Progress 09/20/16 to 08/31/18
Outputs
Target Audience:The target audience for this project was fish immunology researchers and catfish producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The first year data was used to sucessfully submit a USDA grant proposal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
These experiments were initiated to obtain preliminary data for a NIFA submission that requested funding for deep sequencing experiments. Next Generation sequencing, or deep sequencing, will characterize histone modifications that characterize how methylation is 'training' or re-programming macrophages in channel catfish. The first year, we developed and optimized procedures to distinguish 51A and mpeg-1 positive cells in cell preparations for flow cytometry. We also demonstrated a dose-response relationship between increasing beta glucan exposure and increasing lactate dehydrogenase activity in 51A+ and mpeg-1+ leukocytes isolated from channel catfish anterior kidney tissues. We also performed ROS-Glo H2O2 assays demonstrating enhanced activity in response to E. ictaluri in AK cells isolated from channel catfish 14 days after IC injection of beta glucan. In year 2, we exposed 7 day old channel catfish fry to beta glucan by three different routes: oral administration of 1% beta glucan in feed for seven days, and 0.5 mg/L and 2 mg/L immersion for 10 minutes. Three weeks later, at 1 month of age, the fry were challenged with Edwardsiella ictaluri. The PBS exposed control groups averaged 19.3% mortality, the groups fed 1% beta glucan averaged 45.3% mortality, and the groups that were immersion exposed to 0.5 mg/L and 2 mg/L averaged 46% and 67.3% mortality, respectively. These findings suggest that fry that were exposed to beta glucan were more susceptible to bacterial challenge. Immune tolerance may have been induced at 1 week post-hatch. Effectiveness of beta glucan is variable, especially if orally adminstered. We demonstrated consistent results if we intra-peritoneally (IP) injected beta glucan. But this is not a practical delivery method in aquaculture. We designed an experiment to investigate oral delivery. We either gavage delivered or IP injected channel catfish with the same form and amount of beta glucan. Beta-glucan exposure (5 mg/catfish) increased phagocytic uptake of E. ictaluri 2 weeks post administration. Cells from fish that received beta-glucan by IP injection phagocytosed E. ictaluri about 1.2 times greater than control cells. Cells from fish that received beta-glucan by gastric gavage phagocytosed E. ictaluri about 1.5 times greater than control cells. Table 1. Channel catfish fry were exposed to beta glucan at 1 week of age. At 1 month of age, these fry were immersion exposed to E. ictaluri. Treatment and mortality Trial 1 Trial 2 Trial 3 Average PBS control 22 12 24 19.3% 0.5mg/L beta glucan immersion 50 68 20 46% 2mg/L beta glucan immersion 50 68 20 67.3% 1% beta glucan feed for 7 days 36 26 74 45.3%
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience is fish immunology researchers and catfish producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?These data was used for preliminary data in a NIFA grant re-submission. ? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
42TA macrophages were exposed to b glucan 14 days before E. ictaluri phagocytosis assays. FITC adhered represents FITC labeled E. ictaluri that has adhered to the macrophage cell surface. FITC internalized represents FITC labeled E. ictaluri that has been phagocytized. ROS-Glo H2O2 assay demonstrating enhanced activity in response to E. ictaluri in AK cells isolated from channel catfish 14 days after IC injection of b glucan. LDH assay demonstrating enhanced activity in response to E. ictaluri in AK cells isolated from channel catfish 14 days after IC injection of b glucan. To determine if β glucan affects cells other than macrophages, we needed to label channel catfish neutrophils separately from other granulocytes that occur together in the same gate when analyzed by side scatter and forward scatter in flow cytometry. Using flow cytometry procedures routinely performed in our lab, (Ainsworth et al. 1990; Hohn et al. 2009), we prepared single cell suspensions of channel catfish AK leukocytes. Cells were incubated with 51A, an antibody for channel catfish neutrophils (Ainsworth et al. 1990), and Mpg, a commercially available antibody that we use for zebrafish macrophages (unpublished data). Macrophage expressed gene, or Mpg is expressed in macrophages. These two antibodies labeled different channel catfish leukocyte populations (Figure 6). We plan to further characterized the Mpg+ cells in channel catfish. In this grant application, we plan to use 51A and Mpg to elucidate how β glucan affects channel catfish neutrophils and macrophages. ?
Publications
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Progress 09/20/16 to 09/30/16
Outputs
Target Audience:The target audience for this project is fish health researchers, aquaculture extension personnel and fish producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Again, for this reporting period, there have been no opportunities for training and professional development as of yet. How have the results been disseminated to communities of interest?No results have been produced and delivered to communities of interest for this 10 day reporting period. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will be a full year, so we anticipate some of the major goals to be accomplished by then therefore providing the fish producers with fish health information.
Impacts
What was accomplished under these goals?
Since this reporting period is only for the first 10 days of this project, there have been no major accomplishments to report as of now.
Publications
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