World Aquaculture - September 2023

36 SEPTEMBER 2023 • WORLD AQUACULTURE • WWW.WAS.ORG A. hydrophila (Figure 3). However, A. hydrophila was still able to grow at the highest salt concentration. We allowed A. hydrophila cells to form biofilm on glass slides under static culture conditions for 48 hours and examined the slides using a scanning electron microscope. At 5 g/L salt, normal cells of A. hydrophila formed biofilm and were arranged in an intact monolayer covering the entire surface of the slide (Figure 4). However, at 45 g/L salt, elongated bacterial cells were present but no biofilm formation occurred. Instead, irregular masses of secreted substances, polysaccharides to protect the cells from external stressors, (Wang et al. 2023) were occasionally observed. Some of these cells were more than 15 µm in length. Summary and Implications When bacteria are exposed to environmental stressors, such as high salinity, their physiological and phenotypic characteristics may be altered. Presence of salt can inhibit bacterial growth and/ or attachment and may have beneficial effects on fish health. Results of this study indicated that A. hydrophila has high salt tolerance. While high salinity conditions reduced growth and biofilm formation of A. hydrophila, this stress triggered formation of elongated cell forms. These elongated cells can be adaptive/ resistant forms to cope with the adverse conditions and repopulate in post-stress favorable environments. Currently, we are looking into the viability and infectivity of A. hydrophila cells under high salt stress. Preliminary data suggests that high salt is associated with low virulence. The findings of this study would help understand the mechanisms of A. hydrophila survival in catfish ponds and facilitate research on prevention and control of recurring MAS outbreaks. Further laboratory studies will determine what salt concentrations should be maintained in catfish ponds in order to suppress persistence and multiplication of this pathogen. Salt usage would not impose a drastic change in producers’ pond management practices. Notes Haitham H. Mohammed*, Department of Rangeland, Wildlife and Fisheries Management, Texas A&M University, Horticulture/ Forest Science Building, 495 Horticulture Rd, TAMUS, College Station, TX 77843 * Corresponding author: A recent study analyzed the potential risk factors for MAS outbreaks in farmed food-sized catfish in Alabama by utilizing data from two farmer surveys conducted in 2009 and 2011 (Bebak et al. 2015). The study examined farm-level risk factors associated with MAS outbreaks and concluded that farms maintaining a chloride concentration greater than 135 ppm were significantly less likely to experience outbreaks. Conversely, the likelihood of A. hydrophila outbreaks was found to be higher on farms that added salt (NaCl) routinely to their ponds. Intensively managed catfish farms often add salt to ponds to increase chloride levels as a preventive measure against methemoglobinemia caused by high levels of nitrite in the water (Bowser et al. 1983). Research suggests that maintaining higher chloride concentrations in catfish ponds can have a protective effect on catfish. This is because there is some evidence to indicate that salt can inhibit bacterial growth and attachment, potentially providing beneficial effects for catfish health (O’Neal et al. 2006, Lee et al. 2010). Salt may also provide protection to catfish by compensating for osmotic changes that occur during the initial stages of infection, which can increase the odds of recovery. What Is the Effect of Salt on A. hydrophila? The objective of this study was to investigate the impact of salt on A. hydrophila and to describe the adaptive morphological and structural changes that this bacterium undergoes in response to different salinity conditions. A strain of A. hydrophila was grown in tryptic soy broth medium containing salt concentrations of 5 (low), 15 (medium), and 45 (high) grams/liter (equivalent to 0.5 percent, 1.5 percent, 4.5 percent salt) at 28 ℃. Following incubation, specimens from the bacterial cultures were examined using light and scanning electron microscopy at different intervals. Analysis of salt-stressed A. hydrophila revealed the presence of filamentous cell types under the highest (45 g/L) concentration. Some of these elongated cells exceeded 15 μm in length; however, in cultures with 5 g/L and 15 g/L salt, normal short and straight cells (coccobacilli) ranging from 1.0 to 3.0 μm in length predominated (Figure 2). We also monitored growth of the cultures over time by measuring optical density (OD) and looked at biofilm formation at low salinity vs high salinity. We found a negative correlation between salt concentration in the culture medium and growth of FIGURE 3. Growth curve of A. hydrophila at different salt levels (5 g/L Red, 15 g/L Green, and 45 g/L Blue). The optical density (OD) decreased as salt concentration increased. FIGURE 4. Scanning electron micrograph of A. hydrophila biofilm at low (5 g/L, left) and high (45 g/L, right) salt concentrations. Notice the individual cells arranged side by side and covered by the extracellular polymeric substances, indicating biofilm formation at low salt level.