The Immune Response of Fish to Amyloodinium: A Model for the Protozoan Ectoparasites

Colorni, A. IOLR Eilat  Israel
Diamant, A. IOLR Eilat  Israel
Levy, M.G. NCSU Raleigh NC USA
Noga, E.J. NCSU Raleigh NC USA
Avtalion, R.R. Bar Ilan U Ramat-Gan  Israel
 

Aquaculture $200,000 3

Final Report Abstract
We developed reagents for examining the immune response of fish to Amyloodinium ocellatum, one of the most important parasites affecting warmwater marine and estuarine fishes. This initially involved development of a simplified technique for the isolation and purification of serum immunoglobulin from fish. This method was used to obtain purified immunoglobulin from blue tilapia (Oreochromis auresu), sea braem (Sparus aurata) and hybrid striped bass (Morone saxatilis x Morone chrysops).

As part of our validation of these immunological reagents, we determined that the immunoglobulins were of an IgM-like class. Blue tilapia Ig had a molecular weight estimated by SDA-PAGE analysis to be about 77 kD, while the two distinct L chains aureus serum immunoglobulin did not react with sera from distantly related species of fish, including rainbow trout, largemouthbass, channel catfish, striped bass, goldfish and some Central and South American cichlids; but did cross-react with sera from more closely related  African cichlids, including species in the genera Oreochormis, Haplochromis  and Pseuditripheus.  Immunoglobulin was not detected in mucus collected from the skin of O. audreus.

An enzyme-linked immunosorbent assay (ELISA) using affinity purified antibody of either blue tilapia, sea bream or hybrid striped bass and antigens from the parasitic dinoflagellate Amyloodinium ocellatum  was developed to evaluate the immune response of these fish to the parasite. Fish immunized with antigens of the sonicated dinospore stage produced a specific immune response that was detectable by this ELISA. Combinations of A. ocellatum  antigen and fish anti-A. ocellatum  serum serial dilutions were examined to determine which dilutions provided optimal differentiation of seropositive from seronegative fish. Fresh and heat-inactivated serum had similar optical density values indicating that complement did not significantly affect the assay. Serum from blue tilapia that were immunized intraperitoneally withAmyloodinium ocellatum   dinospores stimulated an immune response that inhibited the infectivity and growth of the parasite in cell culture.

Fresh serum had a greater inhibitory effect than heat-inactivated serum. Live parasites induced a much stronger immune response than dead, sonicated parasites as detected in both a cell culture infectivity assay and an ELISA. Parasite infectivity was inversely related to serum antibody titers. Serum from fish immunized with live parasites immobilized the infective dinospores at serum concentrations of 5% and above and agglutinated the parasite at serum concentrations from 2.5% to 0.156%. Antibody  to Amyloodinium ocellatum  was  also detected in sea brea, and hybrid striped bass after intraperitoneal injection of live dinospores and in the serum of hybrid striped bass after a natural outbreak of anyuloodiniosis.

We also studied the ability of Amyloodinium ocellatum  to infect gill cellcultures in the presence of serum or mucus from immunologically naive bluetilapia. Serum concentrations as low as 1.25% markedly inhibited parasite infectivity. Serum concentrations greater than 10% were completely inhibitory. Mucus had considerably less inhibitory activity than serum. Heating serum to 47oC for 20 min. or 56oC for 30 min., as well as treating serum with zymosan or carrageenan, suggested that complement-like activity was responsible for at least some of the activity, but that other factors may also influence parasite infectivity.

Using this in vitro data, we attempted to vaccinate fish against amyloodiniosis. Studies were mainly carried out with sea bream, due to its demonstrated susceptibility to Amyloodinium ocellatum  and our difficulty in infecting blue tilapia with the parasite. Several types of immunization regimens were used, including injection of fish with dinospores or tomonts alone as well as with dinospores in complete Freund's adjuvat. While strong serum antibody response was present in some experiments, no immunization regimen produced detectable protection from challenge. However, the difficulty in producing a productive infection on test fish with a small parasite inoculum made these results equivocal.

We also documented an outbreak of amyloodiniosis in pond-cultured hybrid stripedbass in North Carolina, USA, which is the northern most documented outbreak due to an endemic source in fishes of the western Atlantic. Dinospores produced by this Amyloodinium ocellatum  isolate were anteroposteriorly flattened, as contrasted with the gymnodinoid dinospore homogenates of Amyloodinium ocellatum  isolates DC-1 (Bower isolate), RS-1 (Red Sea isolate) and MS-1 (Mediterranean Sea isolate) suggested a close relationship between these two isolates and the DC-1 isolate. Further work is needed to verify these relationships.