Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Diseases are a significant challenge to the production, economic viability, and sustainability of commercial aquaculture. This proposal seeks to investigate the effectiveness and immune-modulatory properties of the probiotic Enterobacter C6-6 in managing coldwater disease (CWD), columnaris, and related co-infections during early life stages. The project addresses the USDA-NIFA Aquaculture Research Program's FY2022 priority area, focusing on critical disease issues affecting commercial aquaculture species. CWD and columnaris are two of the most devastating diseases impacting the production of salmonids and other cold and warmwater species. Early life stages of fish are particularly susceptible to these diseases due to an underdeveloped immune system, with losses exceeding 70% in some cases. The U.S. aquaculture development plan aims to foster advanced, environmentally sustainable, and safe production methods. To achieve this, novel and eco-friendly solutions are needed to combat diseases and improve fish health. Enterobacter C6-6 has shown promise in inhibiting pathogenic Flavobacterium spp. and reducing mortality from CWD. This study builds on these findings by conducting lab and field trials to evaluate the oral delivery of C6-6 at first feeding as a strategy to minimize early life stage losses due to CWD, columnaris, and co-infections. The study demonstrated the efficacy and scalability of this probiotic, providing critical data to support its potential as a new tool for the aquaculture industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A full time MS and two part time undergraduate students have been working on this study with mentoring and training provided by Dr. Jie Ma and Dr. Ken Cain. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?For the microbiome, further analysis is processing,includingmetrics of alpha and beta diversity that allow us to better understand the statistical differences present between our control and treatment groups. We hope that our sequencing data will provide increased insight into the effects of our probiotic in the context of its potential benefits conferred to the fishsamples taken from fish that were fed the probiotic C6-6. We plan to focus on wrapping up the project by finalizing data analysis and completing the final report. This will include synthesizing all findings, addressing any remaining gaps, and ensuring the project objectives are fully addressed. We also aim to draft and submit manuscripts for publication in peer-reviewed journals to disseminate the results to the scientific community and industry stakeholders. Additionally, we will prepare presentations for conferences or stakeholder meetings to share key outcomes and their implications for aquaculture practices.

Impacts
What was accomplished under these goals? 1. Viability of Probiotic C6-6 in Fish Feed Pellet.Two feed sizes were utilized in the first experiment to measure viability of C6-6 bacteria when stored in -20 ?C and 4 ?C - 1.5 mm and #1 crum (Skretting, UT, USA).Bacterial counts across both diets were consistently similar, but overall concentrations of the probiotic were higher when feed was kept in -20 ?C. When stored at -20 ?C, the concentration of C6-6 on the #1 crum size feed decreased from 1.56 x 106CFUs/gto 6.7 x 105CFUs/g; at the same temperature, the concentration of C6-6 on the 1.5 mm feed decreased from 3.65 x 105CFUs/g to 2.49 x 105CFUs/g. When stored at 4 ?C, C6-6 concentration on the #1 crum size feed decreased from 9.00 x 104CFUs/g to 5.95 x 104CFU/g. Initial concentration of C6-6 in the 1.5 mm feed stored at 4 ?C was higher than the smaller feed size.The second viability experiment examined the effect of preparation methodand introduced several new factor levels as it considered an additional storage temperature, i.e. 15 ?C. Like the relative variation in concentration of C6-6 in the 1stexperiment, temperature was the most significant effecter. The data shows that all feeds stored at -20 ?C produced bacterial counts that were 10-100 times greater than their counterparts stored at 4?C. After 3 weeks, the average number of detectable C6-6 colonies from the straight feed stored at -20 ?C was 2.78 x 108CFU/g compared to 7.22 x 106CFU/g isolated from the straight feed stored at 4?C.A clear linear trend characterized bacterial concentrations over time, suggesting that viability decreases steadily. The original feed prepared by centrifuging the C6-6 and combining the pellet with an equal volume of menhaden oil appeared to contain the smallest number of viable C6-6 cells across the several storage temperatures, though no colonies were detectable in any of the preparations stored at 15 ?C after 1 week.Feed that was made without the incorporation of any fish oil and stored at -20 ?C produced significantly higher quantities of probiotic bacteria throughout the entire testing period (P< 0.05). No significant decline between week 1 and week 10 was noted for this treatment. The feed made using the original methodology and stored at the lowest temperature was the only other feed type with this long-term maintenance of probiotic viability. 2.Effects on Columnaris Disease in Rainbow Trout. Three plates were made to demonstrate the effects of the C6-6 soaked discs on the Fc lawn where each plate had 1 control disc and 3 treatment discs (soaked in diluted C6-6).The average inhibition of the 3 plates was 18.3 mm. In vivo,After the 10 day feed trial, fish were separated into triplicate25 fish groups. Fish were injected either with 100 µL of concentrated F. columnare (1.25 x 10^8CFU/fish-1), or sterile 1 x PBS.Feeding of C6-6 dressed diet and the control diet to respective groups was continued 24 h post challenge.The highest mortality was observed in tanks containing fish fed the control diet (26%). Tanks with fish fed C6-6 before the challenge and during the challenge experienced lower mortality (14%), although this difference was not statistically significant (P<0.05)F. columnarewas reisolated from 90% (18/19) of mortalities. Reisolation of C6-6 was successful for over 80% (6/7) of mortalities from tanks receiving probiotic feed. 3. C6-6 feeding trial at U of ID.An 8-week feed trial consisting of two diets was carried out at the University of Idaho's CNR Aquatic Animal Laboratory (AAL). Diets were fed as the first feed to swim up fry. Eyed eggs were received from Riverence Brood LLC. Each group was randomly split into 3 replicate tanks (23 gal, 2 L flow-through, with 250 fish per tank, or 750 fish total per feed group). Water temperature was maintained at 15 °C. Fish were hand-fed their respective diets at a rate of 1.5-3% body weight per day, 2 times daily for the duration of the 8-week trial. Sample counts were performed weekly to update feed charts. The treatment diet was topcoated with C6-6 at a ratio of 1:10 (v:m) as described. A control diet was made by topcoating of feed at the same ratio and only using menhaden oil. New batches of the probiotic feed were made regularly and viability of each checked to confirm viability of C6-6. Concentrations were checked from 1-day old feed and they ranged from ~ 2.0 x 105- 3.0 x 106CFU/mL-1. Two-sample independent t-tests were carried out on measurements of growth from 1 month, and from the end of the feed trial (2 mo). No significant difference was observed between the two diets for the study period. 4. Commercial feeding trials.C6-6 was provided to Riverence Provisions LLC located in the Magic Valley, Idaho, in order to collect data onthe ability of the probiotic to affect growth metrics in a commercial hatchery setting.This feed trial was conducted to emulate the duration of the feed trial performed at the University of Idaho. 7,020 rainbow trout approximately 0.2 g each were equally split into two separate indoor raceways and the 2-month feed trial began 15 days (150 degree days) after first feeding. Fry in one raceway were administered C6-6 at a dose of 106bacterial cells g-1of feed. The second raceway was fed a standard trout diet with the addition of menhaden oil (Skretting, Toole, UT). Fresh feed was prepared biweekly and subsamples were taken from each batch to ensure viability of the probiotic. 0°C freezers were used for the storage of feed throughout the trial. Feeding rates and sample counts were conducted in accordance with normal operation procedures of the hatchery. Fish were not intentionally exposed to any pathogenic organisms and no outbreaks were expected to occur. As such, the primary concern of this investigation was to measure the influence of C6-6 dressed feed on parameters of growth in a production setting.Total biomass, SGR, and RGR were all elevated in the group receiving C6-6 topcoated-feed. Additionally, the FCR was lower in the fish supplied the C6-6 feed. 5.In a preliminary analysis of intestinal samples across varied time points and treatments, Firmicutes and Proteobacteria were the most dominant phyla represented and within these, the majority of OTUs belong to the classes Bacilli, Gammaproteobacteria, Clostridia and Alphaproteobacteria. It's interesting that Bacilli were more prevalent in that the harmful effects of the disease resulted from their colonization of the gut microbiome based off these data. The family, Enterobacteriaceae (which encompasses the genus to which C6-6 belongs), was detected in 3 of the 10 samples sequenced, Day 28 C6-6, 2 month C6-6 and C6-6 post challenge. As the putative probiotic established a relative abundance > 25% in 50% of the C6-6 samples sequenced, we can assume that the beneficial bacteria is capable of establishing residency in the rainbow trout gut. The sample showing the highest abundance of our target family did belong to the control-diet group. This represents just 16.6% presence across the total number of control samples sequenced, though. In the challenge experiment, despite being injected with flavobacteria, insufficient reads were sequenced for flavobacteriaceae (< 1.0%), so it seems unlikely. Because the data discussed here is representative of only a small subset of the total samples collected, additional sequencing needs to be performed to obtain a more accurate and informative picture of the microbial ecology of rainbow trout fed the probiotic C6-6. This limited data still demonstrates that differences between pre- and post- disease challenge groups, and between probiotic and control diet fed groups likely exist.

Publications

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The target audience at this time is just the research community and industry partners involved in initial experimental aspects of the study. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A full time graduate student has been working on this study with mentoring and training provided by Dr. Jie Ma, Co-PI on the project, and Dr. Ken Cain. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Work will continue as planned to address each objective of the study. The graduate student will continue with laboratory trials and make contact with industry partners to begin planning field trials.

Impacts
What was accomplished under these goals? The primary goal of this research is to extrapolate on the findings made by Burbank et al. to columnaris disease, in addition to coinfection by Flavobacterium psychrophilum and Flavobacterium columnare as concurrent infections play an important role, albeit poorly understood, in the study of fish health. Through accomplishing the objectives of our research, a viable alternative for managing infections from coldwater disease, columnaris and possibly a simultaneous infection by the two can be achieved. Results In Vitro Inhibition: C6-6 showed clear suppression of growth by F. psychrophilum, F. columnare, and a combination of the two. Using both well diffusion and discs assays, growth of the probiotic C6-6 as characterized by creamy white, raised colony morphology (Burbank et al. 2011), could spread from its point of addition and impeding the spread of both Flavobacterium. After subtracting 6 mm from each measurement of diameter for both punched wells and discs, results from this methodology were very similar to those obtained by (Burbank et al. 2012). C6-6 Survival on Feed: Two feed sizes were utilized to measure viability of C6-6 bacteria when stored in -20 ?C, 1.5 mm and a mixture of 0.3 - 0.5 mm. Methods were largely adopted from Burbank et al. (2012) to examine the viability of C6-6 within feed over a month-long period. Bacterial counts across both diets were consistently similar with the smaller diet size exhibiting higher initial and final concentrations of C6-6. Discussion We were able to show successful inhibition of both species of Flavobacterium in vitro by C6-6. Comparing results here to those found by Burbank et al. (2011; 2012), we recreated inhibitory characteristics of C6-6 against F. psychrophilum following the bacterias' streaking on TYES agar, however, the size of the zones of inhibition from our wells were smaller, 4 mm, compared to the 6 mm recorded by Burbank et al. (2012). Results from the disc assay were 1-2 mm higher for our F. pyschrophilum and Fp + Fc (incubated at 15 ?C) plates vs. plates with punched wells. Furthermore, the inhibitory effect of the C6-6 against F. columnare was twice as large as that against F. pyschrophilum. If the enhanced resistance provided by C6-6 laden feed towards F. pyschrophilum following effective suppression of growth of Fp by C6-6 when plated serves as an example of the positive relationship between in vitro and in vivo effect (Burbank et al. 2011, 2012), then the potential for C6-6's treatment of columnare in live fish is high. The same could be postulated for the case of coinfection by F. psychrophilum and F. columnare. The fact that neither coinfection plate showed growth of Fp colonies following incubation for six days at both room temperature and 15 ?C signifies possible out competition of Fp by Fc when the two are evenly mixed (Manna et al. 2022). Intermediate values of C6-6's inhibition of the "coinfection" mixture support this: inhibition of Fp + Fc cultures was stronger than inhibition against Fp alone, but weaker than inhibition against Fc alone. Secondly, our methodology for testing survival of C6-6 on feed when stored at -20 ?C provides a reproducible process to mix the probiotic with feed pellets to create a probiotic-laden feed that maintains viability of the beneficial bacteria over the course of a month. Neither diet at any point exceeded CFU/mL values above 1.0 * 106 which were observed by Burbank in his unpublished thesis work. This difference may be attributed to several causes: 1. The drop plate method of enumerating bacteria as performed by Burbank can result in higher CFU's/mL when compared to counts from serial dilutions 2. When resuspending the C6-6 pellet in 15 mL menhaden oil (Step 5. of "Testing Viability Methods"), the pellet was extremely sensitive, therefore it's possible that a small amount of the pellet was poured off or that a small amount of supernatant remained after decanting 3. It was very difficult to remove all the homogenized mixture of feed and 1 * PBS when using the Griffith tube to prepare our serial dilutions (Step 11. of "Testing Viability Methods"). Consequently, we should attempt the drop plate method and make minor adjustments to steps 5 and 11 of our feed viability methods to achieve counts nearer to 1.0 * 106 before in vivo trials begin (Nikoskelainen et al. 2003; Brunt and Austin 2005; Merrifield et al. 2010; Burbank et al. 2012). It's significant to note that no notable decline in bacterial concentration on the feed occurred after one month with storage at -20 ?C. This finding corresponds to that of Burbank - who tested probiotic viability over a nine-day period - while also providing evidence of longer-term viability. Conclusion and Future Plans Infections from F. pyschrophilum and F. columnare represent two of the most severe diseases limiting commercial production of numerous species of cold and warmwater fish (Nikoskelainen et al. 2003; Nicolas et al. 2008). These findings support the limited research that currently exists on C6-6 and advance this bacterium's potential as a successful probiotic based off its capacity to antagonize virulent pathogens in vitro in addition to exhibiting longer-term viability after mixing with commercial trout diets. Positive results from in vitro testing alone are insufficient in qualifying a probiotic for use as a treatment in an aquaculture setting (Spanggaard et al. 2001; Suomalainen et al. 2005). We hope to clarify the specific modes of action of C6-6 in conferring protection by applying the methods for making probiotic feed detailed in this report to carry out in vivo studies this fall. Although some research has already identified specific toxins employed by C6-6 in countering pathogens such as coldwater disease (Schubiger et al. 2015), we plan to better elucidate the functioning of C6-6 by measuring activity of both the innate and adaptive immune systems, as well as evaluating changes in the gut microbiome of fish fed feed containing cells of C6-6. C6-6 continues to exhibit strong potential to address issues in aquaculture caused by coldwater and other diseases. Through further research of how this bacterium affects fish immune response and intestinal composition following exposure to two very severe diseases, it's possible to establish a sound case for the probiotic, C6-6, as a natural and safe mode of addressing a significant issue facing the aquaculture industry.

Publications