Synergistic Short-Term Effects of Humic Substances, Butyric Acid, and Yeast Cell Walls as Dietary Additives on Health, Immunity, and Susceptibility to Edwardsiella ictaluri and Flavobacterium covae Coinfection in Juvenile Channel Catfish

1Abdulmalik A. Oladipupo, 1Anita M. Kelly, 2Benjamin R. LaFrentz, 1D. Allen Davis,

1Luke A. Roy, and 1Timothy J. Bruce

1School of Fisheries, Aquaculture, and Aquatic Sciences, 203 Swingle Hall, Auburn University, Auburn, AL 36849 ; 2USDA-ARS Aquatic Animal Health Unit, Auburn, AL 36830

Dynamic pathogen interactions influence mortality in catfish ponds and may complicate treatment interventions. The incidence and potential of coinfections in ponds heighten the vulnerability of catfish to diseases, which may reduce the efficacy of antibiotics and vaccinations. The application of organic bio-stimulants, such as probiotics, prebiotics, and organic substances, within aquaculture has proven effective in promoting non-specific immune responses against a broad spectrum of aquatic diseases, yielding sustainable benefits. A short-term study was conducted to evaluate the potential health benefits of dietary humic substances combined with butyric acid (HumiSyn IFC4), either as a single supplement or in combination with yeast cell wall (YCW), against bacterial coinfection in catfish aquaculture. Channel catfish fingerlings (19.7±0.2g) were fed practical diets (32% crude protein) for 45 days in a recirculating aquaculture system. Fish were offered either a control diet, control supplemented with HumiSyn IFC4 (HSB; 4 lb/U.S. ton), control supplemented with YCW (YCW; 1 lb/ U.S. ton), or control supplemented with both HumiSyn IFC4 (2 lb/U.S. ton) and YCW (0.5 lb/ U.S. ton; HSB+YCW). Following the feeding trial, Fulton’s condition factor differed across groups (P = 0.014), with an increase in HSB catfish compared to the control group (P = 0.009). Investigation of post-feeding immune gene expressions (il1b, il8, tnfa, tgfb) in the kidney and spleen tissues showed no differences across treatments (P > 0.05). Meanwhile, antimicrobial protease (cstd1) was upregulated in the HSB catfish group (P = 0.018), while antimicrobial peptides (defb) were downregulated in the gills of the control group compared to YCW (P = 0.036), HSB (P = 0.003), or HSB+YCW (P = 0.045).  Subsequently, catfish were pooled by treatment and transferred into a biosecure wet lab for an in vivo pathogen challenge. All treatment groups were contested in an immersion challenge using triplicate tanks with either a single dose of Edwardsiella ictaluri (S97-07; 7.4 x 106 CFU mL-1), F. covae (ALG-00-530; 1.2 x 106 CFU mL-1), or simultaneous co-infection with half-doses of both bacteria. After the 14-day bacterial challenge, the cumulative percent mortality (CPM) following E. ictaluri exposure was: Control (48%), YCW (30%), HSB (22%), and HSB+YCW (38%). However, CPM increased (Control [70%], YCW [70%], HSB [28%], and HSB+YCW [55%]) with significantly lower CPM in HSB than the Control (P < 0.001) and YCW groups (P  = 0.008) in the co-infection group. Kaplan-Meier survival analysis revealed differences for both single E. ictaluri (P < 0.001) and the co-infection group (P < 0.001), with improved survival observed in the HSB-fed catfish compared to the control group. No mortality was observed in the single F. covae groups, likely due to a low immersion dose.  A dietary blend of humic substances and butyrate applied for 45 days may improve the survival of channel catfish due to single and co-infections. These findings provide the catfish industry with additional insight into potential immunostimulants for developing disease management strategies.

Evaluating the Application of Peracetic Acid in Pacific White Shrimp (Litopenaeus vannamei) Aquaculture Systems

 James T. Tuttle1,2, Timothy J. Bruce2, Luke A. Roy1,2, Hisham A. Abdelrhaman3, Julio C. García4, Benjamin H. Beck4, and Anita M. Kelly1,2

1Alabama Fish Farming Center, Auburn University, Greensboro AL, 36744; 2School of Fisheries Aquaculture and Aquatic Sciences, Auburn University, Auburn AL, 36849; 3Feinstein School of Social and Natural Sciences, Roger Williams University, Bristol RI, 02809; 4Aquatic Animal Health Research Laboratory, United States Department of Agriculture-Agriculture Research Service, Auburn AL, 36832

The Pacific White Shrimp (Litopenaeus vannamei) is one of the most commercially important aquaculture species domestically and globally. From 2020 to 2024, the United States imported a minimum of 748 million metric tons of L. vannamei each year. This major imbalance necessitates expanding and empowering domestic shrimp aquaculture production. One of the biggest challenges to L. vannamei culture is the extensive list of bacterial, parasitic, and viral pathogens that can cause severe losses. Some strategies that have been adopted to mitigate the effects of pathogens are to culture specific pathogen-free (SPF) L. vannamei at low salinities (2.5-10ppt) and maintain optimal aqueous potassium and magnesium (K+ and Mg2+) concentrations. However, these strategies do not guarantee prevention or reduction of disease outbreaks, and novel disease mitigation options are necessary. Shrimp do not have an adaptive immune system and are a benthic species, making vaccination against viruses impossible and antibiotic feed applications unreliable. Much research and progress has been made regarding applying the indiscriminate peracetic acid (PAA) disinfectant. The objectives of this study include determining the acute toxicity of PAA on different life stages of L. vannamei in differing salinities, investigating any potential physiological damage suffered by the L. vannamei exposed to PAA, and determining the minimum concentration of PAA required to inhibit the growth or eradicate Vibrio spp. To accomplish the first objective, individual 5g juveniles and post-larvae (at both 4 ppt and 30 ppt) were subjected to “range-finding tests” in which 3-shrimp treatment groups were subjected to geometrically increasing concentrations of PAA, and mortality was monitored over 48 hours. Once a PAA concentration that resulted in 100% mortality was determined, an LD50 test was run on each shrimp size at the two different salinities. These LD50 tests consisted of 180 shrimp, evenly divided across 18 containers and exposed to 6 different doses of PAA in triplicate, being monitored for mortalities at 2-, 4-, 24-, and 48-hours post-exposure. At those same time intervals, pH and PAA concentration were measured. After 48 hours, any surviving individuals were placed in Davidson’s Fixative and then submerged in 70% ethanol solutions in preparation for histological analysis. In a separate test, a pure colony of a virulent Vibrio parahemolyticus was isolated and placed in Tryptic soy broth containing a NaCl concentration of either 4 g/L or 30 g/L. Both broth cultures were then subjected to geometrically increasing concentrations of PAA starting at 0.25 ppm for 30 minutes. Viable colonies of V. parahemolyticus were enumerated via the drop plate method on Vibrio selective CHROMAgar.  After determining the PAA concentrations for each LD50 test, the estimated LD50 values based on a statistically plotted dose-response curve were 67.1 ppm of PAA for both juveniles in 4 ppt water and PLs in 30 ppt water, and 44.1 ppm of PAA for PLs in 4 ppt water. Histopathology of shrimp gill tissues revealed moderate to little damage in shrimp exposed to their respective LD50 estimated values. No V. parahemolyticus cultures exposed to any concentration of PAA produced any colonies on the selective media, regardless of media broth salinity. These findings are greatly encouraging and can help progress the further application of peracetic acid across a variety of shrimp aquaculture systems.

Optimization of Prime-Boost Intervals Enhances IHNV Vaccine Efficacy in Rainbow Trout (Oncorhynchus mykiss)

Ali Abouismail1, Benjamin Garcia1, Ryan Maxwell2, Megan Cox2, Jie Ma2, Irene Salinas1.

1University of New Mexico, Department of Biology, NM, USA; 2University of Idaho, Department of Fish and wildlife science, Idaho, USA

Vaccination is the most effective way to prevent diseases in aquaculture. Long-lasting immunity often requires the delivery of booster vaccines, yet the optimal interval time between prime and boost vaccination for fish vaccines is often not experimentally determined. The goal of this study was to compare three different prime-boost intervals (4, 8, and 12 weeks) in rainbow trout (Oncorhynchus mykiss) using a live attenuated infectious hematopoietic necrosis virus (IHNV) vaccine. Two independent experiments were conducted when fish were either 1000 or 1500 degree-days, dd. Fish received an immersion prime followed by intranasal boost at designated intervals and were challenged with IHNV 5 months after primary vaccination. Two different challenge pressures were used. For the 1000 dd fish, we used an LD50 for the mock-vaccinated group whereas for the 1500 dd fish it was an LD90 Following challenge at 1000 dd, the 8-week interval elicited optimal protection (95% survival), outperforming both shorter (90% at 4 weeks) and longer (75% at 12 weeks) intervals. Similarly, at 1500 dd, survival was highest (65%) in the 8-week interval group, compared to the 4- and 12-weeks interval groups (55 and 50%, respectively). Serum specific IgM titers as measured by ELISA supported these findings, revealing significantly higher IgM specific titers in 8-week the interval groups for both age classes. Our results demonstrate that an 8-week prime-boost interval maximizes the efficacy of mucosal vaccines against IHN regardless of the challenge dose and highlights the importance of testing interval doses in aquaculture vaccinology.