Christopher A. Ottinger

Aroclor 1248 exposure leads to immunomodulation, decreased disease resistance and endocrine disruption in the brown bullhead, Ameiurus nebulosus.

The brown bullhead Ameiurus nebulosus is a species of the family Ictaluridae commonly used as a sentinel of environmental contamination. While these fish have been utilized for this purpose in areas contaminated with polychlorinated biphenyls (PCBs), few controlled, laboratory-based studies have been designed to document the effects of PCB mixtures in this species. Here, brown bullhead were exposed to the PCB mixture, Aroclor 1248, via intraperitoneal injection and the effects on immune function, plasma hormones and disease resistance were evaluated. Exposure to this mixture led to a decrease in bactericidal activity and circulating antibodies to Edwardsiella ictaluri present from a previous exposure to this pathogen. A subsequent E. ictaluri disease challenge led to significantly higher mortality in A1248 treated fish compared to vehicle-control fish. The mitogenic response to the T-cell mitogen, phytohemaglutinin-P, was increased compared to vehicle-control fish. The steroid hormone, cortisol, and the thyroid hormone, T3, were also significantly lower in A1248 exposed fish. In summary, we have validated a number of functional immune assays for application in brown bullhead immunotoxicity studies. Additionally, we have demonstrated that the PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead. Such data may compliment the interpretation of data yielded from applied field studies conducted in PCB contaminated aquatic ecosystems.

Mycobacterium-Inducible Nramp in Striped Bass (Morone saxatilis)

ABSTRACT In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.

Histologic, immunologic and endocrine biomarkers indicate contaminant effects in fishes of the Ashtabula River.

The use of fish as sentinels of aquatic ecosystem health is a biologically relevant approach to environmental monitoring and assessment. We examined the health of the Ashtabula River using histologic, immunologic, and endocrine biomarkers in brown bullhead (BB; Ameiurus nebulosus) and largemouth bass (Micropterus salmoides) and compared fish collected from a reference site (Conneaut Creek). Seasonal analysis was necessary to distinguish differences in fish between the two rivers. Overall BB from the Ashtabula River had a lower condition factor and significantly more macrophage aggregates than those from the reference site. Reduced bactericidal and cytotoxic-cell activity was observed in anterior kidney leukocytes from both BB and largemouth bass from the Ashtabula River. Lower plasma thyroxine and triiodo-l-thyronine in both species in the Ashtabula River indicated disruption of the thyroid axis. Differences in physiological biomarker responses were supported by body burden chemical concentrations when data were analyzed on a seasonal basis. The use of two fish species added a level of rigor that demonstrated biological effects were not exclusive to a single species. The results provide strong evidence that contaminants have affected fish in the Ashtabula River, a Great Lakes Area of Concern, and provide a baseline by which to evaluate remediation activities.

Correlation of Transforming Growth Factor-β Messenger RNA (TGF-β mRNA) Expression with Cellular Immunoassays in Triamcinolone-Treated Captive Hybrid Striped Bass

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative-competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis × male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

Immunomodulation and Disease Resistance in Postyearling Rainbow Trout Infected with Myxobolus cerebralis, the Causative Agent of Whirling Disease

Abstract Myxobolus cerebralis, the myxosporean parasite that causes whirling disease, has a number of deleterious effects on its salmonid host. Although it is well established that juvenile salmonids in the active stages of whirling disease mount an immune response to the pathogen, the occurrence and longevity of any related immunomodulatory effects are unknown. In this study, postyearling rainbow trout Oncorhynchus mykiss infected with M. cerebralis were examined for leukocyte functions and for resistance to Yersinia ruckeri, a bacterial pathogen of salmonids. Compared with uninfected controls, M. cerebralis-infected fish showed lower proliferative lymphocyte responses to four mitogens (concanavalin A, pokeweed mitogen, phytohemagglutinin, and lipopolysaccharide). Conversely, M. cerebralis-infected fish displayed greater bactericidal activity of anterior kidney macrophages than did uninfected fish. After bath challenges with Y. ruckeri, M. cerebralis-infected fish had slightly lower survival and a more r...

Impact of Thiamine Deficiency on T-cell Dependent and T-cell Independent Antibody Production in Lake Trout

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbeccos phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

Calcein AM release-based cytotoxic cell assay for fish leucocytes

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P</=0.013) or an equal amount of an unrelated antibody (P</=0.001). Average maximum observed percent cytotoxic cell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

In vitro immune functions in thiamine-replete and -depleted lake trout ( Salvelinus namaycush )

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP--biologically inactive form) to thiamine pyrophosphate (TPP--biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state.

Mycobacterial Infections in Striped Bass from Delaware Bay

Eighty striped bass Morone saxatilis were obtained from Delaware Bay using commercial gill nets set adjacent to Woodland Beach (n = 70) and Bowers Beach (n = 10) in December 2003. Fish were examined for gross lesions. Total lengths (TLs) and eviscerated weights were determined to calculate condition factors (K). Portions of spleens were aseptically harvested for bacterial culture, and portions of spleens, kidneys (anterior and posterior), livers, and gonads were obtained for histological examination. The size distribution of the striped bass was relatively homogeneous; the mean TL was about 600 mm for all samples. Mean K exceeded 0.95 in all samples and was not significantly different (P > 0.05) among samples. Significant differences in mycobacterial infection prevalence (P < or = 0.05) were observed among samples; samples obtained at Woodland Beach (WB) on December 10 (53.8%, n = 13) and December 17 (7.1%, n = 42) exhibited the most striking differences in prevalence. Mycobacterial infection intensity ranged from 1 X 10(2) to 1 X 10(7) colony-forming units per gram of spleen. Acanthocephalan infection prevalence and intensity, non-acid-fast bacterial infection prevalence, and fish sex ratio were also significantly different among the samples (P < or = 0.05). Similar to the mycobacterial infections, differences in sex ratio, acanthocephalan infection, and non-acid-fast bacterial infection were observed between the WB samples taken on December 10 and 17. However, no significant associations (P > 0.05) were observed between sex ratio or these infections and mycobacterial infection. The differences in bacterial and parasite infection prevalence and intensity and fish sex ratio in some samples indicate that these fish had a different history and that the epizootiology of mycobacterial infection in striped bass from Delaware Bay may be relatively complex.

Low Pathogenic Avian Influenza Viruses in Wild Migratory Waterfowl in a Region of High Poultry Production, Delmarva, Maryland

SUMMARY Migratory waterfowl are natural reservoirs for low pathogenic avian influenza viruses (AIVs) and may contribute to the long-distance dispersal of these pathogens as well as spillover into domestic bird populations. Surveillance for AIVs is critical to assessing risks for potential spread of these viruses among wild and domestic bird populations. The Delmarva Peninsula on the east coast of the United States is both a key convergence point for migratory Atlantic waterfowl populations and a region with high poultry production (>4,700 poultry meat facilities). Sampling of key migratory waterfowl species occurred at 20 locations throughout the Delmarva Peninsula in fall and winter of 2013–14. Samples were collected from 400 hunter-harvested or live-caught birds via cloacal and oropharyngeal swabs. Fourteen of the 400 (3.5%) birds sampled tested positive for the AIV matrix gene using real-time reverse transcriptase PCR, all from five dabbling duck species. Further characterization of the 14 viral isolates identified two hemagglutinin (H3 and H4) and four neuraminidase (N2, N6, N8, and N9) subtypes, which were consistent with isolates reported in the Influenza Research Database for this region. Three of 14 isolates contained multiple HA or NA subtypes. This study adds to the limited baseline information available for AIVs in migratory waterfowl populations on the Delmarva Peninsula, particularly prior to the highly pathogenic AIV A(H5N8) and A(H5N2) introductions to the United States in late 2014.