Comparison of diagnosis techniques for the protozoan parasite Bonamia ostreae in flat oyster Ostrea edulis

Aquaculture 261 (2006) 1135 – 1143 www.elsevier.com/locate/aqua-online

Comparison of diagnosis techniques for the protozoan parasite Bonamia ostreae in flat oyster Ostrea edulis Pablo Balseiro a , Ramón Fernández Conchas b , Jaime Montes c , Javier Gómez-León a , Beatriz Novoa a , Antonio Figueras a,⁎ a

b

Instituto de Investigaciones Marinas, CSIC, C/Eduardo Cabello, 6 C.P.36208 Vigo, Pontevedra, Spain Instituto Tecnolóxico para o Control do Medio Mariño de Galicia (INTECMAR), Peirao de Vilaxoán, C.P. 36611, Vilagarcía de Arousa, Pontevedra, Spain c Centro de Investigacións Mariñas (CIMA), Pedras de Corón, C.P. 36620, Vilanova de Arousa, Pontevedra, Spain Received 17 December 2005; received in revised form 8 May 2006; accepted 14 May 2006

Abstract In recent years, the cultured production of the flat oyster Ostrea edulis has suffered a dramatic decrease in Europe partially attributed to the protozoan parasite Bonamia ostreae, the causative agent of the Bonamiosis. In this paper the results of a PCR assay for the diagnosis of B. ostreae were compared with those obtained using two classical methods of diagnosis recommended by the OIE (Office International des Epizooties) and the European Union, histology and cytology. The same samples were analyzed by two different laboratories, showing that the results obtained with the PCR method have high sensitivity and good correlation between laboratories. This method is cheaper and faster than histopathology and cytology, with no need of specifically trained personnel to perform the diagnoses. It is appropriate for fast screening of stocks of great numbers of oysters. © 2006 Elsevier B.V. All rights reserved. Keywords: Bonamia; Oyster importations; Aquaculture; Ostrea edulis; Bivalve; Haplosporidia

1. Introduction Due to the epizootics of two protozoans, Marteilia refringens (Grizel et al., 1974) and Bonamia ostreae (Pichot et al., 1980) the production of the flat oyster Ostrea edulis in Europe has dropped since the 1960s. Both protozoa are included as notifiable diseases for the O.I.E. (O.I.E., 2003) and the European Union (E.U., 1991). As consequence of these epizooties, the European aquaculture production of flat oyster fell spectacu-

⁎ Corresponding author. Tel.: +34 986214462; fax: +34 986292762. E-mail address: [email protected] (A. Figueras). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.05.014

larly from 29.595 tons in 1961 to 5.921 in 2000 (F.A.O. Fisheries Department, 2002). In Europe, B. ostreae was first detected in France in 1979 (Pichot et al., 1980). The disease extended quickly to the Netherlands, Denmark, Spain, UK and Ireland (Van Banning, 1982; Balouet et al., 1983; Polanco et al., 1984; Culloty and Mulcahy, 2001). It has been suggested that there were two sources of the disease in Europe with movements of infected oysters from California to Brittany (NW France) and to Asturias (N Spain) (Cigarría and Elston, 1997). The global distribution of B. ostreae comprises the countries listed above and the United States, both Pacific (California and Washington) and Atlantic Coast (Maine) (Elston

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et al., 1986; Friedman and Perkins, 1994; Bower and McGladdery, 2003). Other species from the genus Bonamia are Bonamia exitiosa, which causes high mortalities in Ostrea chilensis from New Zealand and Chile (Hine et al., 2001; Berthe and Hine, 2003; Bower and McGladdery, 2003), Bonamia roughleyi, formerly named Mikrocytos roughleyi (Carnegie and Cochennec-Laureau, 2004), which is the causative agent of Australian Winter Disease in Saccostrea glomerata (Farley et al., 1988) and Bonamia sp. of Ostrea angasi in Australia (Hine and Jones, 1994). Small Subunit Ribosomal gene (SSU rRNA) and internal transcribed spacer (ITS) sequences confirmed the existence of at least two new Bonamia spp., one from O. chilensis from Chile (Campalans et al., 2000), and one more from Crassostrea ariakensis from North Carolina (Burreson et al., 2004). Bonamia sp. is also present in Ostrea puelchana from Argentina, but its correct taxonomy is not determined yet (Kroeck and Montes, 2005). Phylogenetically, the genus Bonamia is placed in the phylum Haplosporidia, by the presence of haplosporosomes (Pichot et al., 1980; Hine et al., 2001) and by the molecular analysis of the SSU rRNA gene (Farley et al., 1988; Carnegie et al., 2000; Cochennec et al., 2000; Cochennec-Laureau et al., 2003; Reece et al., 2004). It is interesting to point out the lack of sporulated stage in its life cycle. The hybridization of parasites that belong to the phylum Haplosporidia with specific probes for B. ostreae (Cochennec et al., 2000; Diggles et al., 2002, 2003) is other indication that the genus Bonamia is part of this phylum, even when it is the only parasite of this phylum with a direct transmission (Poder et al., 1982; Tigé et al., 1982; Bachère and Grizel, 1985; Culloty et al., 1999). This phylum includes several protozoan species that cause heavy mortalities in mollusks as Haplosporidium nelsoni and B. exitiosa in addition to B. ostreae (Haskin et al., 1966; Pichot et al., 1980; Hine et al., 2001; Bower and McGladdery, 2003). As the eradication of the parasite, once it is introduced, is not feasible (Van Banning, 1987), monitoring to confirm freedom from Bonamia spp. in flat oysters to be introduced into non-contaminated zones is critical to limit the spread of the disease. Although cytology and histology may lack sensitivity and skilled personnel are needed, they are routinely used as diagnostic methods for the diagnoses of this pathogen (Bachère et al., 1982; Culloty et al., 2003; Diggles et al., 2003; Mirella da Silva and Villalba, 2004). Several techniques have been developed in addition to the classical diagnostic methods: specific PCR for Bonamia spp. amplification (Carnegie et al., 2000; Cochennec et al., 2000; Diggles

et al., 2003), immunoassays with poly and monoclonal antibodies (Mialhe et al., 1988; Boulo et al., 1989; Rogier et al., 1991; Cochennec et al., 1992) or in situ hybridizations (Cochennec et al., 2000; Carnegie et al., 2003; Diggles et al., 2003). The aim of this study was to compare several diagnostic techniques for Bonamia spp. For this purpose the method of the PCR performed with a previously reported primer pair for B. ostreae (Cochennec et al., 2000) was compared with both histology and cytology simultaneously at two different laboratories, the Laboratorio Nacional de Referencia de Enfermedades de Moluscos Bivalvos at the Instituto de Investigaciones Marinas (C.S.I.C.) and the Unidade de Patoloxía of the Instituto Tecnolóxico para o Control do Medio Mariño de Galicia (INTECMAR), both in Galicia (NW Spain). 2. Materials and methods 2.1. Sampling Eight stocks of 30 O. edulis were collected on November of 2001. Each oyster was simultaneously processed by people of the two participating laboratories in the facilities of the laboratory A for histology, cytology and molecular biology and each participating laboratory examined the same individual oysters by the three methods. For histopathological detection of B. ostreae the soft parts of the oysters were fixed in Davidson's fixative for 24 h (Shaw and Battle, 1957). Oblique transverse sections, approximately 5 mm thick, were taken from each specimen including mantle, gills, gonad, digestive gland, nephridia, and foot. Tissue samples were embedded in paraffin and 5 μm sections were stained with haematoxylin and eosin. For the cytological detection, imprints of gills were made over histological slides. They were fixed in ethanol 96° for 1 min and stained with the Hemacolor kit (Merck) following the manufacturer's instructions. 2.2. DNA isolation and specific PCR analysis DNA from the oysters was extracted with DNAzol kit (Invitrogen) following the manufacturer's instructions. PCR analyses were performed with primers Bo and BoAs, which amplified 300 bp from SSU rRNA gene (Cochennec et al., 2000). The reaction was carried out in a volume of 25 μl with 10 mM of Tris, 50 mM KCl (pH 8.3), 2 mM of MgCl2, 0.4 mM of dNTPs, 0.4 μM of each primer and 1.25 U of Taq polymerase and amplificated for 5 min at 95 °C for denaturation, thirty cycles of 1 min at 94 °C for denaturation, 1 min at 55 °C for annealing and 1 min at 72 °C for elongation

P. Balseiro et al. / Aquaculture 261 (2006) 1135–1143 Table 1 Arbitrary benchmarks for the evaluation of κ-value following Fegan (2000) κ-value

Evaluation

>0.81 0.61–0.80 0.41–0.60 0.21–0.40 0.01–0.20 0.00

Almost perfect agreement Substantial agreement Moderate agreement Fair agreement Slight agreement Poor agreement

with a final extension step of 10 min at 72 °C in a PCR 9700 thermocycler (Applied Biosystems). All PCR assays included a positive control with DNA

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from a heavily infected oyster and a negative control without DNA. PCR products were resolved in 1% agarose gel (w/v) in TAE buffer gel stained with ethidium bromide. 2.3. DNA sequencing PCR products were cut from the agarose gel, purified with the columns Microcon PCR (Millipore) and ligated into a P-GEM T vector (Promega) following the manufacturer's protocol. Competent Escherichia coli cells Top 10F′ (Invitrogen) were transformed. Cloned PCR products were sequenciated in an ABI Prism 377 DNA sequencer (Applied Biosystems). The sequences

Fig. 1. Heavily parasitized oyster histological slide (A, B) and gill smear (C). Bonamia ostreae cells are usually observed within the cytoplasm (arrows). Bar scale: 10 μm (A, C), 3 μm (B).

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Table 2 Comparison among the different techniques employed in this study, PCR, histology, cytology and the combination of histology and cytology data integrated PCR

Histology

Positive Negative Total

Cytology

Positive Negative Total

Combination of histology and cytology

Positive Negative Total

Cytology

Positive

Negative

Total

Positive

Negative

Total

51–50 (21.3–20.8) 23–28 (9.6–11.7) 74–78 (30.8–32.5) 24–33 (10–13.8) 50–45 (20.8–18.8) 74–78 30.8–32.5) 51–50 (21.3–20.8) 23–28 (9.6–11.7) 74–78 (30.8–32.5)

4–3 (1.7–1.3) 162–159 (67.5–66.3) 166–162 (69.2–67.5) 1–1 (0.4–0.4) 165–161 (68.8–67.1) 166–162 (69.2–67.5) 4–4 (1.7–1.7) 162–158 (67.5–65.8) 166–162 (69.2–67.5)

55–53 (22.9–22.1) 185–187 (77.1–77.9) 240–240 (100–100) 25–34 (10.4–14.2) 215–206 (89.6–85.8) 240–240 (100–100) 55–54 (22.9–22.5) 185–186 (77.1–77.5) 240–240 (100–100)

25–33 (10.4–13.8) 0–1 (0–0.4) 25–34 (10.4–14.2) NA

30–20 (12.5–8.3) 185–186 (77.1–77.5) 215–206 (89.6–85.8)

55–53 (22.9–22.1) 185–187 (77.1–77.9) 240–240 (100–100)

NA

In each cell, the number on the left corresponds to laboratory A and the number of the right to laboratory B. Above: number of samples; Below: percentage.

obtained were compared with those previously published in Gen Bank using BLASTn (Altschul et al., 1990).

coefficient which gives a measure of similarity considering that a negative result gives the same information as a positive one (Dunn and Everitt, 1982):

2.4. Epidemiological parameters

sij ¼ ða þ dÞ=p

The sensitivity of each assay was calculated as the proportion of samples “gold standard” positive for B. ostreae found as positive by the evaluated assay. A combination of both histology and cytology against PCR and the PCR against cytology and histology was used as “gold standard”. In the same way, the specifity is the proportion of oysters “gold standard” negative certified as negative by the evaluated assay. Positive Predictive Value (PPV) was calculated as the proportion of unequivocal positives divided by the number of positives found by each assay tested. It gives the probability that an animal that tests positive actually has the disease. In a similar way, Negative Predictive Value (NPV) was calculated as the proportion of unequivocal negatives divided by the number of negatives found by each evaluated assay. It gives the probability that an animal which tests negative actually does not have the disease. κ-value statistic (Fegan, 2000) was used to measure the level of agreement of the PCR assay and the combination of the classical diagnostic methods in both laboratories and the result was evaluated comparing with arbitrary benchmarks (Table 1). For the comparison between laboratories and between methods, we used the simple matching

where a is the total number of matches, d is the total number of mismatches and p is the total number of characters. 3. Results Following histopathological analysis, B. ostreae was observed as cytoplasmatic inclusions with a diameter ranging from 1 to 3 μm (Fig. 1A, B). It was possible to observe these inclusions with their own nucleus and cytoplasm, perfectly differentiated from the cytoplasm of the haemocyte host (Fig. 1B). B. ostreae was detected in 22.9% (55/240) and 22.1% (53/240) of the oysters in laboratories A and B, respectively. B. ostreae appeared on cytological smears (Fig. 1C) with a slightly greater size than on histological preparations. Light microscope examination of gill smears revealed 10.4% (25/240) and 14.2% (34/240) of oysters positive for B. ostreae in laboratories A and B, respectively. Combining classical methods, B. ostreae was detected in 22.9% (55/240) and 22.5% (54/240) of the analyzed samples in laboratories A and B. Histological analysis revealed that between 12.5 and 8.3%

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Fig. 2. Agarose gel electrophoresis of products of PCR with Bo/BoAs primers. Lane 1 and 6, 100 bp DNA ladder (Invitrogen), Lane 2 and 3, oyster positives by the three techniques. Lane 4: oyster positive by PCR assay and negative by histology and cytology. Lane 5: Oyster negative by all the analysis. Bonamia ostreae band is pointed out with an arrowhead and Ostrea edulis with an arrow.

(30/240 and 20/240) of the samples recorded as negative by cytology were in fact positive (Table 2) and only one sample (0.4%) was diagnosed as negative by histology and positive by cytology in laboratory B. Between 9.6 and 11.7% (23/240 and 28/240) of the samples diagnosed as negative by histological analysis were positive for the PCR assay and between 20.8 and 18.8% (50/240 and 45/240) of the oysters catalogued as negative by gill smear analysis were positive by the PCR

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assay in laboratories A and B, respectively. Comparison with the combination of histological and cytological analyses resulted in between 9.6 and 11.7% more positive oysters detected by PCR than for the standard methods alone (Table 2). Electrophoresis of the PCR products showed a band of 300 bp that belongs to B. ostreae as it was retrieved from BLASTn after being ligated, transformed in E. coli and sequenced (Fig. 2). This band was detected in 30.8% (74/240) samples in the laboratory A and 32.5% (78/240) in the laboratory B. The sensitivity of the technique was higher for the PCR assay (92.7 in laboratory A and 92.6 in laboratory B) than for the combination of both classical diagnostic methods (68.9 and 64.1). The specifity of the combination of histology and cytology was higher than PCR assay (97.6 and 97.5 against 89.7 and 84.9). Instead, the percentage of false negatives for the PCR assay was the lowest of all (7.3 and 7.4), while histology produced 31.1 and 35.9 and cytology reached 67.6 and 57.7 in laboratories A and B respectively (Fig. 3). PPV of the PCR assay ranged between 68.9 and 64.1 and NPV from 97.6 to 97.5. The PPV of the combination of the histological and cytological analysis were 92.7 and 92.6 and the NPV reached 87.6 and 84.9 in laboratories A and B, respectively. κ-value for the laboratory A was 0.72 and for laboratory B was 0.67, showing substantial agreement according to arbitrary benchmarks (Table 1). The agreement between laboratories was calculated as

Fig. 3. Sensitivity, specifity and false negative measures of the PCR assay and the classical methods combined in the two participating laboratories. The values are showed as percentage.

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Table 3 Similarity comparison of the different techniques among laboratories Similarity among techniques

PCR A PCR B Histology A Histology B Cytology A Cytology B

PCR A

PCR B

Histology A

Histology B

Cytology A

Cytology B

1 0.9167 0.8875 0.8875 0.7875 0.825

1 0.8625 0.8708 0.7708 0.8083

1 0.9667 0.875 0.8792

1 0.875 0.9125

1 0.9458

1

Similarity is given by the simple matching coefficient (Dunn and Everitt, 1982).

0.94 and the agreement between techniques is summarized in Table 3. 4. Discussion The need of sanitary control in the movement of stocks of live mollusks around the world, even at local scale, is unquestionable. The extension of some diseases from affected places to free areas has caused serious mortalities in the past (Bower and Figueras, 1989; Alderman, 1996; Bartley and Subasinghe, 1996; Renault, 1996; Yoshimizu, 1996; Friedman and Finley, 2003). The PCR method showed a higher sensitivity than cytological and histological methods. If these traditional techniques are only used as diagnostic methods, false negative results can be obtained with consequences for the commercial trade of live oysters (Bucke, 1988; O'Neill et al., 1998; Culloty et al., 2003). Histology and cytology are widely used as diagnostic tools in mollusk diseases laboratories. The most important advantage of histology is the possibility of detecting other different parasites and the possibility of assess the health status of the mollusk and the presence of lesions (Diggles et al., 2003). Cytology is cheap and fast, and it is also widely used (Bachère et al., 1982; Rogan et al., 1991; Cochennec et al., 1998; Culloty et al., 1999, 2003; Diggles et al., 2003; Mirella da Silva and Villalba, 2004). Gill smears are widely employed in the literature (Bachère et al., 1982; Boulo et al., 1989; Carnegie et al., 2000) and there are not significant differences in the results obtained compared with imprints from other tissues (Mirella da Silva and Villalba, 2004). The need of a skilled observer, high cost and slowness in completion of diagnosis, a lack of sensitivity at early stages of infection are some of the disadvantages of these methods (Culloty et al., 2003; Diggles et al., 2003). Usually, the diagnosis of B. ostreae in histological analysis is linked to the presence of inflammatory changes of the tissues as infiltrations, which are not detected with the cytological method. However, the

presence of lesions in oysters does not confirm the presence of the parasite since they can be caused by other pathogens or even by pollutants (Anderson et al., 1995; Renault and Cochennec, 1995; Weinstein, 1997; Oliver et al., 2001; Kleeman et al., 2002). Using histology and cytology as the only method of control of the movements of stocks between regions, the introduction of an infected stock in a zone free of Bonamia spp. may occur (Balouet et al., 1983; Bucke, 1988; O'Neill et al., 1998; Culloty et al., 1999, 2003; Diggles et al., 2003; Carnegie and Cochennec-Laureau, 2004; Mirella da Silva and Villalba, 2004). The high sensitivity of the PCR assay, higher NPV than classical methods and lower number of false negative results for the B. ostreae diagnosis makes this especially suitable for the screening of the movements of stocks and eradication programs on bonamiosis (Van Banning, 1987). On the other side, high specifity of the classical methods (histology and cytology) and higher PPV make these two others more suitable for confirmatory analysis. However, as none of the techniques is 100% accurate, it remains the risk of movement of Bonamia cells. Therefore, it should be desirable to quarantine the oysters at the site of introduction and, prior to release to the environment the oysters, to repeat PCR screening. Different samples taken from the same individual were analyzed in parallel in two separate laboratories. Percentages of similarity are measures of agreement among techniques, and do not give information about the accuracy of a particular test (Fegan, 2000) The percentage of similarity between laboratories was over 90% despite of the diagnostic methods employed and κ-value showed substantial agreement between techniques in both laboratories, which could reveal good skilled personnel for the detection of B. ostreae in both laboratories. This comparison between laboratories was not carried out in previous studies comparing diagnosis techniques in mollusk pathogens (Stokes et al., 1996; O'Neill et al., 1998; Carnegie et al., 2000; Diggles et al., 2003; Mirella da Silva and Villalba, 2004; Meyer et al., 2005). Some of these

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studies have stated the more sensibility of the PCR analysis. Employment of cytological and histological techniques were more controversial, since different papers found cytological imprints more sensitive than histological slides (Bachère et al., 1982; Diggles et al., 2003) and vice versa (Balouet and Poder, 1983; Zabaleta and Barber, 1996). Mirella da Silva and Villalba (2004) stated that there were not significant differences between histological and cytological diagnostic methods. PCR diagnostic methods have four major advantages: First, PCR is cheaper than histopathology. Calculations made in our laboratory shows that the price of processing a stock of thirty oysters by PCR is about half that processing it for histopathology. Second, PCR is faster than histopathology. The technique of histology takes at least five days, against between one or two days for the DNA extraction and PCR assay. Third, PCR is more sensitive than the histopathological and the cytological analysis and, finally, it is easy to train a technician to perform PCR tests. Other important application of PCR is the possibility of detection of the parasite in water. In this way, PCR is suitable for monitoring B. ostreae in the effluents of a mollusk depuration facility; or for monitoring the ballast water, being an useful tool for limiting the spread of the disease (Harvell et al., 1999). Acknowledgements The authors wish to thank E. Amorín, D. Pose, M. Pazos, B. Villaverde and L. Amo for their technical assistance and Dr. A. Ordás for his statistical advice. This research was supported by Plan Galego de Investigación e Desenvolvemento Tecnológico (Ref. PGIDT01MAR40203PR). Secretaría Xeral de Investigación e Desenvolvemento. Xunta de Galicia. Spain. We wish to acknowledge also Sanidad Animal, Ministerio de Agricultura y Pesca, Spain. P.B. is supported by I3P fellowship of the Consejo Superior de Investigaciones Científicas (C.S.I.C.). The helpful comments and suggestions of the reviewers were greatly appreciated. References Alderman, D.J., 1996. Geographical spread of bacterial and fungal diseases of crustaceans. Rev. Sci. Tech. 15, 603–632. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Anderson, T.J., Hine, P.M., Lester, R.J.G., 1995. A Steinhausia-like infection in the ovocytes of Sydney rock oysters Saccostrea commercialis. Dis. Aquat. Org. 22, 143–146. Bachère, E., Grizel, H., 1985. Réceptivité de trois populations naturelles d'huîtres plates Ostrea edulis L. au protozoaire Bona-

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