Detection of antibody response against Amyloodinium ocellatum (Brown, 1931) in serum of naturally infected European sea bass by an enzyme-linked immunosorbent assay (ELISA)

Blood samples of nineteen sea bass from a naturally infected cultured population were analysed in order to evaluate specific antibody response against the parasitic dinoflagellate Amyloodinium ocellatum. The specific antibody response was evaluated by an indirect enzyme-linked immunosorbent assay (ELISA), using whole parasite crude antigen and rabbit IgG anti sea bassIgM. Experimental data showed that some of sampled specimens developed an adaptive immunological response against the parasite, detectable by indirect ELISA. Results let to conjecture that sea bass, showing an acquired immunity against A. ocellatum, could develop a partial resistance against new infections of the dinoflagellate. Introduction Amyloodiniosis is due to the non-specific dinoflagellate Amyloodinium ocellatum; the parasite affects the host during the summer season, when the high temperatures favour its life cycle (Paperna, 1984), producing disease outbreaks and causing severe losses in cultured fish (Ghittino et al., 1980; Paperna, 1980). Some authors pointed out an acquired immunity against A. ocellatum mediated by specific antibody response in tilapia Oreochromis aureus (Smith et al., 1992; Smith et al., 1993; Smith et al., 1994) and in tomato clown fish Amphiprion frenatus (Cobb et al., 1998a; Cobb et al., 1998b). Tomato clown fish develops strong resistance to the infection following repeated but not lethal challenges although specific antibodies were not always detectable in immune fish (Cobb et al., 1998a). According to Cobb et al. (1998b), the protective response of tomato clown fish is long-lived and it is directed against trophont stage of the dinoflagellate. Our experiment was performed in order to evaluate the presence of a specific antibody response in a cultured sea bass population in which an outbreak due to A. ocellatum was observed 6 weeks before sampling. Materials and Methods Blood samples were collected at the beginning of August 1999, with an environmental temperature of 25°-27°C, from 19 sea bass aged 1+ reared in the same tank of a land-based fish farm where amyloodiniosis is endemic and always present during the summer season. Among the sampled population, an outBull. Eur. Ass. Fish Pathol., 21(3) 2001, 105 break of A. ocellatum causing mortality was shown during the middle of June of the same year. After the outbreak, the parasitosis was controlled by submitting the fish population to cupper sulphate treatments scheduled every 3-5 days. After collection, blood samples were allowed to clot overnight at 4°C and sera were obtained by centrifugation and stored at -20°C until analysis. At the moment of sampling, the intensity and the prevalence of infection was very low due to the repeated antiparasitic treatments, operated in order to avoid new outbreak events. Specific immune response was detected using an indirect enzyme-linked immunosorbent assay (ELISA) procedure, using sonicated trophonts as antigen. In order to obtain antigens for the indirectELISA, trophonts were isolated from infected fish gills by gently scraping and put into 100 litres aquarium where ten sea bass, ranging 50-80 grams, were kept. High infection intensity was obtained within 48 hours, when infected fish were killed and the new generation of trophonts was isolated by gently scraping the infected gills. Trophonts were than washed three times in cold sterilised physiological solution containing penicillin (100 IU ml-1) and streptomycin (100 μg ml-1). Parasites were successively purified from cellular debris by a discontinuous density gradient made with 30%, 60% and 80% Percoll (Pharmacia Biotech®) in HBSS. After centrifugation (100 g for 10 min), purified trophonts where harvested from the bottom of the tube, whereas mucous, bacteria, cells, cellular debris and small clots formed layers at the upper interfaces. Purified parasites were washed three times in cold sterilised physiological solution containing the same antibiotics and three times in cold sterilised solution antibiotic free. After purifications, parasites were counted under stereoscope and successively sonicated with a mini-probe for 3 minutes with a cycle of 15 seconds on and 15 seconds off. Sonicated parasites were centrifuged (4000 rpm for 15 min) and finally protein content of the supernatant was determined using a BCA protein assay kit (Pierce®). Approximately 220,000 sonicated trophonts were equivalent to 420 μg of protein. A flat-bottomed 96-well microtitre plate (Maxisorp, Nunc®, Roskilde, Denmark) was coated with whole parasite crude proteins (5 μg ml-1) in 50 mM carbonate coating buffer, pH 9.6, and incubated overnight at 4°C. For each washing step, the plate was washed 3 times with PBS containing 0.1% Tween 20 (PBS-T) and 3 times with PBS. Uncoated sites were blocked with 3% bovine serum albumin in PBS-T for 1 h at room temperature. Test fish sera, diluted 1:40 in PBS-T, were incubated in triplicate overnight at 4°C. Five negative control sera at the same dilutions as the test sera were included in the assay. The diluition 1/ 40 was chosen becouse in preliminary tests (data not shown) we obtained the highest absorbance values from the positive samples compared to the lowest absorbance values from negative controls. Subsequently, rabbit anti-sea bass purified IgG, diluted 1:400 in PBS-T, was incubated for 1 h at 37°C in a moist chamber. Moreover, the plate was incubated for 1 h at 37°C with Mab anti-rabbit IgG (γchain specific) peroxidase conjugate (Sigma®), diluted 1:2000 in PBS-T. The enzymatic reaction was obtained with the substrate orthoBull. Eur. Ass. Fish Pathol., 21(3) 2001, 106 phenylenediamine and the reaction was stopped with 3M H2SO4. ELISA readings of optical density (O.D.) were performed at 492 nm on a microplate reader (Model 550, BioRad®). In order to obtain the upper limit of negativity with a 99.9% confidence limit, positive value was set at the mean optical density value of five negative control sera plus three standard deviations of the mean, as reported by Crowther (1995). Negative control sera were obtained from fish held in a recirculation system supplied with artificial salt water where any species of parasite was never observed. Results and Discussion As shown in figure 1, blood samples of eleven of nineteen specimens showed positive titres against A. ocellatum, comparing to optical density values obtained from the five not-exposed fish. In fact, optical density values greater than 0.08, obtained from the mean optical density value plus three standard deviation of the negative control sera, should be considered positive for anti-A. ocellatum antibody. The remaining part of sampled specimens showed lower optical densities values than positive threshold. Among the positive fish, only three specimens (the first, the sixth and the thirteenth, respectively) showed high optical densities, being 0.194, 0.146 and 0.141, whereas the remaining eight positive blood samples showed lower optical densities, included between 0.086 and 0.105, very close to the border between positive and negative titres. Infectious resistance of fish against Protozoa of external surfaces was shown for some of the most important parasites of cultured fish. Figure 1. Serum immune response of sea bass naturally infected by Amyloodinium ocellatum. The dotted line indicates the border betwen positive and negative titres (mean ± 3SD). Each spot represents optical density of in ELISA of sera from individual sea bass. Bull. Eur. Ass. Fish Pathol., 21(3) 2001, 107 Among them, the protection against infections due to A. ocellatum and Ichthyophthirius multifiliis involves both the innate and the acquired immune systems (Woo, 1996; Dickerson and Clark, 1996). Moreover, the acquired immune system can be stimulated either by specific vaccinations (Smith et al., 1993; He-Jiang et al., 1997; El-Bahy and Gado, 1999), or by repeated but not lethal parasitic challenges, as shown by Cobb et al. (1998a) in tomato clown fish, or by a parasitic epizootic (Smith et al., 1994). As regards to our results, humoral antibody response of sea bass against A. ocellatum was detectable in some of the sampled specimens cultured in a fish farm where the parasitic dinoflagellate was endemic. At the same time, the level of antibody response of the positive fish, as shown by low optical densities, seems to show a poor acquired immunity of sea bass against the parasite. This could be due to the repeated treatments against A. ocellatum that reduced the contact between host and parasite, as resulted by the low intensity and the low prevalence of infection at the sampling time. Moreover, according to Cobb et al. (1998a), specific serum antibodies could not be always detectable in immune tomato clown fish. The same authors suggested that there might be a localised antibody response in external epithelial tessues, although they rarely detected specific antibodies in skin mucus. According to our earlier “on field” experience (Cecchini, unpublished data), such association could be hypothesised for sea bass as well. In fact, an increased resistance to the parasitosis is generally observed among older fish, compared to 0+ ones and this could be interpreted as an acquired immune response, following natural occurrence of repeated con-tact with the parasitic dinoflagellate. ReferencesCobb, C.S., Levy, M.G., Noga, E.J. (1998a) Ac-quired immunity to amyloodiniosis is associ-ated with an antibody response. Dis. Aquat.Org. 34: 125-133. Cobb, C.S., Levy, M.G., Noga, E.J. (1998b)Development of immunity by the tomatoclownfish Amphiprion frenatus to thedinoflagellate parasite Amyloodiniumocellatum. J. Aquat. Anim. Health 10: 259-263. Crowther J.R. (1995) ELISA. Theory and Prac-tice. Methods in Molecular Biology. Vol. 42.Pp 223. Humana Press, Totowa, New Jersey. Dickerson, H.W., Clarck, T.G. (1996) Immuneresponse of fishes to ciliates. Annual Reviewof Fish Diseases 6: 107-120. El-Bahy, N.M., Gado, M.M. (1999) Immuniza-tion trials against Ichthyophthirius multifiliisFouquet (Ciliophora) in fish. Assiut VeterinaryMedical Journal 41: 185-199. Ghittino, P., Bignani, S., Annibali, A., Boni, L.(1980) First record of serious oodiniasis in seabass (Dicentrarchus labrax) intensively rearedin brackish water. Riv. Ital. Piscicult. Ittiopatol.15: 122-127. He-JiangYan, Zhan-Yin, Xu-GuoLiang, Gong-ZhiYuan, Toong-JinLam, Yoke-Min-Sin (1997)Protection of goldfish against Ichthyophthiriusmultifiliis by immunization with arecombinant vaccine. Aquaculture 158: 1-10. Paperna, I. (1980) Amyloodinium ocellatum(Brown, 1931) (Dinoflagellida) infestations incultured marine fish at Eilat, Red Sea: epiz-ootiology and pathology. J. Fish Biol. 3: 363-372. Bull. Eur. Ass. Fish Pathol., 21(3) 2001, 108 Paperna, I. (1984) Reproduction cycle and tol-erance to temperature and salinity ofAmyloodinium ocellatum (Brown, 1931)(Dinoflagellida). Ann. Parasitol. Hum. Comp.59: 7-30. Smith, S.A., Levy, M.G., Noga, E.J. (1992) De-velopment of an enzyme-linkedimmunosorbent assay (ELISA) for the detec-tion of antibody to the parasitic dinoflagellateAmyloodinium ocellatum in Oreochromis aureus.Vet. Parasitol. 42: 145-155.Smith, S.A., Levy, M.G., Noga, E.J. (1994) De-tection of anti-Amyloodinium ocellatum anti-body from cultured hybrid striped bass(Morone saxatilis x M. chrysops) during an epi-zootic of amyloodiniosis. J. Aquat. Anim.Health 6: 79-81. Smith, S.A., Noga, E.J., Levy, M.G., Gerig, T.M.(1993) Effect of serum from tilapia Oreochromisaureus immunised with dinosporeAmyloodinium ocellatum on the motility, infec-tivity and growth of the parasite in cell cul-ture. Dis. Aquat. Org. 15: 73-80. Woo, P.T.K. (1996) Protective immune re-sponse of fish to parasitic flagellates. AnnualReview of Fish Diseases 6: 121-131.