Structure-activity relationships of piscidin 4, a piscine antimicrobial peptide.

Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 µM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.

Evidence for synergism of the antimicrobial peptide piscidin 2 with antiparasitic and antioomycete drugs.

Piscidins are potent, broad-spectrum, host-produced antimicrobial peptides (AMPs) that appear to constitute the most common AMP family in teleost fish. Here, we show that piscidin 2 has potent activity against the water mould Saprolegnia, one of the most important pathogens of freshwater fish. The minimum oomyceticidal concentration (MOC100) of piscidin 2 against zoospores of three pathogenic isolates of Saprolegnia ranged from 12.5 to 25.0 µg mL⁻¹. This piscidin concentration is well within levels that have been estimated to be present in at least some fish (1-32.5 µg mL⁻¹). In the presence of either copper or malachite green, two drugs commonly used to treat water moulds, there was evidence for partial synergism (PSYN) with piscidin 2. There was also evidence for PSYN after exposure of the ciliate parasite Tetrahymena pyriformis to piscidin 2 plus copper. Our data provide further evidence that piscidins may be an important host defence against skin and gill pathogens and that the piscidin levels in host tissue might influence the success of drug treatments.

Histopathology of culture-associated skin erosions and papillary hyperplasia of Atlantic halibut, Hippoglossus hippoglossus (L.).

Atlantic halibut, Hippoglossus hippoglossus (L.), that were cultured in tanks with a smooth bottom (gel-coated fibreglass) substrate developed papillary hyperplasia and skin erosions on the blind (ventral) side. No viruses, bacteria or parasites were observed in any sections of affected skin. Comparison of microscopic pathology with that of skin from normal, wild-caught halibut showed severe epidermal proliferation with foci of severe mucous cell hyperplasia. Both epidermal thickness and mucous cell density were significantly greater in fish held on a smooth substrate compared to skin of healthy fish. Spongiosis was present, especially at the base of the papillary, hyperplastic epidermis, and there was a chronic inflammatory infiltrate in the scale pockets composed of lymphocytes, histiocytes and erythrocytes. Skin erosions had various degrees of epidermal loss, in some cases to the basement membrane. A predisposing factor for the epidermal sloughing may have been related to the spongiosis, and the vacuolated, degenerated basal cells. When affected halibut were cultured for an additional 28, 62 or 97 days on a sand substrate, which is conducive to skin lesion healing, there was no apparent change in epidermal thickness over time. However, mucous cell density significantly increased from day 0 to 97 during the healing process.

Antiparasitic activity of the antimicrobial peptide HbbetaP-1, a member of the beta-haemoglobin peptide family.

A family of antimicrobial peptides (AMPs) derived from the beta-subunit of haemoglobin was recently isolated from channel catfish, Ictalurus punctatus, infected with Ichthyophthirius multifiliis (ich), an important freshwater fish parasite that causes ichthyophthiriosis. We previously discovered that one of these AMPs, HbbetaP-1, had strong cidal activity against ich as well as another ectoparasite, Tetrahymena pyriformis. HbbetaP-1 toxicity was specific, primarily affecting the trophozoite (trophont) stage of ich. Here, we show that HbbetaP-1 acts more rapidly to kill smaller (presumably less mature) trophonts of ich, taking almost twice as long to kill larger trophonts (P < 0.0001). It acts more rapidly than an unrelated AMP, piscidin 1, which is haemolytic and also lethal to ich trophonts. HbbetaP-1 is potently and selectively lethal to the trophont stage of the dinoflagellate ectoparasite, Amyloodinium ocellatum, one of the most important pathogens of warmwater marine fish. HbbetaP-1 has no effect on the fish gill cell line feeder layer (G1B cells) used to propagate Amyloodinium, further suggesting a highly selective action. These findings suggest that HbbetaP-1 or related AMPs might function in protecting marine as well as freshwater fish and that HbbetaP-1 has highly selective activity against specific life stages of important fish ectoparasites.

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.

Development of an ELISA for quantification of the antimicrobial peptide piscidin 4 and its application to assess stress in fish.

Antimicrobial peptides (AMP) are an integral component of innate immunity. One of the most widespread AMP in fish are the piscidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. The widespread phylogenetic distribution of piscidins suggests that they might play an important host defense role in many fish. Quantifying their expression is important in understanding how and where they function. Using a novel piscidin (piscidin 4) that we recently isolated from commercially cultured hybrid striped bass (white bass, Morone chrysops Rafinesque, female symbolxstriped bass, Morone saxatilis Walbaum male symbol), we optimized the conditions for measuring this piscidin via sandwich ELISA. We used an antibody to the highly conserved amino terminus of all piscidins as the capture antibody and a peroxidase-labeled antibody specific for the carboxy terminus of piscidin 4 as the detecting antibody. Specificity of the detecting antibody was confirmed by lack of cross-reactivity with other piscidins in ELISA, as well as specificity for piscidin 4 in tissue extracts via Western blotting. The accuracy of the test, defined as piscidin 4 recovery, was 96-103%. Precision, measured by the coefficient of variation, was 13-19%, and parallelism, determined by linearity of the response, had an r(2)>0.99. The ELISA paralleled the results obtained via Western blotting. Piscidin 4 levels expressed in gill tissue of healthy hybrid striped bass were well within concentrations that are lethal to important fish pathogens. Mean gill piscidin 4 in healthy hybrid striped bass was significantly greater than in either nutritionally stressed fish or in diseased (ectoparasite-infested) fish, suggesting that piscidin 4 can be significantly downregulated with stress or disease. These data suggest that the piscidin 4 ELISA might be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health.

Lateral line depigmentation (LLD) in channel catfish, Ictalurus punctatus (Rafinesque).

Head and lateral line erosion (HLLE) is a chronic dermatopathy affecting a number of fish that presents as depigmented skin along the lateral line system of the trunk and head. We present microbiological, immunological and histopathological features of this lesion in channel catfish, Ictalurus punctatus (Rafinesque), that developed after exposure to a chronic nutritional stress. Depigmention was limited to skin that was adjacent to the lateral line. The epidermis of affected fish was thin and reduced to a one-cell-thick layer over the lateral line. Melanocytes were depleted at the dermo-epidermal junction and formed aggregates in the epidermis. Innate immunity was weaker in affected fish than that previously measured in well-fed channel catfish. Because the pathology and apparent aetiology of HLLE described in various fish species are highly variable, HLLE appears to be a clinical sign, rather than a disease or syndrome. Thus, we propose that this clinical sign be referred to as lateral line depigmentation (LLD), because this description more accurately encompasses all cases of this presentation reported in fish. As nutritional requirements of channel catfish and lateral line neuroanatomy are well-known, the ability to reproducibly induce LLD in this species could provide a useful model for understanding its pathogenesis.

Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical component of the host's defense against infection. Several types of AMPs have been recently identified from mucosal tissues or immune cells of a number of teleosts. Among these are the piscidins, which are 22 residue, alpha-helical AMPs that were originally isolated from mast cells of hybrid striped bass Morone saxatilis male x Morone chrysops female. Using an antibody specific for the conserved N-terminal amino acid sequence of piscidin 1, we used immunohistochemistry to probe skin, gill, and gastrointestinal tract of 39 teleosts representing 7 different orders. Nine fish species were piscidin-positive, with all of these species being in the Perciformes, the largest and most evolutionarily advanced order of teleosts. Piscidin-positive cells were identified in species belonging to the families Moronidae, Serranidae, Sciaenidae, Siganidae and Belontidae. Immunopositive cells were usually most consistent with mast cells, although in some species, the granule appearance and tinctorial properties diverged somewhat from those of a typical piscine mast cell. In addition, rodlet cells were piscidin-positive in one member of the family Cichlidae; to our knowledge, it is the first time that a host-associated chemical biomarker has been identified in rodlet cells. Our data suggest that piscidins are present in many evolutionarily advanced teleosts. Piscidin-immunoreactive cells were most common at sites of pathogen entry, including the skin, gill and gastrointestinal tract. These results strongly suggest that piscidins are a widespread and important component of many fishes' defense against disease.

Whole blood assay for examining lymphocyte blastogenesis of percichthyid bass (Morone) [corrected].

A simple and reproducible method was developed for the measurement of blastogenesis of peripheral blood lymphocytes using whole blood of hybrid bass (striped bass [Morone saxatilis] female x white bass [M. chrysops] male) stimulated with Concanavalin A, phytohemagglutinin-P, lipopolysaccharide or pokeweed mitogen. Compared to traditional methods which use leucocyte separation procedures, whole blood culture is faster and less expensive. Only small aliquots of blood (10 microliters per culture well) were needed, which would be beneficial for sampling small fish as well as for taking multiple samples from single animals. Optimal culture conditions for hybrid bass, including mitogen concentration, incubation temperature and incubation period, were determined. This is the first report to demonstrate a blastogenic response of whole blood cells in fish.

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.

Histone-like protein: a novel method for measuring stress in fish.

We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health.

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.

Invasion of Tilapia mossambica (cichlidae) viscera by the monogenean Enterogyrus cichlidarum.

The monopisthocotylean monogenean Enterogyrus cichlidarum, which normally inhabits the stomach and anterior intestinal lumina of tilapine cichlids, invaded the internal organs of juvenile Mozambique tilapia (Tilapia mossambica). Adult worms infected the intestinal mucosa, peritoneal cavity, liver, heart, blood vessels, swimbladder, and braincase. Most of the parasites were adults and contained eggs. Both adults and eggs incited a mononuclear inflammatory response. Infections were associated with chronic morbidity and mortality. Although affected fishes were overcrowded, the precise stress responsible for such an unusually invasive event is unknown.

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.

Some unusual features of mycobacteriosis in the cichlid fish Oreochromis mossambicus.

Adult, aquarium-reared Oreochromis mossambicus (Tilapia mossambica), which were naturally infected with Mycobacterium marinum, displayed non-healing skin ulcers and other clinical signs considered to be typical of piscine mycobacteriosis. However, in addition, they frequently had melanotic foci in the skin and spleen, due to the presence of pigment cells surrounding the cutaneous and splenic inflammation. Such melanotic foci have never been reported in response to mycobacteriosis. All fish also have variable numbers of melanomacrophages which appeared to replace pancreatic acini. The relationship of the pancreatic melanomacrophages to the pathogenesis of mycobacteriosis is uncertain, but such lesions may have contributed to the chronic cachexia associated with this case.

Etiologies, observations and reporting of estuarine finfish lesions.

Lesions in estuarine finfish are associated with a variety of organisms including parasites and bacterial, viral, and fungal infectious agents. In addition, trauma, suboptimal water quality, and other abiotic stress factors may result in the loss of homeostasis. We have observed solitary ulcerative lesions on menhaden sampled from the Chesapeake Bay, Maryland, the Pimlico River, North Carolina, and the St. Johns River, Florida. Histologically, the lesions demonstrated a marked chronic inflammatory infiltrate and granulomas in response to fungal hyphae throughout large areas of exposed necrotic muscle. Gram-negative rod-shaped bacteria were also observed in the lesions, a common finding in ulcers of aquatic organisms. Similar observations in menhaden and other species have been described previously in the literature as ulcerative mycosis, mycotic granulomatosis, red spot disease, and epizootic ulcerative syndrome. Despite the many different known causes of fish lesions, the popular press and the scientific literature have recently emphasized Pfiesteria piscicida and other Pfiesteria-like dinoflagellates (and their bioactive compounds) as the primary causative agent for finfish lesions, particularly mycotic granulomatous ulcers in Atlantic menhaden. While some laboratory data suggest that Pfiesteria may play a role in field-observed lesions, much more cause-and-effect evidence is needed to determine the importance of other risk factors, both alone or and in combination with Pfiesteria. In order to better understand the etiology of lesion initiation and progression in estuarine finfish, accurate assessments of environmental conditions collected on appropriate temporal and spatial scales, and fish morphological indicators consistent with gross and histological pathologic terminology, should be used for reporting fish lesion observations and kills. Further, this outlook will help to avoid bias and may foster a broader perspective for examining the health of estuarine systems in general.

Quantitative comparison of the stress response of striped bass (Morone saxatilis) and hybrid striped bass (Morone saxatilis x Morone chrysops and Morone saxatilis x Morone americana).

Striped bass (Morone saxatilis) exposed to a standardized confinement stress had markedly different clinical and endocrinologic responses, compared with hybrid striped bass exposed to the same stress. Plasma cortisol concentration increased at a faster rate and appeared to reach a higher value in striped bass than in hybrid bass. Mean plasma cortisol concentration was 742 +/- 43 ng/ml in striped bass, compared with 490 +/- 37 and 531 +/- 40 ng/ml in striped bass x white perch (M americana) and striped bass x white bass (M chrysops) hybrids, respectively, after a 45-minute net confinement. Plasma cortisol concentration also remained significantly (P = 0.003) higher in striped bass for at least 48 hours after the net confinement. These hormonal differences were associated with a markedly lower survival and resistance to infection in striped bass, compared with the hybrids.

Kidney biopsy: a nonlethal method for diagnosing Yersinia ruckeri infection (enteric redmouth disease) in rainbow trout (Salmo gairdneri).

The sensitivity and specificity of kidney biopsy were 93 and 88%, respectively, for detecting Yersinia ruckeri infection in rainbow trout (Salmo gairdneri). There was no statistically significant difference between results obtained by kidney biopsy and those obtained by necropsy, the standard method for isolation of this agent from the kidney. One hundred percent of conscious fish that were tested survived the procedure.

Epidemic oral ulceration in largemouth bass (Micropterus salmoides) associated with the leech Myzobdella lugubris.

An epidemic of severe ulcerations of the tongue and buccal cavity was documented in subadult and adult largemouth bass (Micropterus salmoides) from Currituck Sound, North Carolina (USA). Other external clinical signs were not present. Between November 1986 and May 1987, reports from fishermen indicated that as many as 90% of large (greater than 300 mm total length) bass were affected. Older fish were the most commonly involved. The leech, Myzobdella lugubris (= Illinobdella moorei), was consistently present on or near the lesions. Lesions were heavily infected with several different bacteria that were apparently secondary invaders. Stressful (high) salinity or an interruption in the normal migratory cycle of the parasite are suggested as possible causes for this condition in these bass.

Cataracts in cichlid fish.

Bilateral cataracts were detected in a group of aquarium-raised tilapia (Sarotherodon mossambicus). Vision impairment was manifested as random swimming into objects, but the fish appeared to function normally otherwise. Most of the lens was opaque, with the outer extent of the cortex and subcapsular region remaining transparent. Skeletal abnormalities, ie, deformed fins, abnormal jaws, and curvature of the spine, were observed in affected individuals. Histologically, there was progressive degeneration of the cataractous portion as the affected fish matured. Preliminary breeding experiments indicated that the condition may be inherited.