Lora Petrie-Hanson

Ontogeny of channel catfish lymphoid organs.

Previously, we showed that catfish could not mount a detectable antibody response after bacterial exposure until 21 days post-hatch (ph). In order to evaluate the changes associated with the development of a functional humoral response, we evaluated the temporal and spatial distribution of immune cell populations in developing catfish. Cells functioning in nonspecific immunity were present in the renal hematopoietic tissue (rht) and thymus at hatch and in the spleen by day 3 ph. Immunoglobulin (Ig) positive lymphocytes were first detected on day 7, 10, and 14 in the rht, thymus and spleen, respectively. Mature thymocytes were first detected on day 10 ph. Distinct thymic regionalization and splenic lymphoid tissue organization were not observed until day 21 ph. We suggest that the reason for a lack of antibody production until day 21 ph is the poor organization of secondary lymphoid tissue until that age.

Characterization of rag1 mutant zebrafish leukocytes

BackgroundZebrafish 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 (rag 1 mutants).ResultsDifferential counts of peripheral blood leukocytes (PBL) showed that rag 1 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 rag 1 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 rag 1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag 1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag 1 mutants and 26% in the wild-types.Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag 1 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.ConclusionOur study demonstrates that in comparison to wild-type zebrafish, rag 1 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. Rag 1 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.

Persistence of Largemouth Bass Virus Infection in a Northern Mississippi Reservoir after a Die-Off

Abstract Approximately 3,000 adult largemouth bass Micropterus salmoides were found dead in September 1998 in Sardis Reservoir, Mississippi. Investigation of largemouth bass electrofished 1 month after the reported losses revealed a high level of infection with largemouth bass virus (LMBV) in the population. Subsequent sampling on five separate occasions during the following year revealed that approximately one-half of the fish were positive for LMBV 7 months after the die-off. On the last sampling, 13 months after the die-off, approximately one-third of bass cultured positive for LMBV. The most common finding associated with LMBV infection was a yellow waxy substance in the swim bladder that consisted of erythrocytes and eosinophils in a fibrin clot indicating previous hemorrhage. In nearly every case, fish that had the swim bladder lesion cultured positive for LMBV. Predisposition to infection did not correlate with the gender or size of the fish. Sympatric white bass Morone chrysops, white crappies Pom...

Differential cytochemical staining characteristics of channel catfish leukocytes identify cell populations in lymphoid organs

This is one of the first characterizations of channel catfish (Ictalurus punctatus) leukocytes by enzyme cytochemistry. Leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, alpha-naphthyl butyrate esterase, beta-glucuronidase, alpha-naphthyl acetate esterase, Sudan Black B and anti-immunoglobulin specific immunohistochemistry. Lymphocytes, monocytes, macrophages, neutrophils, and surface immunoglobulin positive (surface Ig+) cells were present in channel catfish renal hematopoietic tissue and spleen and demonstrated distinctive cytoplasmic foci staining patterns, cytoplasmic blushing or cell membrane staining. Monocytes, macrophages, lymphocytes and surface Ig+ cells were present in the thymus. Thymic and splenic cellular organization appeared very similar to these same mammalian tissues. In the thymus, acid phosphatase positive cells were distributed throughout the parenchyma, while alpha-naphthyl butyrate esterase and beta-glucuronidase positive cells were concentrated in the cortex and the medulla, respectively. Surface immunoglobulin positive cells occurred in the cortex. In the spleen, acid phosphatase positive cells were scattered throughout the parenchyma, while alpha-naphthyl butyrate esterase positive cells were scattered throughout the parenchyma and adjacent to splenic arterioles. Beta-glucuronidase and surface immunoglobulin positive cells were restricted to immediately adjacent to splenic arterioles. Sudan Black B positive cells were scattered throughout the parenchyma, while alpha-naphthyl acetate esterase positive cells occurred adjacent to peri-arteriole lymphoid sheaths and appear very similar to mammalian metallophils.

Evaluation of Zebrafish Danio rerio as a Model for Enteric Septicemia of Catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.

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.

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-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 Ca2+-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.

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).

Summary 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.

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.