Nancy J. Szabo

Vaccination-Induced Systemic Autoimmunity in Farmed Atlantic Salmon

Over half of the salmon consumed globally are farm-raised. The introduction of oil-adjuvanted vaccines into salmon aquaculture made large-scale production feasible by preventing infections. The vaccines that are given i.p. contain oil adjuvant such as mineral oil. However, in rodents, a single i.p. injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome, characterized by autoantibodies, immune complex glomerulonephritis, and arthritis. In the present study, whether the farmed salmon that received oil-adjuvanted vaccine have autoimmune syndrome similar to adjuvant oil-injected rodents was examined. Sera and tissues were collected from vaccinated or unvaccinated Atlantic salmon (experimental, seven farms) and wild salmon. Autoantibodies (immunofluorescence, ELISA, and immunoprecipitation) and IgM levels (ELISA) in sera were measured. Kidneys and livers were examined for pathology. Autoantibodies were common in vaccinated fish vs unvaccinated controls and they reacted with salmon cells/Ags in addition to their reactivity with mammalian Ags. Diffuse nuclear/cytoplasmic staining was common in immunofluorescence but some had more specific patterns. Serum total IgM levels were also increased in vaccinated fish; however, the fold increase of autoantibodies was much more than that of total IgM. Sera from vaccinated fish immunoprecipitated ferritin and ∼50% also reacted with other unique proteins. Thrombosis and granulomatous inflammation in liver, and immune-complex glomerulonephritis were common in vaccinated fish. Autoimmunity similar to the mouse model of adjuvant oil-induced lupus is common in vaccinated farmed Atlantic salmon. This may have a significant impact on production loss, disease of previously unknown etiology, and future strategies of vaccines and salmon farming.

Endothelin 1 levels in the aqueous humor of dogs with glaucoma.

PurposeEndothelin 1 is a small peptide that is involved in regulation of intraocular pressure and modulation of ocular circulation. To investigate the role of endothelin 1 in canine glaucoma, the authors measured aqueous humor levels of endothelin 1 in healthy dogs and in dogs with hypertensive glaucoma. MethodsAqueous humor samples were obtained with general anesthesia from the eyes of healthy dogs (n = 5) and dogs with hypertensive glaucoma (n = 10). Measurements were made by enzyme immunoassay for endothelin 1. ResultsThe endothelin 1 aqueous humor range was 1.12 − 3.63 pg/mL for healthy dogs and 1.97 − 14.97 pg/mL for glaucomatous dogs. The healthy and glaucomatous canine endothelin 1 aqueous levels (mean ± SD) were 2.33 ± 0.90 and 8.11 ± 5.03 pg/mL, respectively. A two-way analysis of variance indicated that this difference was significant (P = 0.0084). The effect of age on endothelin 1 levels was not significant (P = 0.6283). The large variability found within the glaucomatous group could be explained by the degree of damage of the retina (P = 0.0006). There was no significant correlation between intraocular pressure and endothelin 1 aqueous humor levels within the glaucomatous group (P = 0.29). ConclusionsThe aqueous humor of dogs with hypertensive glaucoma contains significantly higher levels of endothelin 1 than that of healthy dogs.

Distinct expression and activity profiles of largemouth bass (Micropterus salmoides) estrogen receptors in response to estradiol and nonylphenol

The estrogen receptor (ER) signaling cascade is a vulnerable target of exposure to environmental xenoestrogens, like nonylphenol (NP), which are causally associated with impaired health status. However, the impact of xenoestrogens on the individual receptor isotypes (alpha, beta a, and beta b) is not well understood. The goal of these studies was to determine the impact of NP on largemouth bass (Micropterus salmoides) ER isotype expression and activity. Here, we show that hepatic expression levels of three receptors are not equivalent in male largemouth bass exposed to NP by injection. Transcript levels of the ER alpha subtype were predominantly induced in concert with vitellogenin similarly to fish exposed to 17beta-estradiol (E(2)) as measured by quantitative real-time PCR. NP also induced circulating plasma levels of estrogen, which may contribute to overall activation of the ERs. To measure the activation of each receptor isotype by E(2) and NP, we employed reporter assays using an estrogen response element (ERE)-luciferase construct. Results from these studies show that ER alpha had the greatest activity following exposure to E(2) and NP. This activity was inhibited by the antagonists ICI 182 780 and ZM 189 154. Furthermore, both beta b and beta a subtypes depressed ER alpha activation, suggesting that the cellular composition of receptor isotypes may contribute to the overall actions of estrogen and estrogenic contaminants via the receptors. Results from these studies collectively reveal the differential response of fish ER isotypes in response to xenoestrogens.

Manifestations of systemic autoimmunity in vaccinated salmon.

The development of systemic autoimmunity may result as an undesired side-effect following vaccination, and this condition was recently shown to occur in farmed salmon (Salmo salar). Several of previously reported side-effects following vaccination of fish should therefore be reviewed in the light of this condition. Here, organs and pathological changes in three separate groups of fish severely affected by vaccination were investigated by different morphological methods (n=84). Granulomas or microgranulomas were observed at the injection site and in several organs. Mott cells were observed in all tissues examined. Pannus-like changes with lymphocyte infiltrates were observed in spines. In conclusion, the reactions following vaccination were of a systemic nature that may be explained by a pathogenetic mechanism caused by systemic autoimmunity.

Predicting maternal body burdens of organochlorine pesticides from eggs and evidence of maternal transfer in Alligator mississippiensis

Few data exist regarding maternal-embryonal transfer of organochlorine pesticides (OCPs) in reptiles. The objective of the present study was to evaluate maternal transfer of OCPs in American alligators (Alligator mississippiensis) from low-, intermediate-, and high-OCP-exposure sites. Overall, total OCP burdens ranged from less than 0.8 ppb in blood to more than 44,000 ppb in abdominal adipose tissue (wet wt concentrations). Lipid-adjusted ratios of maternal adipose burdens (total OCPs) to yolk burdens were close to one (0.94 +/- 0.31:1), suggesting that animals were in steady state and that OCPs in eggs originated from adipose lipids. In contrast, lipid-adjusted muscle and liver OCP burdens were greater than yolk OCP burdens, suggesting that lipids in muscle were not utilized during oogenesis and that nonlipid liver tissue sequesters OCPs. Predictive equations were derived for several tissues and several OCP analytes with r2 values ranging from 0.40 to 0.99 (p < 0.05). We suggest that yolk burdens are predictive of maternal tissue burdens for certain tissues and OCPs and that certain OCPs are maternally transferred in the American alligator. Furthermore, we suggest that future studies should investigate the applicability of these predictive equations for assessing maternal exposure in other crocodilian species.

Comparison between omega-3 and omega-6 polyunsaturated fatty acid intakes as assessed by a food frequency questionnaire and erythrocyte membrane fatty acid composition in young children

Objective:We conducted a dietary validation study in youth aged 1–11 years by comparing dietary intake of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) as assessed by a parent-completed semiquantitative food frequency questionnaire (FFQ) over time to erythrocyte membrane composition of the same fatty acids.Design:The study population included youth aged 1–11 years who were participants in the Diabetes Autoimmunity Study in the Young (DAISY), a longitudinal study in Denver, Colorado that is following a cohort of youth at risk for developing type I diabetes. Four hundred and four children who had erythrocyte membrane fatty acid data matched to an FFQ corresponding to the same time frame for a total of 917 visits (matches) were included. PUFA intake was expressed both as g/day (adjusted for total energy) and as percent of total fat intake. We used mixed models to test the association and calculate the correlation between the erythrocyte membrane estimates and PUFA intake using all records of data for each youth.Results:Intakes of total omega-3 fatty acids (β=0.52, P<0.0001, ρ=0.23) and marine PUFAs (β=1.62, P<0.0001, ρ=0.42), as a percent of total fat in the diet, were associated with percent of omega-3 and marine PUFAs in the erythrocyte membrane. Intakes of omega-6 PUFAs (β=0.04, P=0.418, ρ=0.05) and arachidonic acid (β=0.31, P=0.774, ρ=0.01) were not associated.Conclusions:In these young children, an FFQ using parental report provided estimates of average long-term intakes of marine PUFAs that correlated well with their erythrocyte cell membrane fatty acid status.

Uptake of human pharmaceuticals in bull sharks (Carcharhinus leucas) inhabiting a wastewater-impacted river

The presence of human pharmaceuticals in sewage-impacted ecosystems is a growing concern that poses health risks to aquatic wildlife. Despite this, few studies have investigated the uptake of active pharmaceutical ingredients (APIs) in aquatic organisms. In this study, the uptake of 9 APIs from human drugs was examined and compared in neonate bull sharks (Carcharhinus leucas) residing in pristine (Myakka River) and wastewater-impacted (Caloosahatchee River) tributaries of Floridas Charlotte Harbor estuary. The synthetic estrogen used in human contraceptives (17α-ethynylestradiol) and 6 of the selective serotonin/norepinephrine reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, venlafaxine) used in human antidepressants were observed at detectable and, in some cases, quantifiable levels in plasma of Caloosahatchee River sharks. Comparatively, only venlafaxine was detected in the plasma of a single Myakka River shark at a level below the limit of quantitation. These results suggest that sharks residing in wastewater-impacted habitats accumulate APIs, a factor that may pose special risks to C. leucas since it is one of few shark species to regularly occupy freshwater systems. Further research is needed to determine if the low levels of API uptake observed in Caloosahatchee River bull sharks pose health risks to these animals.

Aquatic contaminants alter genes involved in neurotransmitter synthesis and gonadotropin release in largemouth bass.

Many aquatic contaminants potentially affect the central nervous system, however the underlying mechanisms of how toxicants alter normal brain function are not well understood. The objectives of this study were to compare the effects of emerging and prevalent environmental contaminants on the expression of brain transcripts with a role in neurotransmitter synthesis and reproduction. Adult male largemouth bass (Micropterus salmoides) were injected once for a 96 h duration with control (water or oil) or with one of two doses of a single chemical to achieve the following body burdens (microg/g): atrazine (0.3 and 3.0), toxaphene (10 and 100), cadmium (CdCl(2)) (0.000067 and 0.00067), polychlorinated biphenyl (PCB) 126 (0.25 and 2.5), and phenanthrene (5 and 50). Partial largemouth bass gene segments were cloned for enzymes involved in neurotransmitter (glutamic acid decarboxylase 65, GAD65; tyrosine hydroxylase) and estrogen (brain aromatase; CYP19b) synthesis for real-time PCR assays. In addition, neuropeptides regulating feeding (neuropeptide Y) and reproduction (chicken GnRH-II, cGnRH-II; salmon GnRH, sGnRH) were also investigated. Of the chemicals tested, only cadmium, PCB 126, and phenanthrene showed any significant effects on the genes tested, while atrazine and toxaphene did not. Cadmium (0.000067 microg/g) significantly increased cGnRH-II mRNA while PCB 126 (0.25 microg/g) decreased GAD65 mRNA. Phenanthrene decreased GAD65 and tyrosine hydroxylase mRNA levels at the highest dose (50 microg/g) but increased cGnRH-II mRNA at the lowest dose (5 microg/g). CYP19b, NPY, and sGnRH mRNA levels were unaffected by any of the treatments. A hierarchical clustering dendrogram grouped PCB 126 and phenanthrene more closely than other chemicals with respect to the genes tested. This study demonstrates that brain transcripts important for neurotransmitter synthesis neuroendocrine function are potential targets for emerging and prevalent aquatic contaminants.

Doxycycline levels in preocular tear film of horses following oral administration.

OBJECTIVE To determine the concentration of doxycycline in preocular tear film following oral administration in horses as a possible therapeutic modality for infectious and keratomalacic equine keratitis. PROCEDURE Eight broodmares without ocular disease from a Thoroughbred breeding facility were included in this study. Each mare received 20 mg/kg of doxycycline by mouth once daily in the morning for five consecutive days. Tears were collected 1 h after doxycycline administration starting on day one of administration and continuing for 10 consecutive days. Doxycycline levels in the tears were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). RESULTS Doxycycline was present in the tears of each mare at low microg/mL levels with the highest concentration appearing on the third to fifth days (8.21-9.83 microg/mL). Doxycycline levels had fallen below quantifiable ranges by day 10. No systemic side-effects were noted in any of the horses included in this study. CONCLUSIONS Oral doxycycline is present in preocular tear film of normal horses with noninflamed eyes and may be useful as treatment in equine ulcerative keratomalacia. The oral dose listed was tolerated well by the horses in this study. The drug levels attained at 20 mg/kg once daily orally of doxycycline may aid in the treatment of corneal ulceration in horses, but further study is warranted.

Evaluation of Two Commercially Available TLC Materials as SER Substrates

Two commercially available thin-layer chromatographic plates were evaluated as substrates for routine analysis by surface-enhanced Raman spectrometry (SERS). One plate was purchased precoated with silver; the other was coated in-house via chemical reduction. The use of SERS as a detection method for thin-layer chromatography (TLC) was also considered. To this end, a mixture of commonly used sulfonamides was separated and detected directly from each plate. The mobilities of the sulfa drugs were not strongly affected by the silver, but Raman response was weak and analyte degradation was apparent. Improvement was found through modification of the surface by adding a thin film of water to the substrate. Overall substrate performance was evaluated with the use of crystal violet as the model analyte in both dry and dampened states. Signal vs. concentration curves were linear across at least two orders of magnitude with limits of detection in the low- to sub-ng range (total analyte applied). Under routine preparation and analysis procedures, reproducibility was generally <30%, but improved to ∼10% when greater care was applied. Typical spectra for two compound series having a variety of functional groups were obtained, showing the substrates to be moderately active.