. 42:199-206.

Castro, D., J. A. Santamaria, A. Luque, E. Martinez-Manzanares & J. J. Borrego. 1997. Determination of the etiological agent of brown ring disease in southwestern Spain. Dis. Aquat. Org. 29:181-188.

Castro, D., A. Luque, J. A. Santamaria, P. Maes, E. Martinez-Manzanares & J. J. Borrego. 1995. Development of immunological techniques for the detection of the potential causative agent of the brown ring disease. Aquaculture 132:97-104.

Castro, D., E. Martinez-Manzanares, A. Luque, B. Fouz, M. A. Morinigo, J. J. Borrego & A. E. Toranzo. 1992. Characterisation of strains related to brown ring disease outbreaks in southwestern Spain. Dis. Aquat. Org. 14:229-236.

Clarke, D. 1999. Cultivation of clams in Ireland: the potential for growth. Bord Iascaigh Mhara, Dun Laoghaire, Co. Dublin, Ireland.

Cowan, S. T. 1974. Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, London.

Cuif, J.-P. & Y. Dauphin. 1996. Occurrence of mineralization disturbances in nacreous layers of cultivated pearls produced by Pinctada margaritifera var. cumingi from French Polynesia. Comparison with reported shell alterations. Aquat. Living Resour. 9:187-193.

Cunningham, C. O. 2002. Molecular diagnosis of fish and shellfish diseases: present status and potential use in disease control. Aquaculture 206:19-55.

FAO. 2002. The state of world fisheries and aquaculture (SOFIA) 2002. FAO, Rome. 150 pp.

Figueras, A., J. A. F. Robledo & B. Novoa. 1996. Brown ring disease and parasites in clams (Ruditapes decussatus and R-philippinarum) from Spain and Portugal. J. Shellfish Res. 15(2):363-368.

Harwood, V. J., J. P. Gandhi & A. C. Wright. 2004. Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review. J. Microbiol. Methods 59:301-316.

Jansen, S., O. Samuelsen, K. Andersen, L. Torkildsen, C. Lambert, G. Choquet, C. Paillard & O. Bergh. 2003. Characterisation of strains of Vibrio splendidus and V. tapetis isolated from corkwing wrasse Symphodus melops suffering vibriosis. Dis. Aquat. Org. 53:25-31.

Lee, E. G. H. & M. R. Gordon. 1987. Immunofluorescence screening of Renibacterium salmoninarum in the tissues and eggs of fanned Chinook salmon. Aquaculture 65:7-14.

Lynch, S. A., D. V. Armitage, S. Wylde, M. F. Mulcahy & S. C. Culloty. 2006. The susceptibility of young prespawning oysters, Ostrea edulis, to Bonamia ostreae. J. Shellfish Res. 24:1019-1025.

Maes, P. & C. Paillard. 1992. Effect du Vibrio P1, pathogene de Ruditapes philippinarum, sur d'autres especes de bivalves. Les Mollusques marins, Biologie et Aquaculture. Ifremer, Actes de Colloques 14:141-148.

McPherson, M. J., P. Quirke & G. R. Taylor. 1991. PCR: a practical approach. Oxford: Oxford University Press. 253 pp.

Noel, T., J.-L. Nicolas, V. Boulo, E. Mialhe & P. Roch. 1996. Development of a colony-blot ELISA assay using monoclonal antibodies to identify Vibrio P1 responsible for "brown ring disease": in the clam Tapes philippinarum. Aquaculture 146:171-178.

Novoa, B., A. Luque, D. Castro, J. J. Borrego & A. Figueras. 1998. Characterization and infectivity of four bacterial strains isolated from brown ring disease-affected clams. J. Invert. Pathol. 71:34-41.

Paillard, C. 2004. A short review of brown ring disease, a vibriosis affecting clams, Ruditapes philipinarum and Ruditapes decussatus. Aquat. Living Resour. 17:467-475.

Paillard, C. & P. Maes. 1990. Etiologie de la maladie de l'anneau brun chez Tapes philippinarum: Pathogenicite d'un Vibrio sp. C.R. Acad. Sci. Paris 310:15-20.

Paillard, C. & P. Maes. 1994. The brown ring disease in the manila clam, Ruditapes philippinarum: Establishment of a classification system. Dis. Aquat. Org. 19:137-146.

Paillard, C. & P. Maes. 1995a. The Brown Ring Disease in the Manila clam, Ruditapes philippinarum. I. Ultrastructural alterations of the periostracal lamina. J. Invert. Pathol. 65:91-100.

Paillard, C. & P. Maes. 1995b. The Brown Ring Disease in the manila clam, Ruditapes philippinarum. II. Microscopic study of the brown ring syndrome. J. Invert. Pathol. 65:101-110.

Paillard, C., B. Allam & R. Oubella. 2004. Effect of temperature on defense parameters in Manila clam Ruditapes philippinarum challenged with Vibrio tapetis. Dis. Aquat. Org. 59:249-262.

Paillard, C., P. Maes & R. Oubella. 1994. Brown ring disease in clams. Ann. Rev. Fish Dis. 4:1-22.

Paillard, C., S. Gausson, J.-L. Nicolas, J. P. Le Pennec & D. Haras. 2006. Molecular identification of Vibrio tapetis, the causative agent of the brown ring disease of Ruditapes philippinarum. Aquaculture 253:25-38.

Paillard, C., P. Maes, J. Mazurie, S. Claude, A. Marhic & M. Le Pennec. 1997. Epidemiological survey of the brown ring disease in clams of Atlantic coast: role of temperature in variations of prevalence. Proc. VIIIe Symp. Int. Soc. Veterinary Epidemiology and Economics Vol. 31/32(2). Paris, France: AEEMA Publications. pp. 14031-14033.

Park, K. I. & K. S. Choi. 2001. Spatial distribution of the protozoan parasite Perkinsus sp. found in the Manila clams, Ruditapes philippinarum, in Korea. Aquaculture 203:9-22.

Park, K. I., A. Figueras & K. S. Choi. 2006a. Application of enzyme-linked immunosorbent assay (ELISA) for the study of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia): II. Impacts of Perkinsus olseni on clam reproduction. Aquaculture 251:182-191.

Park, K. I., C. Paillard, P. Le Chevalier & K. S. Choi. 2006b. Report on the occurrence of brown ring disease (BRD) in Manila clam, Ruditapes philippinarum, on the west coast of Korea. Aquaculture 255:610-613.

Parsons, A. 2005. Status of Irish aquaculture 2004. Marine Institute/Bord Iascaigh Mhara/Taighde Mara Teo. 59 pp.

Perkins, F. O. 1996. Shell disease in the gold lip pearl oyster, Pinctada maxima, and the Eastern oyster, Crassostrea virginica. Aquat. Living Resour. 9:159-168.

Plana, S. & M. Le Pennec. 1991. Alteration de la glande digestive et consequences nutritionnelles chez la palourde Ruditapes philippinarum contaminee par un bacterie du genre Vibrio. Aquat. Living Resour. 4:255-264.

Reid, H. I., H. L. Duncan, L. A. Laidler, D. Hunter & T. H. Birkbeck. 2003a. Isolation of Vibrio tapetis from cultivated Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 221:65-74.

Reid, H. I., P. Soudant, C. Lambert, C. Paillard & T. H. Birkbeck. 2003b. Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis. Dis. Aquat. Org. 56:249-258.

Robledo, J. A. F., J. Caceres-Martinez & A. Figueras. 1994. The presence of the Brown Ring Disease in clams from Galicia (NW of Spain) and Aveiro (W of Portugal). In: C. P. Vivares, editor. 6th Int. Colloquium on Pathology in Marine Aquaculture. BIOCIM, Montpellier. pp. 28.

Rodriguez, J. M., A. E. Toranzo & J. L. Romalde. 2003. Development and optimization of a new PCR-based protocol for rapid diagnosis of Vibrio tapetis, the causative agent of brown ring disease in clams. In: Book of abstracts of the XIth International Conference of the European Association of Fish Pathologists (EAFP), St. Julians, Malta, 2003.

Rodriguez, J. M., S. Lopez-Romalde, R. Beaz, M. Carmen Alonso, D. Castro & J. L. Romalde. 2006. Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD. Dis. Aquat. Org. 69:175-183.

Romalde, J. L., D. Castro, B. Magarinos, L. Lopez-Cortes & J. J. Borrego. 2002. Comparison of ribotyping, randomly amplified polymorphic DNA, and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis. Syst. Appl. Microbiol. 25:544-550.

Saulnier, D., J. C. Avarre, G. Le Moullac, D. Ansquer, P. Levy & V. Vonau. 2000. Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of Syndrome 93 in New Caledonia. Dis. Aquat. Org. 40:109-115.

Sherperd, S. A. & S. Huchette. 1997. Studies on southern Australian abalone (genus Haliotis): Ring formation in H. scalaris. Mollusc. Res. 18:247-252.

Simon, R. C. & S. Schill. 1984. Tables of sample size requirements for detection of fish infected by pathogens: three confidence levels for different infection prevalence and different population sizes. J. Fish Dis. 7:515-520.

Sindermann, C. J. 1990. Principal diseases of marine fish and shellfish. San Diego: Academic Press. pp. 413-416.

Smibert, R. M. & N. R. Krieg. 1981. General characterisation. In: P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nestor, W. A. Wood, N. R. Krieg & G. B. Phillips, editors. Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. pp. 409-443.

Soudant, P., C. Paillard, G. Choquet, C. Lambert, H. I. Reid, A. Marhic, L. Donaghy & T. H. Birkbeck. 2004. Impact of season and rearing site on the physiological and immunological parameters of the Manila clam Venerupis (= Tapes = Ruditapes) philippinarum. Aquaculture 229:401-418.

Walker, P. & R. Subasinghe. R. 2000. DNA based molecular diagnostic techniques--research needs for standardization and validation of the detection of aquatic animal pathogens and diseases. FAO Fisheries Technical Paper, vol. 395. FAO, Rome, 1-14. 93 pp.

West, P. A. & R. R. Colwell. 1984. Identification and classification of Vibrionaceae. An overview. In: R. R. Colwell, editor. Vibrios in the environment. New York: John Wiley & Sons Inc.

L. C. DRUMMOND, (1)* P. O'REILLY, (2) M. F. MULCAHY (1) AND S. C. CULLOTY (1)

(1) Department of Zoology, Ecology and Plant Science, University College Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland; (2) Department of Microbiology, University College Cork, Cork, Ireland

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Characterization tests for Vibrio tapetis isolates

Growth in:
  0.0% NaCl                             Negative
  1.5% NaCl                             Positive
  2.0% NaCl                             Positive
  6.0%                                  Negative
Growth at:
  4[degrees]C                           Positive
  22[degrees]C                          Positive
  30[degrees]C                          Negative
Gram Reaction                           Negative
Motility                                Positive
Morphology                              Curved Rods
Oxidase                                 Positive
Oxidation/Fermentation                  Fermentative
Catalase                                Positive
Susceptibility to O/129 (150[micro]g)   Susceptible
Amino Acids:
  Arginine Dihydrolase                  Negative
  Lysine Decarboxylase                  Negative
  Ornithine                             Negative
Decarboxylase
Amylase                                 Positive
Gelatine Liquefaction                   Positive
Lipase                                  Positive
Esculine                                Positive/Negative
Nitrate Reduction                       Positive
Indole                                  Positive
Gas from Glucose                        Negative

TABLE 2.
Comparison of positive and negative results between four techniques
for diagnosis of Brown Ring Disease/detection of Vibrio tapetis in a
sample of Manila clams, Venerupis (Ruditapes) philippinarum. The first
column shows the numbers of positives and negatives detected by each
individual technique and the following columns show the comparative
numbers of positives and negatives detected by each of the remaining
techniques. n = 52.

Positives and Negatives         Shell          Shell
Detected by Each Technique    Analysis +     Analysis -

Paillard PCR + (26)               2              24
Paillard PCR - (26)               2              24
Shell Analysis + (4)
Shell Analysis - (48)
Microbiology + (19)               0              19
Microbiology - (33)               4              29
Rodriguez PCR + (8)               1               7
Rodriguez PCR - (44)              3              41

Positives and Negatives      Microbiology   Microbiology
Detected by Each Technique        +              -

Paillard PCR + (26)               13             13
Paillard PCR - (26)                6             20
Shell Analysis + (4)               0              4
Shell Analysis - (48)             19             29
Microbiology + (19)
Microbiology - (33)
Rodriguez PCR + (8)                3              5
Rodriguez PCR - (44)              16             28

Positives and Negatives       Rodriguez      Rodriguez
Detected by Each Technique      PCR +          PCR -

Paillard PCR + (26)               6              20
Paillard PCR - (26)               2              24
Shell Analysis + (4)              1               3
Shell Analysis - (48)             7              41
Microbiology + (19)               3              16
Microbiology - (33)               5              28
Rodriguez PCR + (8)
Rodriguez PCR - (44)

Positives and Negatives        Paillard       Paillard
Detected by Each Technique      PCR +          PCR -

Paillard PCR + (26)
Paillard PCR - (26)
Shell Analysis + (4)               2              2
Shell Analysis - (48)             24             24
Microbiology + (19)               13              6
Microbiology - (33)               13             20
Rodriguez PCR + (8)                6              2
Rodriguez PCR - (44)              20             24

TABLE 3.
A comparison of the efficiency of detection of BRD/Vibrio tapetis in
a sample of 52 Venerupis (Ruditapes) philippinarum by different
techniques, individually and in combination.

                                                           Percentage
Technique/Combination of Techniques                        Detection

BRD shell analysis                                             7.7
Microbiological characterization                              36.5
Rodriguez assay                                               15.4
Paillard assay                                                50
BRD shell analysis + Microbiological characterization         44.2
BRD shell analysis + Rodriguez assay                          21.2
BRD shell analysis + Paillard assay                           53.8
Microbiological characterization + Rodriguez assay            46.1
Microbiological characterisation + Paillard assay             61.5
Rodriguez assay + Paillard assay                              53.8
BRD shell analysis + Microbiological characterization +       51.9
  Rodriguez assay
BRD shell analysis + Microbiological characterization +       65.4
  Paillard assay
BRD shell analysis + Rodriguez assay + Paillard assay         57.7
Microbiological characterization + Rodriguez assay +          65.4
  Paillard assay
BRD shell analysis + Microbiological characterization +       69.2
  Rodriguez assay + Paillard assay