The susceptibility of young prespawning oysters, Ostrea edulis, to Bonamia ostreae.

ABSTRACT Young prespawning oysters, Ostrea edulis, were held over 6 mo at two different Bonamia ostreae-endemic sites in Ireland, to determine to what extent they could become infected with this protozoan parasite. Prevalence and intensity of infection were monitored, using the traditional method of ventricular heart smears and polymerase chain reaction (PCR). Results showed that 0+ and 1+ oysters were susceptible to infection. Infection was observed in the naive and previously exposed oysters 2 months post relaying. Of ventricular heart smears and PCR, PCR was the more sensitive diagnostic technique in detecting B. ostreae in most of the oysters. Current methods recommended by the Office International Epizooties (OIE) and the European Union (EU), histology and screening of heart smears for B. ostreae, may be inadequate because certain low levels of infection may go undetected.

KEY WORDS: prespawning, Ostrea edulis, Bonamia ostreae, diagnostics, ventricular heart smear, PCR, sensitivity

INTRODUCTION

The protist Bonamia ostreae Pichot, Comps, Tige, Grizel and Rabouin is the causative agent of bonamiasis (Pichot et al. 1980), also known as microcell disease, hemocytic parasitosis and hemocyte disease of the European flat oyster, Ostrea edulis Linnaeus. B. ostreae has caused major mortalities among O. eclulis throughout its distribution range in Europe and has been observed in flat oysters on both coasts of North America with no detectable mortalities in the field (Comps et al. 1980, Alderman 1981, Balouet & Poder 1983, Bucke & Feist 1985, Elston et al. 1986, Montes& Figueras 1987, Van Banning 1990, Van Banning 1991, Barber & Davis 1994, Friedman & Perkins 1994). Previous studies indicate that older oysters appear to be more susceptible than younger oysters, with market-sized oysters experiencing the heaviest mortalities (Grizel & Tige 1982, Van Banning 1990, McArdle et al. 1991, Culloty & Mulcahy 1996). Van Banning (1990) suggested that oysters were only susceptible to B. ostreae once they came to spawning and that female follicles were the source of infection. He hypothesized that initial infections occur after the female phase, after phagocytosis of residual infected gametes by hemocytes. However, Culloty and Mulcahy (1996) found that prevalence of infection in males and females was similar. Two- to four-year-old oysters represent the age groups most heavily infected by B. ostreae (Grizel & Tige 1982). Culloty and Mulcahy (1996) monitored two age classes of oysters, 1.5 y and 2.5 y, in the field over 2 years and found that the 2.5-y-old oysters were already infected prior to monitoring and the younger oysters, 1.5 y, became infected when 2 y old, post spawning. To date, many studies have concentrated on older oysters when screening for B. ostreae infection because it is considered that there is a greater chance of diagnosing infection in these older, larger oysters (Grizel & Tige 1982, Van Banning 1990, McArdle et al. 1991). Few studies have looked at the susceptibility of young oysters (Montes et al. 2002). It would seem that B. ostreae can be diagnosed from spat up, but the prevalence of infection is low. For example, Tige & Grizel (1982) examined 25,988 oyster sections, including some seed oysters; overall 2,517 (9.68%) of the oysters in the study were infected with B. ostreae. The two age groups most heavily infected were 2-y olds and 3-4-y olds. Seed oysters had a prevalence of infection of 0.12% (3 animals out of 2852 seed oysters screened). Previous epidemiologic studies have used conventional histologic and cytologic techniques to diagnose B. ostreae, however molecular based techniques such as PCR and in situ hybridization are also now available. Studies have indicated that when screening for B. ostreae a higher prevalence of infection has been observed when using PCR (Carnegie et al. 2000).

The aim of this study is to determine when young oysters can become infected and to what extent. Prevalence and intensity of infection were monitored over a 6-mo period. Two different diagnostic techniques were used to detect the parasite: the results from traditional ventricular heart smears and polymerase chain reaction (PCR) were compared.

MATERIALS AND METHODS

Oysters

0+ oysters (mean weight 1.08 g) and 1+ oysters (mean weight 20.67 g) were used in the study and were from 3 locations: (1) Rossmore, Cork Harbour, Ireland (code R0+, R1+): native oysters that have been exposed to B.ostreae for over 15 y. Two age groups were used: 0+ oysters were 2-3 mo old and the 1+ approximately 18 mo old. (2) Tralee, Ireland (code T1+): a public fishery where B. ostreae has never been diagnosed. 1+ oysters were approximately 18 mo old at the beginning of the study. Seasalter hatchery, England (code SS0+): a hatchery where B.ostreae has never been detected. 0+ oysters were approximately 1 mo old.

Trial Sites and Holding Conditions

Field trials were set up in July 2003 and conducted for 6 mo at two sites where B. ostreae is present: Rossmore, Cork Harbour on the south coast of Ireland (51[degrees]85'N, 8[degrees]26'W) and Clarenbridge, Galway Bay on the west coast of Ireland (53[degrees]16'N, 9[degrees]3'W).

At both trial sites the young oysters were placed intertidally, on the midlower shore, in oyster bags, on trestles. The trestles held the oyster bags approximately 40 cm off of the muddy substrate. The following configurations were used: Rossmore, Cork Harbour, 1,000 SS0+; 200 R0+; 1,000 R1+ and 1,000 T1+. All were deployed at a density of 200 oysters/bag; Clarenbridge, Galway Bay: 1,000 SS0+, 1,000 R1+ and 1,000 T1+. All were deployed at a density of 200 oysters/bag. Insufficient 0+ Rossmore oysters were available for relaying in Galway.

Monitoring of Oysters

Prior to relaying, an initial sample of 60 oysters from each source and age group was screened to ensure that B. ostreae was not present in either group of naive oysters, and to determine the prevalence of infection in the Rossmore (Cork Harbour) oysters. Samples (n = 60) from each age group were subsequently collected from Rossmore and Clarenbridge at 2-too, 4-mo and 6-mo. Certain samples could not be taken from Clarenbridge because of high mortality in the oyster bags.

Diagnostic Methods

Ventricular heart smears were prepared immediately (Bachere et al. 1982). Each oyster was then cut in two. For PCR screening one small sample of gill tissue (5 [mm.sup.2]) was frozen for DNA extraction. A back up sample was stored in 95% ethanol. The remaining oyster tissues (gill and digestive gland) were fixed in Davidson solution and preserved in 70% ethanol for histology and in-situ hybridization if required.

Monitoring for Infection With B. ostreae

All oysters were screened for B. ostreae infection by ventricular heart smears as previously described (Bachere et al. 1982, Culloty et al. 1999): Class 0: no B. ostreae cells observed after 5 min of screening; Class 1: 1-10 B. ostreae cells observed within 5 min of screening; Class 2: 11-100 B. ostreae cells observed within 5 min of screening; Class 3: B. ostreae cells observed in all fields of vision and Class 4: a heavy parasite burden in all host hemocytes.

Polymerase Chain Reaction

Oyster DNA from the frozen gill tissue was extracted using the phenol-chloroform extraction method (Sambrook et al. 1989). Two sets of primers are available for B. ostreae (Carnegie et al. 2000, Cochennec et al. 2000). Both sets of primers were used in this study. PCR 1 using the Cochennec primer set and PCR 2 using the Carnegie c primer set. Duplicate PCR reactions were carried out on each sample for each primer set, and positive and negative controls were used in each PCR reaction. Positive controls were obtained from oysters heavily infected with the parasite. Negative controls consisted of double distilled water (dd[H.sub.2]O). Modifications were made to the PCR master mix concentrations and PCR reaction cycle. PCR reactions were carried out in a total volume of 50 [micro]L, including 100 ng DNA, lx Green Go Taq Reaction buffer (PROMEGA), 0.25 mm dNTPs, 1.25 U Taq DNA Polymerase (Advanced Biotechnologies), 2.5 mm Mg[Cl.sub.2] and 1 [micro]m of forward and reverse primers. In the PCR reaction cycle, using the Cochennec primer set, amplifications were carried out on a Hybaid thermal cycler with 1 cycle of denaturation at 95[degrees]C for 180 s, 34 cycles of annealing at 95[degrees]C for 30 s, 55[degrees]C for 30 s, 72[degrees]C for 30 s and 1 cycle of extension at 72[degrees]C for 300 s. In the PCR reaction cycle, using the Carnegie c primer set, amplifications were carried out on a Hybaid thermal cycler with 35 cycles of annealing at 94[degrees]C for 60 s, 60[degrees]C for 60 s, 72[degrees]C for 60 s and 1 cycle of extension at 72[degrees]C for 600 s. PCR products were checked on 2% agarose gels after electrophoresis. PCR results were graded as either positive or negative.

The results of screening by heart smear and by PCR were then compared.

RESULTS

Prevalence of Infection

Initial screening of the different oyster groups indicated that B. ostreae was not present in the SS0+, TI+ or R1+ groups, using either heart smears or PCR for diagnosis. B. ostreae was observed in one heart smear in the R0+ initial sample (1/52), and was also detected using PCR (3/52). Over the course of the study 0+ and 1+ oysters in each group developed Bonamia ostreae infections at both trial sites.

From the results of screening of heart smears over the 6-mo field trial period in Rossmore (Table 1), R0+ showed the lowest prevalence of infection (3%), whereas R1+ had the highest (9%). The prevalence for SS0+ was 4% and T1+ was 6%. However from the PCR results the naive strains had the lowest prevalence (SS0+ = 5%; T+ = 8%) and the Rossmore strain had the highest (R0+ = 10%; R+ = 26%). The oysters were all infected by 2 mo and of infection by 6 mo was 9% (H.S. & PCR1) and 3% (PCR 2) in the SS0+, 6% (H.S.), 0% (PCR1) and 12% (PCR 2) in the R0+, 14% (H.S.), 6% (PCR1) and 23% (PCR 2) in the T1+ and 7% (H.S.), 14% (PCR) and 32% (PCR 2) in the R1+.

Over the 6-mo field trial (Table 2) in Clarenbridge using the results for both diagnostic techniques, all groups were found to have developed infection. A sample from T1+ and R1+ was not collected at 2 mo. The naive strains had the lowest prevalence of infection (SS0 + H.S. = 1%; T1+ H.S. = 4%). The R1+ group had the highest prevalence (H.S. = 20%). In the PCR results the SS0+ had the lowest prevalence of infection (2%) and T1+ had a prevalence of 22%. R1+ had the highest prevalence of infection (41%).

The overall prevalence of infection observed in Rossmore combining both techniques and sample times for each oyster group was as follows; R0+ 14.9%, SS0+ 10.1%, T1+ 12.2% and R1+ 28.7%. In Clarenbridge, the overall prevalence of infection observed for each oyster group was as follows: SS0+ 7.1%, T1+ 25.3 % and R1+ 59.3%.

This difference in prevalence of infection was more obvious in the R1+ group at both sites than in the SS0+ and T1+. Prevalence of infection in R1+ oysters was higher in Clarenbridge, than in Rossmore.

Intensity of Infection

In the initial sample in Rossmore, the only infected R0+ animal had a class 2 infection (Fig. 1b). In the 2-mo R0+ sample both infected oysters had class 1 infections. In the 4 and 6-mo R0+ samples class 1, class 2 and class 3 infections were observed (Fig. 1a, b, c, d). SS0+ and T1+ developed class 2 and 3 infections in Rossmore over the 6-mo field trial.

[FIGURE 1 OMITTED]

In Clarenbridge, no infection was observed in the oyster groups in the T0 sample. No sample was taken from the T1+ and R1+ oyster groups in the 2-mo sample. Infection was not observed in the SS0+ group in the 2-mo sample. In the 4-mo sample class 1 infections were observed in all groups, and class 2 and class 3 infections were also observed in the R1+ group. In the 6-mo sample in all groups, classes 1, 2 and 3 infections were also observed.

In Clarenbridge, a higher prevalence of class 1 infection in the R1+ oysters was observed than in Rossmore. R1+ developed class 2 and 3 infections at both sites. The naive strains had a similar prevalence of class 1 infection at both sites. T1+ developed class 2 and 3 infections in Clarenbridge, Galway Bay. SS0+ did not develop a class 2 or a class 3 infection in Clarenbridge.

Comparison of Techniques

The sensitivity of detection by the two techniques differed in most samples. A higher prevalence of infection was seen in the PCR result for SS0+ 2-mo sample than in the heart smear screening (H.S = 4%; PCR = 12%). Prevalence of infection was similar for both techniques in the 4-mo and 6-mo samples. Results from the two techniques differed for the R0+ oysters at each sample period with a significant difference in the 4-mo sample (H.S. = 5%; PCR = 17%). A similar prevalence of infection was detected by the two methods in the 2-mo T1+ sample (H.S. & PCR = 3%) but no infection was observed in the 4-mo sample using PCR (H.S. = 9%). In the 6-mo sample a higher prevalence was detected using PCR (H.S. = 14%; PCR = 29%). In the R1+ oysters a higher prevalence of infection was found at each sample time period using PCR compared with the heart smear screening. Both techniques revealed a decrease in prevalence in the 6-mo sample.

At the Clarenbridge site, infection was first detected by both PCR and heart smears in the SS0+ group in the 4-mo sample (PCR = 10%) (H.S. = 3%). In both the T1+ and R1+ oysters, infection was also detected in the 4-mo sample. A significant difference in detection was seen between heart smears and PCR in the T1+ 6-mo sample (H.S. = 5%; PCR = 54%). A decrease in infection was seen in the R1+ group between the 4 and 6-mo samples using both techniques.

The sample size for each oyster group was lower than planned at certain sample times. Sample size was reduced in the 0+ groups over the trial period, caused by high mortality; in Clarenbridge, no sample was available for the SS0+ group at 6-mo.

In the Rossmore study 141 individuals (17%) from the four oyster groups were found to be positive for B. ostreae using heart smear and PCR screening. Of the individuals who were observed to have class 1 infections, when screened using the heart smear technique, only 35.2% were shown to be positive when screened by PCR. The results for individuals with Class 2 and 3 infections were 89% and 88% respectively.

In the Clarenbridge, study 136 individuals (28.9%) from the three oyster groups were found to be positive for B. ostreae in the heart smear and PCR screening. Of the individuals who were diagnosed with class 1 infections using heart smear screening infection 48.9% of individuals were also detected by PCR. The results for individuals with class 2 and 3 infections were 71.4% and 50% respectively. A diagnostic comparison for detection of B. ostreae using a combination of trial sites and all oyster groups (Table 3) resulted in a greater number of infected individuals being diagnosed with a combination of three methods (H.S. + PCR Cochennec + PCR Carnegie c = 27%) and the least number of infected individuals being detected with the hear smear screening technique alone (10%).

DISCUSSION

The results of this study demonstrate that young prespawning oysters from spat size up can become infected with B. ostreae quite quickly after exposure and can demonstrate a high prevalence and moderate intensity of infection. Infection was observed in 0+ and 1+ oysters during the study. B. ostreae was not detected in the initial sample of the naive oyster groups, SS0+ and T1+. One individual oyster was found to be infected in the initial sample of the R0+ group using the heart smear screening and three individuals were found to be positive in the PCR. B. ostreae was not detected in the R1+ initial sample. These oysters had been exposed to B. ostreae since settling because they were in a B. ostreae-endemic area, however prevalence of infection does vary among oyster groups. Individual variation in susceptibility to infection may explain the single R0+ oyster that was infected with a class 2 B. ostreae infection. All prespawning oyster groups in the study became infected with the parasite by the fourth month of exposure in the field. Both naive and previously exposed oysters were susceptible to infection. Prevalence of infection differed between the two age groups: older oysters had a higher prevalence of infection. High mortality was observed in the SS0+ oysters, but stress of transportation to a new site may have contributed to this mortality.

The susceptibility of young bivalve species to water-borne pathogens has been investigated in other studies. It has been demonstrated that juvenile (seed) eastern oysters, Crassostrea virginica, from nursery systems exposed to raw water pumped from a Perkinsus marinus and Haplosporidia nelsoni-endemic area, are highly susceptible to infection, although infections may be very light in intensity and low in prevalence (Ford et al. 2000, Ford et al. 2001).

The overall prevalence of infection observed in this study was lower in the naive strains and higher in the previously exposed oyster groups at both sites. This would indicate an increase in prevalence of infection over the period of exposure with longer periods of exposure resulting in higher prevalence of infection. Whereas most previous age-related studies investigating B. ostreae prevalence and intensity of infection have used older oysters (18+ months), Grizel (1985) reported that prevalence of infection in oysters <18 mo of age was less than 10%, whereas in older oysters, from 30 mo up, infection reached a 30% prevalence of infection. In this study, earliest infection of naive oysters was first detected in SS0+ in the 2-mo sample (HS: 4%; PCR: 12%). The mean length of the SS0+ oysters at that time was 2.9 cm. Infection was detected in the initial sample of the previously exposed R0+ oysters (HS: 2%; PCR: 6%), the mean length of which was 1.2 cm. Caceres-Martinez et al. (1995) found the presence of B. ostreae to be better related to size than to age of Ostrea edulis. In that study hatchery-reared stock were cultured in a B. ostreae-infected area. Once the stock reached 18 mo of age it was divided into 3 length groups (Group 1 [1.76 cm], Group 2 [2.52 cm] and Group 3 [3.68 cm]). Prevalence of infection and mortality were measured monthly over the following 9-mo e period. Results indicated that infection occurred when the oysters reached 4 cm in length. Mortality was found to be positively correlated with age and with the presence of B. ostreae. Montes et al. (2002) investigated the effects of bonamiasis on three hatchery-spawned oyster populations in different culture areas in Galicia (NW Spain). The stocks were placed on raft cultures at 4 mo of age and were sampled every 3 months over a 24-mo period. B. ostreae was detected in Cambados, a B. ostreae-endemic area, when the oysters were 16 mo old. In the other culture area, Bueu, B. ostreae was not detected until 24 mo of culture (28 mo old).

In this study, infection intensity remained low throughout the trial in all oyster groups; however, higher intensities occurred in both previously exposed and naive oysters during the 4 and 6-mo sampling time periods: R1+ and T1+ (Fig. 1 and Fig. 2) both developed infection and also high intensities of infection. Heaviest intensities and prevalence of infection occurred in the winter months from November 2003 to January 2004. Winter stressors such as low food availability and environmental conditions may have contributed to increased stress levels and reduced immuno-competence. In a study undertaken by Culloty and Mulcahy (1996) 2 age groups, 1.5 y and 2.5 y, of flat oysters were monitored in Rossmore, Cork Harbour over a 2-y period. Eleven months post relaying (January 1991) the 1.5-y-old oysters had the higher prevalences and intensities of infection. It was concluded that winter stressors such as adverse environmental conditions and an inadequate food supply might have contributed to an increase in susceptibility to the parasite. A build up of infection from time zero up until the winter may also have contributed to this result.

[FIGURE 2 OMITTED]

In this study the presence of B. ostreae was detected earlier by molecular techniques than by heart smears, indicating that infection may be present before diagnosis is possible by OIE recommended diagnostic techniques (OIE, 2003). For the detection of B. ostreae, PCR may be a more suitable and sensitive technique than heart smears (Carnegie et al. 2000: Cochennec et al. 2000). The results of this study suggest that it is a more sensitive method than heart smears, especially for detecting infection in younger oysters. However, any one method of detection is less effective than two. The use of any 2 of the 3 methods (heart smears, PCR Cochennec and PCR Carnegie) increased the likelihood of picking up B. ostreae and the likelihood of picking up the parasite is equally good with each combination. There may be an advantage, depending on the objective of the detection, in using a third method in addition, as more individuals were found to be positive using all three methods. Each of the diagnostic methods has their limitations, which may result in false positives and false negatives (Table 4). False negatives may be more common in the subjective heart smear technique especially with a lower class of infection. False positives may occur in the PCR because of the low specificity of the primer groups. Such positives would need to be verified by sequencing. Optimization of molecular techniques is still required. In a study comparing the effectiveness of light microscopic techniques in detecting B. ostreae it was found that hemolymph cell monolayers were more sensitive than tissue imprints (heart, gills, digestive gland and gonad) however heart imprints provided the highest sensitivity among the tissue imprints. Histologic sections were the least sensitive technique in this study (Da Silva & Villalba 2004). In a study to compare diagnostic techniques for B. exitiosa Hine, Cochennec-Laureau and Berthe in flat oysters, O. chilensis Hutton (Hine et al. 2001, Berthe & Hine, 2003), Diggles et al. (2003) concluded that PCR was a more sensitive technique than either heart smears or histology. Intensity of infection was not quantified using real-time PCR in this study; however in comparison with the heart smear results, low intensity B. ostreae infections (observed in heart smears) were detected using traditional PCR in most samples. The sensitivity of PCR in detecting different classes of intensity of infection was examined and PCR was found to detect classes 2 and 3 more effectively than class 1. Using a combination of heart smear results from both trial sites a greater percentage of individuals with a class 2 infection (81.25%) were more likely to be detected by PCR. Individuals with class 3 (75%) also had a high percentage of detection by PCR, whereas less than half of oysters with class 1 infection (41.8%) were detected by PCR. A reason for PCR not detecting light infections might be a failure to target sufficient numbers of parasites in the oyster gill tissue used in the DNA extraction. Carnegie et al. (2000) found that 37.9% of oysters, in which B. ostreae was not detected using cytology, were positive using PCR. There are several possible reasons to explain why a sample was positive for the PCR and negative in the heart smear. The parasite may have been present in the gills but not in the heart. B. ostreae cells in heart smears are easily observed when they are numerous, however in light infections small B. ostreae cells may be mistaken for routine intracytoplasmic inclusions, and a margin of error must be taken into account when detecting low levels of infection. Furthermore, though it is known that transmission can occur directly from oyster to oyster, the complete life cycle is unclear and may contain other as yet unidentified stages but with detectable DNA (Kleeman et al. 2002). Molecular techniques are highly sensitive and enable the rapid detection of all life-cycle stages of a parasite that may otherwise be unrecognizable using traditional detection methods.

Early, rapid detection of bonamiasis is crucial for effective control of the disease. Although the movement of oysters within the European Union (EU) is regulated, and transfers are not permitted from B. ostreae-infected to uninfected areas, it can be seen from this study, as from a previous study (O'Neill et al. 1998), that current prescribed diagnostic methods (i.e., examination of stained tissue sections and tissue imprints of susceptible organs do not always detect B. ostreae). Some infections may not be diagnosed using EU- and Office Internationale d'Epizootic (OIE)- prescribed methods, and prevalence of infection in some samples may be underestimated. The monitoring of O. edulis populations and B. ostreae is critical to prevent and limit associated risks. Currently under EU legislation twice yearly sampling of 150 oysters per site takes place from oyster growing regions. Samples are screened using heart smears and tissue sections.

Pre-transfer sampling of O. edulis has not usually involved younger, smaller oysters but has concentrated on larger, older oysters that are believed to be more susceptible to infection and more likely to show infection. The results of this study suggest that screening of young oysters would be advisable before spat and young oysters are transferred to B. ostreae-free growing sites. A further consideration is that other bivalves such as Crassostrea gigas are moved freely within Europe from bonamiasis-infected areas to uninfected areas. Because C. gigas is not deemed to be a carrier of B. ostreae (Culloty et al. 1999), it is not prescreened before movement. However, O. edulis spat are often found in consignments of C. gigas. The results of the present study indicate that such O. edulis spat could be infected, and could therefore transfer the parasite.

ACKNOWLEDGMENTS

The authors thank the anonymous reviewers for improving this manuscript. This work was supported by EU CRAFT PROJECT-1999-72338.

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S. A. LYNCH, * D. V. ARMITAGE, S. WYLDE, M. F. MULCAHY AND S. C. CULLOTY

Department of Zoology, Ecology, Plant Science, University College Cork, Lee Maltings, Cork, Ireland 

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

TABLE 1.
Comparison of screening by heart smears and PCR. Assays for the
detection of Bonamia ostreae in young oysters at Rossmore,
Cork Harbour.

                Initial Sample           2nd Month

                           PCR (%)                 PCR (%)

Population   n    HS (%)   1    2    n    HS (%)   1    2

SSO+         56     0      0    0    55     4      0    12
R0+          52     2      0    6    38     0      3     5
T1+          58     0      0    0    60     3      0     3
R1+          60     0      0    0    59     5      0    19

                  4th Month               6th Month

                           PCR (%)                 PCR (%)

Population   n    HS (%)    1    2    n    HS (%)   1     2

SSO+         46      2      0    2   32      9      9     3
R0+          60      5     10   17   17      6      0    12
T1+          60      9      0    0   59     14      6    23
R1+          60     25     43   52   59      7     14    32

SSO+, Bonamia-free Site 2; R0+, Rossmore 0+; T1+, Bonamia-free
Site 1; R1+, Rossmore 1+; n, sample size.

HS (%), the percentage of heart smears that were found to be
positive in the sample; PCR Cochennec & PCR Carnegie c (%), the
percentage of PCR-positive oysters in replicate samples.

Table 2.
Comparison of screening by heart smears and PCR. Assays for the
detection of Bonamia ostreae in young oysters at Clarenbridge,
Galway Bay.

                Initial Sample          2nd Month

                           PCR (%)                 PCR (%)

Population   n    HS (%)   1    2    n    HS (%)    1    2

SSO+         56     0      0    0    41     0       0    0
T1+          58     0      0    0    NS     NS     NS   NS
R1+          60     0      0    0    NS     NS     NS   NS

                  4th Month               6th Month

                           PCR (%)                 PCR (%)

Population   n    HS (%)   1    2    n    HS (%)   1    2

SSO+         30      3      3    7   NS     NS     NS   NS
T1+          49      6      8    2   59      5     54   20
R1+          59     34     53   53   57     26     50   31

SSO+, Bonamia-free Site 2; T1+, Bonamia-free Site 1; R1+, Rossmore
1+; n, sample size.

HS (%), the percentage of heart smears that were found to be positive
in the sample; PCR Cochennec & PCR Camegie c (%), the percentage of
PCR-positive oysters in replicate samples.

NS, no sample.

TABLE 3.
A comparison of the efficiency of detection of B. ostreae in all
oyster groups by different diagnostic techniques.

                                           Rossmore     Clarenbridge

Heart smears (H.S.)                     8.5% (71/830)    13% (60/470)
PCR Cochennec                             7% (58/830)    21% (97/470)
PCR Carnegie c                           10% (86/830)    14% (64/470)
PCR Cochennec + PCR Camegie c            13% (106/830)   24% (115/470)
H.S. + PCR Cochennec                     16% (129/830)   33% (157/470)
H.S. + PCR Carnegie c                    19% (156/830)   26% (124/470)
H.S. + PCR Cochennec + PCR Carnegie c    21% (177/830)   37% (175/470)

                                            Total

Heart smears (H.S.)                       10% (131/1300)
PCR Cochennec                             12% (155/1300)
PCR Carnegie c                          11.5% (150/1300)
PCR Cochennec + PCR Camegie c             17% (221/1300)
H.S. + PCR Cochennec                      22% (286/1300)
H.S. + PCR Carnegie c                   21.5% (280/1300)
H.S. + PCR Cochennec + PCR Carnegie c     27% (352/1300

TABLE 4.
Comparative analysis of reproducibility of the three methods, heart
smear, PCR Cochennec and PCR Carnegie c.

                 Number                          Number
  Total      Common to H.S.,     Number         Positive
Number of    PCR Cochennec +    Positive         by PCR
Positives    PCR Carnegie c      by H.S.        Cochennec

   262             48              131             155

  Number         Number          Number          Number
 Positive      Positive by     Positive by     Positive by
  by PCR       H.S. + PCR      H.S. + PCR    PCR Cochennec +
Carnegie c      Cochennec      Carnegie c     PCR Carnegie c

   150             62              53              84