Pyrethroid-associated nephrotoxicity in channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus, at a public aquarium.

Over the course of an approximately 11-month period, an outdoor, freshwater, mixed species, recirculating, display system at a public aquarium experienced intermittent mortalities of channel catfish (Ictalurus punctatus) and blue catfish (I. furcatus). Catfish acutely presented for abnormal buoyancy, coelomic distention, and protein-rich coelomic effusion. Gross lesions typically involved massive coelomic distension with protein-rich effusion, generalized edema, and gastric hemorrhage and edema. Microscopically, primary lesions included renal tubular necrosis, gastric edema with mucosal hemorrhages, and generalized edema. Aerobic culture and virus isolation could not recover a consistent infectious agent. Intracoelomic injection of coelomic effusion and aspirated retrobulbar fluid from a catfish into naïve zebrafish (bioassay) produced peracute mortality in 3 of 4 fish and nervous signs in the fourth compared with 2 saline-injected control zebrafish that had - no mortality or clinical signs. Kidney tissue and coelomic effusion were submitted for gas chromatography tandem mass spectrometry by multiple reaction monitoring against laboratory standards, which detected the presence of multiple pyrethroid toxins, including bioallethrin, bifenthrin, trans-permethrin, phenothrin, and deltamethrin. Detection of multiple pyrethroids presumably reflects multiple exposures with several products. As such, the contributions of each pyrethroid toward clinical presentation, lesion development, and disease pathogenesis cannot be determined, but they are suspected to have collectively resulted in disrupted osmoregulation and fluid overload due to renal injury. Pesticide-induced toxicoses involving aquarium fish are rarely reported with this being the first description of pyrethroid-induced lesions and mortality in public aquarium-held fish.

Evaluating the Effects of Biotoxins on Immune Cell Functions in Zebrafish.

A variety of biological toxins can be present at harmful levels in the aquatic environment. Cyanobacteria are a diverse group of prokaryotic microorganisms that produce cyanotoxins in the aquatic environment. These biotoxins can be hepatotoxins, dermatoxins, or neurotoxins and can affect fish and mammals. At high levels, these compounds are fatal. At non-lethal levels, they act insidiously and affect immune cell functions. Algae-produced biotoxins include microcystin and anatoxin A. Aquatic animals can also ingest material contaminated with botulinum neurotoxin E (BoNT/E) produced by Clostridium botulinum, also resulting in death or decreased immune functions. Zebrafish can be used to study how toxins affect immune cell functions. In these studies, toxin exposures can be performed in vivo or in vitro. In vivo studies expose the zebrafish to the toxin, and then the cells are isolated. This method demonstrates how the tissue environment can influence leukocyte function. The in vitro studies isolate the cells first, and then expose them to the toxin in culture wells. The leukocytes are obtained by kidney marrow extraction, followed by density gradient centrifugation. How leukocytes internalize pathogens is determined by endocytic mechanisms. Flow cytometry phagocytosis assays demonstrate if endocytic mechanisms have been altered by toxin exposure. Studies using isolated leukocytes to determine how toxins cause immune dysfunction are lacking. The procedures described in this article will enable laboratories to use zebrafish to study the mechanisms that are impacted when an environmental toxin decreases endocytic functions of immune cells.

Flavobacterium covae is the predominant species of columnaris-causing bacteria impacting the Channel Catfish industry in the southeastern United States.

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

Trained Immunity Provides Long-Term Protection against Bacterial Infections in Channel Catfish.

Beta glucan exposure induced trained immunity in channel catfish that conferred long-term protection against Edwardsiella ictaluri and Edwardsiella piscicida infections one month post exposure. Flow cytometric analyses demonstrated that isolated macrophages and neutrophils phagocytosed higher amounts of E. ictaluri and E. piscicida. Beta glucan induced changes in the distribution of histone modifications in the monomethylation and trimethylation of H3K4 and modifications in the acetylation and trimethylation of H3K27. KEGG pathway analyses revealed that these modifications affected expressions of genes controlling phagocytosis, phagosome functions and enhanced immune cell signaling. These analyses correlate the histone modifications with gene functions and to the observed enhanced phagocytosis and to the increased survival following bacterial challenge in channel catfish. These data suggest the chromatin reconfiguration that directs trained immunity as demonstrated in mammals also occurs in channel catfish. Understanding the mechanisms underlying trained immunity can help us design prophylactic and non-antibiotic based therapies and develop broad-based vaccines to limit bacterial disease outbreaks in catfish production.

Fin healing and regeneration in sturgeon.

Pectoral fin healing in fin spines and rays were examined in juvenile Atlantic sturgeon Acipenser oxyrinchus oxyrinchus following three different sampling techniques (n = 8-9 fish per treatment): entire leading fin spine removed, a 1-2 cm portion removed near the point of articulation, or a 1-2 cm portion removed from a secondary fin ray. Also, to determine whether antibiotic treatment influences healing, an additional group of fish (n = 8) was not given an injection of an oxytetracycline (OTC)-based antibiotic following removal of the entire leading fin spine. Following fin sampling, fish from different treatments were mixed equally between three large (4,000 I) recirculating systems and fin-ray healing and mortality were monitored over a 12 month period. To assess healing, blood samples were collected at 4 months to measure immune system responses, radiographs were taken at 4, 8 and 12 months to assess the degree of calcification in regions of damaged fins and fins were analyzed histologically at 12 months. Fish grew from a mean weight of 1.8 to 3.2 kg during the experiment and survival was near 100% in all treatments, with only one fish dying of unknown causes. Leukocyte counts, an indication of health status and survival were similar among treatments and in groups with or without antibiotic injection. Radiographs revealed mineralization took longer in fish with the entire leading fin spine removed and was the slowest near the point of articulation, presumably due to the greater structural support for the pectoral fin at this location. Histological sampling indicated spines and rays had similar healing patterns. Following injury, an orderly matrix of collagen bundles and many evenly spaced scleroblasts were present, transitioning to Sharpey fibres, with concentric layers forming lamellar bone. Healing and mineralization were characterized as periosteal osteogenesis and included embedded osteocytes surrounded by an osteoid seam. Chondroid formation was apparent in a few fractures not associated with treatments. The duration of time for external wound healing and internal mineralization of spines and rays depended on the fin treatment, with the slowest healing observed in fish with the most tissue removed, the entire leading fin spine.

Differential gene expression following TLR stimulation in rag1-/- mutant zebrafish tissues and morphological descriptions of lymphocyte-like cell populations.

In the absence of lymphocytes, rag1-/- mutant zebrafish develop protective immunity to bacteria. In mammals, induction of protection by innate immunity can be mediated by macrophages or natural killer (NK) cells. To elucidate potential responsive cell populations, we morphologically characterized lymphocyte-like cells (LLCs) from liver, spleen and kidney hematopoietic tissues. In fish, these cells include NK cells and Non-specific cytotoxic cells (NCCs). We also evaluated the transcriptional expression response of select genes that are important indicators of NK and macrophage activation after exposure to specific TLR ligands. The LLC cell populations could be discriminated by size and further discriminated by the presence of cytoplasmic granules. Expression levels of mx, tnfα, ifnγ, t-bet and nitr9 demonstrated dynamic changes in response to intra-coelomically administered ß glucan (a TLR2/6 ligand), Poly I:C (a TLR3 ligand) and resiquimod (R848) (a TLR7/8 ligand). Following TLR 2/6 stimulation, there was a greater than 100 fold increase in ifnγ in liver, kidney and spleen and moderate increases in tnfα in liver and kidney. TLR3 stimulation caused broad up regulation of mx, down-regulation of tnfα in kidney and spleen tissues and up regulation of nitr9 in the kidney. Following TLR 7/8 stimulation, there was a greater than 100 fold increase in ifnγ in liver and kidney and t-bet in liver. Our gene expression findings suggest that LLCs and macrophages are stimulated following ß glucan exposure. Poly I:C causes type I interferon response and mild induction of LLC in the kidney and R-848 exposure causes the strongest LLC stimulation. Overall, the strongest NK like gene expression occurred in the liver. These differential effects of TLR ligands in rag1-/- mutant zebrafish shows strong NK cell-like gene expression responses, especially in the liver, and provides tools to evaluate the basis for protective immunity mediated by the innate immune cells of fish.

Zebrafish Sensitivity to Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins.

Tissue PAH, blood cell and tissue changes following exposure to water accommodated fractions of crude oil in alligator gar, Atractosteus spatula.

Alligator gar Atractosteus spatula acclimated to brackish water (9 ppt) were exposed to water accommodated fraction oil loadings (surrogate to Macondo Deepwater Horizon, northern Gulf of Mexico) of 0.5 and 4.0 gm oil/L tank water for 48 h. The surrogate oil was approximately 98% alkanes and alkynes and 2% petroleum aromatic hydrocarbons. The 2% petroleum aromatic hydrocarbons were predominately naphthalene. After 48 h, naphthalene levels in fish liver exposed to 0.5 or 4 gm oil/L were 547.79 and 910.68 ppb, while muscle levels were 214.11 and 253.84 ppb. There was a significant decrease in peripheral blood lymphocyte numbers and a significant reduction of granulocytes in the kidney marrow of the same fish. Tissue changes included hepatocellular vacuolization and necrosis, necrotizing pancreatitis, renal eosinophilia, and splenic congestion. After 7 days recovery, liver naphthalene levels decreased to 43.59 and 43.20 ppb, while muscle levels decreased to 9.74, and 16.78 ppb for oil exposures of 0, 0.5 or 4 g/L. In peripheral blood and kidney marrow, blood cell counts returned to normal. The severity of liver and kidney lesions lessened after 7 days recovery in non-oiled water, but splenic congestion remained in all gar.

Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E.

Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

The effects of oil exposure on peripheral blood leukocytes and splenic melano-macrophage centers of Gulf of Mexico fishes.

In August and November 2010 we collected and examined peripheral blood and tissues from three species of Gulf of Mexico fish. Findings were compared to non-exposed control fish. The leukocyte counts of exposed alligator gar were not significantly different from controls, while exposed Gulf killifish and sea trout had significantly decreased lymphocyte counts. Liver ethoxyresorufin-O-deethylase (EROD) values from sea trout were significantly greater than control sea trout EROD values, suggesting poly aromatic hydrocarbon exposure. Splenic melano-macrophage centers (MMCs) from exposed sea trout and Gulf killifish showed a significant increase in number compared to non-exposed fish. Sea trout splenic MMCs were also significantly greater in size. These findings suggest that Gulf fish sampled were exposed to crude oil from the Macondo well and were in a lymphopenic or immuno-compromised state.

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio).

The use of the visible implant elastomer (VIE) tagging system in zebrafish (Danio rerio) was examined. Two tag orientations (horizontal and vertical) at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1-4 month post hatch). Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus) and 5 tag colors (yellow, red, pink, orange, blue) were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year) and multiple tagging sites (right and left of dorsal fin and pectoral fin base) were examined in adult zebrafish. Lastly, survival of recombination activation gene 1(-/-) (rag1(-/-)) zebrafish was evaluated after VIE tagging. The best tag location was the dorsal fin base, and the most visible tag color was pink. Growth rate of juvenile zebrafish was not affected by VIE tagging. Horizontal tagging is recommended in early stages of fish growth (1-2 months post hatch). VIE tags were retained for 1 year and tagging did not interfere with long-term growth and survival. There was no mortality associated with VIE tagging in rag1(-/-) zebrafish. The VIE tagging system is highly suitable for small-sized zebrafish. When familiar with the procedure, 120 adult zebrafish can be tagged in one hour. It does not increase mortality in adult zebrafish or interfere with growth in juvenile or adult zebrafish.

Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure.

BACKGROUND: Recombination activation gene 1 deficient (rag1(-/-)) mutant zebrafish have a reduced lymphocyte-like cell population that lacks functional B and T lymphocytes of the acquired immune system, but includes Natural Killer (NK)-like cells and Non-specific cytotoxic cells (NCC) of the innate immune system. The innate immune system is thought to lack the adaptive characteristics of an acquired immune system that provide enhanced protection to a second exposure of the same pathogen. It has been shown that NK cells have the ability to mediate adaptive immunity to chemical haptens and cytomegalovirus in murine models. In this study we evaluated the ability of rag1(-/-) mutant zebrafish to mount a protective response to the facultative intracellular fish bacterium Edwardsiella ictaluri. METHODOLOGY/PRINCIPAL FINDINGS: Following secondary challenge with a lethal dose of homologous bacteria 4 and 8 weeks after a primary vaccination, rag1(-/-) mutant zebrafish demonstrated protective immunity. Heterologous bacterial exposures did not provide protection. Adoptive leukocyte transfers from previously exposed mutants conferred protective immunity to naïve mutants when exposed to homologous bacteria. CONCLUSIONS/SIGNIFICANCE: Our findings show that a component of the innate immune system mounted a response that provided significantly increased survival when rag1(-/-) mutant zebrafish were re-exposed to the same bacteria. Further, adoptive cell transfers demonstrated that kidney interstitial leukocytes from previously exposed rag1(-/-) mutant zebrafish transferred this protective immunity. This is the first report of any rag1(-/-) mutant vertebrate mounting a protective secondary immune response to a bacterial pathogen, and demonstrates that a type of zebrafish innate immune cell can mediate adaptive immunity in the absence of T and B cells.

Characterization of rag1 mutant zebrafish leukocytes.

BACKGROUND: Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). RESULTS: Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag1 mutants and 26% in the wild-types. Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression. CONCLUSION: Our study demonstrates that in comparison to wild-type zebrafish, rag1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Zebrafish kidney phagocytes utilize macropinocytosis and Ca+-dependent endocytic mechanisms.

BACKGROUND: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. METHODOLOGY/PRINCIPAL FINDINGS: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. CONCLUSIONS/SIGNIFICANCE: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca(2+)-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.

Low-cost aquatic lab animal holding system.

We have constructed a low-cost aquatic animal holding system that provides an alternative to expensive, commercially available systems. Our flow-through system is especially useful for programs that are limited in space and funding. The easy assembly and maintenance of the system are advantages for the researchers who may be unfamiliar with aquatic animals.

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus.

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion.

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.

Susceptibility of channel catfish fry to Channel Catfish Virus (CCV) challenge increases with age.

Susceptibility of channel catfish to Channel Catfish Virus Disease (CCVD) has been generally considered to be inversely related to age. However, in experimental immersion challenges, we found that channel catfish fry, 3 to 8 d post hatch (dph), are most resistant to CCV and susceptibility increases with age. Initial studies involved 2 spawns that had high CCV carrier percentage. To determine if the resistance seen in the fry was related to the CCV carrier status of the parents, we selected 4 spawns from CCV negative parents and 2 spawns from CCV positive parents and immersion challenged them at 8, 23, 36 and 60 dph with 0, 2.5 x 10(4) or 2.5 x 10(6) plaque forming units (PFU) of CCV l(-1). Survivors of the low-dose exposed groups were rechallenged at 120 dph with 2.5 x 10(6) PFU CCV l(-1). Each brood demonstrated increasing susceptibility to CCVD with age and only the fish that were initially exposed at 60 dph developed protective immunity. Time course assays evaluating tissue levels of virus in channel catfish exposed to CCV at 7, 21 and 42 dph suggested that the resistance was an early event in the infection process. The resistance in fry was most pronounced in fish from CCV positive spawns and was correlated to neutralizing antibody titers in the maternal parent in the 8 dph challenge. However, other factors may be involved because all groups displayed the initial resistance and subsequent susceptibility to CCVD. The age effect may be an important influence on the progression of CCVD outbreaks and indicates the need to consider age for experimental challenges. Additionally, we documented the level of vertical transmission of CCV. Fry from the 4 positive spawns had a CCV prevalence of 40 to 75 %.