Amyloodinium Research Articles

Development of Immunity by the Tomato ClownfishAmphiprion frenatusto the Dinoflagellate ParasiteAmyloodinium ocellatum

The dinoflagellate Amyloodinium ocellatum, which causes amyloodiniosis or “marine velvet disease,” is one of the most serious ectoparasitic diseases affecting warmwater marine fish culture worldwide. We demonstrated that tomato clownfish Amphiprion frenatus can develop strong immunity to infection following repeated nonlethal parasitic challenges. The protective response is long-lived and directed against the trophont stage of the parasite.

Acquired immunity to amyloodiniosis is associated with an antibody response.

The dinoflagellate Amyloodinium ocellatum, which causes amyloodiniosis or 'marine velvet disease', is one of the most serious ectoparasitic diseases plaguing warmwater marine fish culture worldwide. We report that tomato clownfish Amphiprion frenatus develop strong immunity to Amyloodinium ocellatum infection following repeated nonlethal challenges and that specific antibodies are associated with this response. Reaction of immune fish antisera against dinospore and trophont-derived antigens in Western blots indicated both shared and stage-specific antibody-antigen reactions. A mannan-binding-protein affinity column was used to isolate IgM-like antibody from A. frenatus serum. The reduced Ig consisted of one 70 kD heavy chain and one 32 kD light chain with an estimated molecular weight of 816 kD for the native molecule. Immunoglobulin (Ig) isolated from immune but not non-immune fish serum significantly inhibited parasite infectivity in vitro. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal rabbit antibody produced against affinity-purified A. frenatus Ig. Anti-Amyloodinium serum antibody was not always detectable in immune fish, although serum antibody titers in immune fish increased after repeated exposure to the parasite. These results suggest that there may be a localized antibody response in skin/gill epithelial tissue, although antibody was rarely detected in skin mucus.

V5 2:45 Development of immunity by the tomato Clownfish (amphiprion frenatus) to the dinoflagellate parasite amyloodinium ocellatum as evidenced by in vivo and in vitro analysis

V5 2:45 Development of immunity by the tomato Clownfish (amphiprion frenatus) to the dinoflagellate parasite amyloodinium ocellatum as evidenced by in vivo and in vitro analysis

Improved cell culture propagation of Amyloodinium ocellatum

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 24:173-178 (1996) - doi:10.3354/dao024173 Improved cell culture propagation of Amyloodinium ocellatum Oestmann DJ, Lewis DH A system for cultivating Amyloodinium ocellatum was developed using aggregates of red drum Sciaenops ocellatus dorsal fin cells in suspension. Tomonts harvested from infected fish using density gradient centrifugation in Percoll were placed in saltwater media (30 ppt) containing anti-biotics (penicillin 100 IU ml-1, streptomycin 100 mg ml-1). Microbe-free infective dinospores emerging from the tomonts were used to infect red drum cell aggregates. Dinospores attached and transformed into trophonts on the cell aggregates, developing into normal size (80 um) trophonts in saltwater media (10 ppt). Trophont infected cell aggregates were placed in increased saltwater concentration (30 ppt) at 48 h incubation to induce the release of trophonts and their encystment as tomonts. Tomonts were separated from the cellular debris by saltwater media washing and Percoll density gradient centrifugation. Tomonts were then incubated to produce a second generation of dinospores. The parasite was maintained in vitro for 10 complete life cycles with no observed decreased viability using this system. Amyloodinium . Protozoa . Propagation . Cell culture Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 24, No. 3. Publication date: March 07, 1996 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1996 Inter-Research.

Protective immune response of fish to parasitic flagellates

The piscine immune system is normally quite efficient in protecting the host (innate and acquired immunity) from parasitic infections. Innate immunity may occur at two distinct levels — between host species and within a host species. If the resistance is at the host species (or a higher taxonomic group) level, then it is inter-host innate immunity. For example, Oncorhynchus mykiss can be infected with the pathogenic hemoflagellate, Cryptobia salmositica isolated from Oncorhynchus spp. but cannot be infected with Cryptobia catostomi from Catostomus commersoni. At the next level, there are individuals within a susceptible host species that are resistant to infection — this is intra-host innate immunity; e.g. some Salvelinus fontinalis are resistant to C. salmositica infection while others are not. This resistance to infection is not dependent on age or size of the fish; it is inherited and is controlled by a dominant gene. Protection at both levels of innate immunity is via the activation of the alternative pathway of complement activation to lyse the parasite. Also, S. fontinalis can be infected with the pathogenic C. salmositica have very high parasitaemias but they do not suffer from the disease as O. mykiss. This resistance to disease is related to high levels and rapid production of α2-macroglobulin which is one of two natural antiproteases. The α2-macroglobulin in the blood neutralises the metallo-protease secreted by the pathogenic C. salmositica. Acquire immunity was shown in fish that survived infections of pathogenic flagellates. Fish that have recovered from Amyloodinium ocellatum, C. salmositica, Cryptobia bullocki, and Trypanosoma danilewskyi are protected. This protection requires prior exposure to the pathogen and/or its antigens. Humoral (e.g. complement fixing antibodies to lyse the parasite) and cell-mediated (e.g. T-cell cytotoxicity, phagocytosis) are part of the protective mechanism in acquired immunity. Also, an attenuated live C. salmositica vaccine has been developed and it protects juvenile and adult salmonids from cryptobiosis for at least 2 years.

Communications: Clearance ofAmyloodinium ocellatumDinospores byArtemia salina

The potential for using nauplii of brine shrimp Artemia salina to remove the dinoflagellate Amyloodinium ocellatum from aquaculture systems for red drum Sciaenops ocellatus was investigated. Dinospores of A. ocellatum were dispensed in cell culture plates at a concentration of approximately 10,000/mL and were exposed to brine shrimp nauplii at concentrations of l, 2, 4, or 8 nauplii/mL over a 24-h period. 1n the presence of 8 nauplii/mL, dinospores were eliminated in 8 h. In another study, 300,000 dinospores were placed in 3 L of saltwater in two tanks, and l,000 nauplii were added to one tank. After 12 h, three 202-g red drums were placed in both tanks for an additional 12-h-exposure period, after which the fish were euthanized and the trophont load on gill filaments was assessed. Fish from the tank with brine shrimp nauplii had 65% fewer trophonts on their gills than those from the nontreated tank (10.75 versus 3.75 trophonts/filament). These data and the observation that dinospore remains could be observed in naupliar fecal casts suggest the potential value of brine shrimp as a bioremediation measure for this serious gill parasite.

A method for producing microbe-free Amyloodinium ocellatum (Brown) with Percoll®

A method for collecting microbe-free Amyloodinium ocellatum from infected red drum ( Sciaenops ocellatus) is described. Surface contamination of trophonts removed from fish by osmotic shock was reduced by washing the preparation with antibiotic containing saltwater and Percoll® density gradient purification. The resulting tomont preparation was free of external contamination. Sterile, infective dinospores excysted from the tomonts after 72 h incubation at 26°C in antibiotic containing 30 ppt saltwater.

Communications: Detection of Anti-Amyloodinium ocellatumAntibody from Cultured Hybrid Striped Bass (Morone saxatilis×M. chrysops) during an Epizootic of Amyloodiniosis

After an epizootic of amyloodiniosis (caused by the protozoan Amyloodinium ocellatum) in a commercial aquaculture facility for hybrid striped bass (female striped bass Morone saxatilis × male white bass M. chrysops), sera from these fish, as well as from others that had been experimentally immunized with the parasite, were evaluated by an enzyme-linked immunosorbent assay for antibody specific for the parasite. Titers were similar between the fish infested in culture and the experimentally immunized fish, and were significantly higher in both the cultured and artificially exposed fish than in unexposed fish. These results suggest that an infestation of A. ocellatum can stimulate the production of humoral antibodies to the parasite, providing further evidence that natural infestations of the parasite may confer protective resistance in fish that survive the initial parasitic infestation.

The effects of hyposalinity on eggs, juveniles and adults of the marine monogenean, Neobenedenia melleni Treatment of ecto-parasitosis in seawater-cultured tilapia

The effects of hyposalinity on survival of eggs, juveniles and adults of the marine monogenean, Neobenedenia melleni, were studied as a basis for treatment of ecto-parasitosis in seawater-cultured tilapia. Eggs, collected from adult monogeneans at 38 g·l −1, were exposed in vitro to salinities of 6, 12, 15, 18, 24 and 38 g·l −1 (control) for periods ranging from 5 to 14 days, then returned to 38 g·l −1 and post-treatment development and hatching were observed. Hatching was prevented by exposure to salinities of 15 g·l −1 and below for 5 days and 18 g·l −1 and below for 7 days. Seawater-cultured Florida red tilapia infected with N. melleni were exposed to salinities of 12, 15, 18 and 36–38 g·l −1 (control) for periods ranging from 2 to 7 days. Exposure to salinities of 15 g·l −1 or below for a minimum of 2 days eliminated juvenile and adult monogeneans in situ, while exposure to 18 g·l −1 for 7 days was not 100% effective. Under commercial-scale conditions, hyposalinity was tested as a therapeutant for N. melleni parasitosis during seawater growout of Florida red tilapia. Fingerlings (avg. wt.=22 g) were stocked into six 10-m 3 flow-through seawater pools at a density of 30 fish·m −3 and infection levels monitored every 28 days. Based on conditions determined to prevent hatching of N. melleni eggs, fish were treated with brackishwater (18 g·l −1 for 7 days) when average infection level reached ≥ 5 monogeneans/fish. An average of 1.3 (range=0–2) treatments per pool was effective in controlling parasitosis during growout over 210 days. Final survival, body weight, and biomass density of fish in five pools averaged 84.2%, 589 g, and 11.6 kg·m −3, respectively. Survival in one pool was impaired (17.8%) by secondary infection with the dinoflagellate Amyloodinium ocellatum during brackishwater treatment. Based on the eradication of egg, juvenile and adult stages of N. melleni by these conditions, a 5-day treatment with 15 g·l −1 salinity, begun at incipient stages of infection, is recommended for optimum control of parasitosis in practical culture.

Effect of serum from tilapia Oreochromis aureus immunized with the dinospore Amyloodinium ocellatum on the motility infectivity and growth of the parasite in cell culture

Effect of serum from tilapia Oreochromis aureus immunized with the dinospore Amyloodinium ocellatum on the motility infectivity and growth of the parasite in cell culture

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 dinoflagellate Amyloodinium ocellatum 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 sonicated, produced a specific immune response that was detectable by this ELISA. Combinations of serial dilutions of A. 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.

Immunological responses of fish to parasitic organisms

The piscine immune system is well developed and is normally quite efficient in protecting healthy free ranging fish from parasitic diseases. However, when fish are cultured in high numbers and are stressed by adverse environmental factors (e.g. heavy metal pollution, low dissolved oxygen, nutritional deficiencies, and/or overcrowding), parasites may have the advantage and the risk of disease outbreak increases in the fish population. We know very little about innate immunity against parasites. Hence it has not been considered a viable strategy to protect fish from diseases. The alternative pathway of complement activation is the protective mechanism in certain fish species against hemoflagellates (Cryptobia salmositica and Cryptobia catostomi). This mechanism also operates in some resistant individuals (in a susceptible fish species) against C. salmositica. Complement is also involved in innate parasiticidal activities against adult intestinal tapeworms (Acanthobothrium quadripartitum) and encysting larval stages of digeneans (Diplostomum spathaceum and Cryptocotyle lingua). It is suggested here that more studies be conducted to elucidate the mechanism(s) of innate immunity in nonsusceptible host species, and also to follow the heredity of parasite resistant factor(s) in individuals that belong to an otherwise susceptible host fish species.The innate nonspecific cytotoxicity of leukocytes of flounders, infected with the copepod, Phrixocephalus cincinnatus, was significantly depressed compared to those from uninfected flounders. The target cells for the leukocytes were murine cells; future studies should include parasites as target cells to determine the importance of the depression on disease resistance in copepod-infected fish.Fish that survive Amyloodinium ocellatum, C. salmositica, Cryptobia bullocki, Trypanosoma danilewskyi, Ichthyophthirius multifiliis, Myxidium lieberkuehni, a myxosporean parasite (PKX), and D. spathaceum infections are generally protected from disease when they are later exposed to the same parasite. Complement fixing antibodies and/or cell-mediated immunity are important in acquired immunity against C. salmositica, C. bullocki, T. danilewskyi, I. multifiliis, Diplostomum spathaceum, Cryptocotyle lingua, and Posthodiplostomum minimum. A live C. salmositica-vaccine has been developed. The vaccine has fewer polypeptide bands and a few of the remaining polypeptides are antigenically different from those in the virulent strain. It does not cause disease in fish but has remained protective. Fish are assumed to, or are known to, respond immunologically to numerous other parasitic infections. These include: Cryptocaryon irritans, Myxosoma cerebralis, Gyrodactylus bullatarudis, Dactylogyrus vastator, Neobenedenia melleni, Telogaster opisthorchis, Ligula intestinalis, Diphyllobothrium, Caryophyllaeus laticeps, Pomphorhynchus laevis, Anguillicola crassus, and Larnaea cyprinacea. However, the precise role of the immune system in these infections has yet to be determined.In general, there are little antibody and cell-mediated responses during early myxosporean infections because the parasite antigenically mimics host tissues. The proliferative type of inflammation is the principal defense when spores are formed, resulting in parasite encapsulation. Melanomacrophages ingest spores in tissue and transport them to kidneys, spleen, or liver where they are encapsulated and destroyed. Also, cestodes are thought to adsorb host antigens on their body surface to evade the host immune system. The extent and the mechanism of host tissue mimicry by parasites, presumably to avoid or reduce the host immune response, are both fascinating and in need of more careful study in the future.Not much is known about immunodepression due to parasitic diseases in fish. This is an important phenomenon because it generally increases the susceptibility and mortality of infected fish when they are exposed to another pathogen. Also, it will adversely affect vaccination programs carried out against other pathogens. Immunodepression has been found in C. salmositica, Glugea stephani, and PKX infections. It is suggested here that other parasitic infections should be closely examined for their effects on the piscine immune system. It is also beneficial to more closely evaluate the interactions of multiple infections (microbial and parasitic) and their effects on the fish.

Effect of Serum and Mucus of Blue Tilapia, Oreochromis aureus on Infectivity of the Parasitic Dinoflagellate, Amyloodinium ocellatum in Cell Culture.

We studied the ability of Amyloodinium ocellatum to infect gill cell cultures in the presence of serum or mucus from naive blue tilapia Oreochromis aureus. 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 47°C for 20 min or 56°C for 30 min, as well as treating serum with zymosan or carrageenan, suggested that a complement-like factor was responsible for at least some of the activity, but that other factors may also influence parasite infectivity.

Amyloodiniosis in Cultured Hybrid Striped Bass (Morone saxatilis×M. chrysops) in North Carolina

Abstract An outbreak of amyloodiniosis in pondcultured hybrid striped bass × white bass (female Morone saxatilis × male M. chrysops) is the northernmost documented outbreak caused by an endemic source in fish of the western Atlantic. Dinospores produced by this isolate of Amyloodinium ocellatum were anteroposteriorly flattened, as contrasted with the gymnodinoid dinospores of the A. ocellatum-type species, suggesting that more than one strain or species of A. ocellatum may exist.

Culture conditions affecting the in vitro propagation of Amyloodinium ocellatum

Culture conditions affecting the in vitro propagation of Amyloodinium ocellatum

Bacterial involvement in a saddleback disease of the reef silverside, At henna harringtonensis Goode (Pisces: Atherinidae) from Bermudan waters

Saddle‐shaped skin lesions commonly occurring in both wild and captive Bermudan reef silversides yielded a mixed infection of motile and non‐motile Gram‐negative cells of Acinetobacter calcoaceticus, Aeromonas hydrophila, Pseudomonas sp., Vibrio parahaemolyticus and a myxobacterial‐like strain. Bacteria occurred as almost pure microcolonies and mixed clumps on proximal scale and fin ray surfaces, in intercellular epidermal spaces, on perimysium of fin erector muscle myomeres and on eroded dorsal fin rays and their intact membranes, but neither on healthy areas of the skin nor within muscle cells. Effects were epithelial destruction and liquefactive necrosis of myomeres, and raising and sloughing of Malpighian cells, scales and dermis. Host response was minimal, consisting of infiltration of a few lymphocytes and macrophages. The fish pathogenic dinoflagellate Amyloodinium ocellatum was seen in one fish with a saddle lesion and caused muscle cell lysis, as evidenced by cloudy cell swelling. Antibiotic sensitivity tests showed increased drug resistance with mixed bacterial types compared to that of single pure isolates. This suggests that alternatives to chemotherapy for control of bacterial fish diseases is appropriate, because most are probably caused by mixed infections.

Propagation of the Marine Dinoflagellate Amyloodinium ocellatum under Germ-Free Conditions

Germ-free infections of Amyloodinium ocellatum were produced on both living fish and in organ cultures. Exposing gnotobiotic guppies (Poecilia reticulata) to 125 dinospores in multiwell tissue culture plates produced nonlethal infections that could be serially propagated. Exposure to 250 or more parasites killed the fish during the first infection cycle, but if the dead fish were incubated in a cell culture medium/seawater mixture, the parasites could survive and reproduce for up to 2 wk in these organ cultures. Organ cultures containing only seawater or those containing bacteria did not support the prolonged survival of Amyloodinium.

Propagation in Cell Culture of the Dinoflagellate Amyloodinium , an Ectoparasite of Marine Fishes

Amyloodinium ocellatum, a common dinoflagellate ectoparasite of marine fishes, was successfully propagated on a fish gill cell line. In vitro infections were similar in cytopathology and development to those reported on natural hosts, and large numbers of parasites could be produced. Exposure of parasites in cell culture to an antiprotozoal drug produced a dose-dependent inhibition of infectivity that was much more sensitive than a motility assay previously used to assess the toxic effects of a drug on protozoan ectoparasites. This propagation system may be a useful model for studying the biology and control of protozoan skin parasites of fishes and for quantitatively studying hostparasite interaction at cellular interfaces.

A Standardized Method of Propagating the Marine Fish Parasite, Amyloodinium ocellatum

The peridinian dinoflagellate Amyloodinium ocellatum was propagated by serial passage in clownfish (Amphiprion ocellaris) and hybrid striped bass (Morone chrysops X Morone saxatilis). Each 25-50-mm fish was exposed to 4,000-6,000 dinospores in 400 ml of artificial seawater for 30 min. Two days after exposure, trophonts were harvested by immersing the fishes in fresh water. After encystment, tomonts were axenized by multiple washes with sterile distilled water and sterile artificial seawater containing penicillin and streptomycin, and then incubated in the antibiotic solution. High yields of both tomonts and dinospores of the same sizes and ages were obtained, and host mortalities were eliminated. Microbial growth in incubating cultures was inhibited until after dinospores had emerged from tomonts, and dinospores remained infective for at least 4 days at 26 C.

Solving parasite-related problems in cultured marine fish

1. 1. Our knowledge of parasite born e diseases of cultured marine fish is restricted by the limited extent of practiced mariculture. 2. 2. Several parasites, pathogenic to fish including capsaloid, microcotylid and diclidophoran monogeneans and caligid copepods are ubiquitous in mariculture systems in diverse species of fish and geographical regions. 3. 3. The alga Amyloodinium ocellatum and the ciliated protozoan Cryptocaryon irritons are not as ubiquitous, but nevertheless present the most serious risk to farmed fish of most species due to their high pathogenicity, fecundity and resistance to conventional means of control. 4. 4. Infections in maricultured fish are dominated by monoxenous parasites. Occurence of heteroxenous parasites is restricted by their requirement for additional hosts to maintain transmission. 5. 5. Cage culture allows free exchange of infection between caged and native fishes; in inshore systems infections have to be introduced primarily from external sources. 6. 6. Water supply does not appear to be an efficient route of entry of infection into culture systems; parasites are introduced either by wild fry or wild breeders collected into the system, or with wild fish infiltrating via water supply or drainage. 7. 7. Adverse growth conditions are the most common circumstances in outbreak of epizooites. Over-crowding does not appear to promote primarily epizooites, its impact is indirect by contributing to the deterioration of the growth conditions. 8. 8. Epizooites are likely to occur also in juvenile, immunologically naive fish as well as following introduction of parasites into unexperienced fish population. 9. 9. Control has been implemented thus far through an empirical approach. Recent research is geared towards the development of methodology specifically designed to the needs of mariculture.