Veterinary Parasitology, 42 ( 1992 ) 145-155 Elsevier Science Publishers B.V., Amsterdam

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Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to the parasitic dinoflagellate Arnyloodinium ocellatum in Oreochromis aureus S.A. Smith a'~, M.G. L e v y b a n d E.J. N o g a a ~Departtnent of Companion Anirnal and Special Species Medicine bDepartment ~f Microbiology, Pathology and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA (Accepted 13 August 1991 )

ABSTRACT Smith, S.A., Levy, M.G. and Noga, E.J., 1992. Development of an enzyme-linked immunosorbent assay ( ELISA ) for the detection of antibody to the parasitic dinoflagellate Amyloodinium ocellatum in Oreochromis aureus. Vet. Parasitol., 42:145-155. An enzyme-linked immunosorbent assay (ELISA) using antibody to affinity-purified Oreochromis aureus immunoglobulin and antigens from the parasitic dinoflagellate Amyloodinium ocellaturn was developed. The ELISA was then used to evaluate the immune response of the tilapine fish to immunization with the parasite. Fish immunized with antigens of the dinospore stage, either live or sonicared, produced a specific immune response that was detectable by this ELISA. Combinations of serial dilutions ofA. ocellatum antigen and fish anti-A, ocellatum serum were examined to determine which dilutions provided optimal differentiation of seropositive from seronegative fish. Fresh and heat-inactivated serum from both seropositive and seronegative fish produced similar results. INTRODUCTION

With the increasing importance of aquaculture, the study of factors affecting the immunocompetence of aquatic organisms has expanded. During the last few years, various assays have been developed to detect antibodies to bacterial (Busch, 1981 ), viral (Ahne, 1981 ) and parasitic (Dickerson et al., 1986 ) pathogens of fish. With the advancement of assays using immunological principles and techniques, the sensitivity and accuracy of detection have increased. The enzyme-linked immunosorbent assay (ELISA) has been found to be a rapid, accurate, highly sensitive and reproducible technique for both ~Present address of author to whom correspondence should be addressed: Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00

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the detection of specific pathogens and the evaluation of the humoral immune response of fish (Roberson, 1981; Bortz et al., 1984; Hamilton et al., 1987; Thuvander et al., 1987). In addition, the ELISA requires only a small amount of serum that may be obtained by non-lethal methods. Amyloodinium ocellatum is one of the most pathogenic parasites of warm water marine fish (Sindermann, 1990). Epidemics have caused severe morbidity and mortality in aquarium fish (Brown, 1931 ), cultured striped bass (Lawler, 1980), sea bass (Ghittino et al., 1980) and sea bream (Paperna, 1980). As part of an investigation to study the immune response of fish to A. ocellatum, an ELISA was developed to facilitate the evaluation of the specific immune response of the tilapine fish Oreochromis aureus to this parasite. This highly sensitive and specific assay was needed for future studies that would evaluate the optimal method of vaccination for protecting fish against A.

ocellatum. MATERIALS AND METHODS

Experimental animals and maintenance Adult blue tilapia, O. aureus, were obtained from stock maintained by the Department of Zoology, North Carolina State University. The first experimental group consisted of 12 fish of approximately the same size (mean 252 ram, range 220-280 m m ) and weight (mean 313 g, range 205-405 g) which were tagged and then separated into two groups of six fish each. Fish were maintained in a 300 gallon circular tank with a bottom undergravel filtration system at 0 ppt salinity and a temperature of 28 °C. Fish were fed a commercial brand of pelleted feed (3/16 inch trout feed, Zeigler Brothers, Inc., Gardners, PA). The temperature was checked daily and ammonia, nitrite and pH levels of the water were monitored bi-weekly. The second experimental group consisted of 12 fish of approximately the same size (mean 177 mm, range 160-210 m m ) and weight (mean 160 g, range 134-236 g) which were separated into two groups of six fish each. The fish were housed individually at 0 ppt salinity and a temperature of 25 °C in 10 gallon aquaria each with a box filtration system. Fish were fed an artificial diet (Bower, 1983) at body maintenance levels, with temperature and water quality parameters checked as previously described.

Parasite and antigen production Amyloodinium ocellatum (strain 85-1 of DC-1 ), originally isolated from the clownfish (Amphiprion ocellaris), was maintained in cultures of G1B cells (Noga, 1987; Noga and Bower, 1987). Briefly, free-swimming dinospores, less than 48 h old, were collected from cell culture by aspiration and the num-

l M M U N E RESPONSE T O D I N O F L A G E L L A T E AMYLOODINIUM OCELL,ITUM

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bet of dinospores counted in a 0.1 ml sample. Dinospores used for immunizations were either used live or sonicated on ice at a setting of No. 6 with a mini-probe (Branson Sonifier, Cell Disruptor 200, Danbury, CT) for 3 rain with a cycle of 15 s on and 15 s off. The sonicated material was centrifuged at 60 × g for 5 min and the protein content of the supernatant was determined using the Biotrol pyrogallol red method (Biotrol USA, Inc., West Chester, PA). Approximately 600 000 sonicated dinospores were equivalent to 150/tg of protein. The sonicated material was then aliquoted and stored at - 70 ° C. G 1B cells were also similarly sonicated, adjusted to the equivalent amount of protein as the sonicated dinospores and used in the ELISA as described for the A. ocellatum antigen. Fish i m m u n i z a t i o n a n d s e r u m collection

In the first experimental group, six fish were each immunized i.p. with 1.0× 105 live dinospores ofA. ocellatum in 0.5 ml of phosphate-buffered saline (PBS), pH 7.2, per 100 g body weight on Day 0, and boosted i.p. with the same amount of antigen on Days 21 and 42. Six control fish were shamimmunized and boosted i.p. on Days 0, 21 and 42 with 0.5 ml PBS. Blood was collected from both groups of these fish on Day 91 from the caudal tail vessels, allowed to clot at room temperature for 1 h and stored overnight at 4 ° C. Serum was collected the next day by centrifugation in pediatric centrifuge tubes (Microtainer, Becton Dickinson and Company, Rutherford, N J) at 14 0 0 0 × g for 2 min and stored at - 7 0 ° C . In the second experimental group, six fish were each immunized i.p. with 1.0× l0 s sonicated dinospores ofA. ocellatum in 0.5 ml PBS on Weeks 0 and 9. Another six control fish were sham-immunized and boosted i.p. on Weeks 0 and 9 with 0.5 ml PBS. Serum was collected pre-immunization at Week 0 and then every 2 weeks for 18 weeks as previously described. Serum from each of these two groups was pooled and stored at - 7 0 ° C. Aliquots of individual sera from both parasite- and sham-immunized fish were also collected at Week 23 and heat-inactivated at 50°C for 20 min (Sakai, 1981 ). I m m u n ological reagents

Methods for the affinity purification of O. aureus serum immunoglobulin (Ig) and the production of rabbit antiserum have been described previously (Smith et al., 1992a). Briefly, the separation offish Ig from serum of O. aureus immunized with goat anti-mouse IgG (goat IgG) (Cappel Laboratories, West Chester, PA ) was accomplished using an affinity column of agarose beads having covalently linked goat IgG (Sigma Chemical Company, St. Louis, MO). The Ig was eluted with a glycine buffer, neutralized with Tris buffer, dialyzed in PBS and concentrated by centrifugation in a Centricon-30 micro-

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concentrator (Amicon, Danvers, MA). Antiserum to the affinity-purified O. aureus serum Ig was prepared by immunization of laboratory rabbits with 200 #g of the affinity-purified Ig in Freund's complete adjuvant, followed by boosters of 100 ¢tg in Freund's incomplete adjuvant. The rabbits were exsanguinated under anesthesia and the serum collected and stored at - 70 ° C. ELISA

Parasite antigen ( 150/zg m l - 1) was thawed and diluted 1 : 1000 in antigen coating buffer ( 1.6 g sodium carbonate and 2.9 g sodium bicarbonate in 1 1 distilled water, pH 9.6) and 100 ¢tl were placed into each well of a 96-well flat-bottom microtiter plate (E.I.A. Microtitration plate, Flow Laboratories, Inc., McLean, VA). Plates were incubated at 37°C for 2 h and then stored at 4 ° C. Serial doubling dilutions of the parasite antigen ( 1 : 1 0 0 - 1 : 6400) were used to sensitize the microtiter plates, while serial doubling dilutions ( 1 : 5001:16 000) of both fish serum (first antibody) seropositive or seronegative for A. ocellatum and rabbit antiserum (second antibody) seropositive or seronegative for O. aureus serum Ig were evaluated in the ELISA. In addition to comparing various dilutions of parasite antigen, quadruplicate microtiter plate wells were sensitized with a 1 : 1000 dilution of parasite antigen and a 1 : 1000 dilution of G 1B cell antigen. Pre- and post-immunization sera from fish i m m u n i z e d with PBS or live dinospores ofA. ocellatum were compared to determine whether the fish had also produced a specific antibody response to the G 1B cells on which the parasite had been cultured. Antigen-coated microtiter plates were w a r m e d to room temperature ( 2 0 22 °C) and each plate washed four times with approximately 250 ml of ELISA wash solution (8.5 g NaC1 and 0.5 ml Tween 20 in 1 1 distilled water). Two hundred microliters of ELISA blocking buffer (8.5 g NaC1, 0.372 g disodium EDTA and 6.05 g Tris base in 1 1 distilled water (pH 7.4) containing 3% bovine serum albumin) were added to each well and incubated for 1 h at 37°C. Following four washings with ELISA wash solution, 100/~1 of serum diluted 1 : 1000 in ELISA buffer with 0.05% Tween 20 from either parasiteor PBS sham-immunized fish were added to triplicate wells and the plate was incubated for 1 h at 37°C. Following another four washings in ELISA wash solution, 100/zl of rabbit anti-O, aureus Ig serum diluted 1 : 1000 were added to each well and incubated for 1 h at 37°C. After another four washings in ELISA wash solution, 100 ¢tl of peroxidase-conjugated goat anti-rabbit IgG (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, M D ) diluted 1 : 1000 were added to each well and incubated for 1 h at 37°C. The plate was then washed four times with ELISA wash solution and 100 ¢tl of freshly prepared TMB ( 3,3',5,5'-tetra-methylbenzidine ) peroxidase substrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, M D ) were added. The reaction was stopped after approximately 5 min by the addition of 100 #l of 1 M phos-

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phoric acid and the absorbence at 450 nm determined (MR700 Microplate Reader, Dynatech, Sussex, UK). Background non-specific binding was assessed from the optical density (O.D.) values of wells to which all reagents except fish serum had been added. Initially, the minimum positive O.D. value was set at the mean O.D. value of non-exposed individuals plus three standard deviations of the mean (de Savigny and Voller, 1980; Kodoma et al., 1985 ). However, after positive and negative sera from fish in the first experimental group were identified by comparison of O.D. values, ELISA values (EV) of fish in the second experimental group were calculated using a single sample of the previously identified positive and negative sera as controls for the assay. ELISA values were calculated from mean O.D. values of triplicate wells, as follows (Hornitzky and Searson, 1986) EV-

Test fish serum O.D.- ( - )Reference fish serum O.D. ( + )Reference fish serum O.D.- ( - ) Reference fish serum O.D. × 100

An EV greater than two was considered positive (de Savigny and Voller, 1980). RESULTS Using a single rabbit antiserum dilution of 1 : 1000, optimal separation of A. ocellatum-seropositive from A. ocellatum-seronegative fish occurred at a parasite antigen dilution of 1 : 1000 and a fish serum dilution of 1 : 1000. Following evaluation of a population of stock O. aureus (n = 120 ) without prior exposure to A. ocellatum, the minimum positive O.D. value was set at the mean O.D. value of 0.021 (range 0.000-0.070) for non-exposed individuals plus three standard deviations (SD) (0.017) of the mean (de Savigny and Voller, 1980; Kodoma et al., 1985 ). Thus, for this ELISA an O.D. value greater than 0.072 would be considered positive for anti-A, ocellatum antibody. O.D. values for blank wells lacking fish serum, and for wells containing pre- and post-immunization sera from individual fish immunized with either PBS or live dinospores ofA. ocellatum, were compared (Fig. 1 ). Only the post-immunization sera from fish immunized with antigens of the parasite had O.D. values higher than three SD of the mean for non-exposed individuals. O.D. values obtained for both pre- and post-immunization serum samples in wells sensitized with sonicated G 1B cell antigens were slightly higher (mean 0.05 ) than background levels for the A. ocellatum antigen-sensitized wells; however, values were similar for both PBS- and parasite-immunized fish. The ELISA using parasite antigen was then used to examine the kinetics of the immune response of fish in the second experimental group which were

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Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to the parasitic dinoflagellate Amyloodinium ocellatum in Oreochromis aureus.

An enzyme-linked immunosorbent assay (ELISA) using antibody to affinity-purified Oreochromis aureus immunoglobulin and antigens from the parasitic din...
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