July 24, 2025
July 24, 2025
1Kim Jacobsen, 2Eileen Henderson, 3Matt Griffin, and 1Esteban Soto
1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616; 2California Animal Health & Food Safety Laboratory, San Bernardino, CA, 92408; 3Warmwater Aquaculture Research Unit, Agricultural Research Service, U.S. Department of Aquaculture, Stoneville, MS 38776.
Edwardsiellosis is an emerging infectious disease of many cultured fish in North America. The disease is caused by members of the genus Edwardsiella, including Edwardsiella pisicicida. In natural outbreaks, acute-sub-acute septicemias have been associated with high mortalities and significant economic losses for the aquaculture industry. To gain a better understanding of the disease pathogenesis, as well as to test prophylactic and therapeutics in-vivo, a reliable and reproducible method of inducing experimental infection is needed. Currently, intra-coelomic (IC) injection is the primary challenge method used for laboratory induction of E. piscicida infection in fish. Effective IC challenge models for E. piscicida have been developed for several fish species, however these methods do not reflect the natural infection process. Immersion challenge models present an attractive method for experimental infection of fish as they more closely simulate true infection dynamics within the aquatic environment. One of the main difficulties in establishing an immersion model of infection is the low mortality rate and high variability that can occur with this challenge method. Previous studies with other fish pathogens have found that disruption of the skin mucous and/or creation of an artificial wound can facilitate bacterial infection and improve the virulence and reproducibility of the challenge. To investigate whether these methods could be used to develop an effective and reproducible immersion challenge method for E. piscicida in salmonids, challenge experiments with Rainbow trout (O. mykiss) and Chinook salmon were conducted. In the first challenge, yearling rainbow trout (82.8 g ± 26.1 g) and 3-month-old Chinook salmon (2.3 g ± 0.4 g) were exposed via immersion to 107 CFU/mL of E. piscicida for 30-minutes using three different challenge methods: immersion with intact skin/mucous, immersion after removal of mucous coating, and immersion after removal of the adipose fin to create an open wound (n = 10 fish per species per treatment). This trial led to 30% mortality in the salmon challenged after mucous abrasion and 40% mortality in the salmon challenged after fin cut. None of the negative control (NC) or intact immersion salmon developed clinical signs of infection and there were no mortalities in any of the trout. A second challenge experiment was conducted using 5-month-old trout (4.48 ± 1.38 g) and salmon (5.86 ± 1.43 g) in which fish had their adipose fins cut and were then immediately immersed with 107 CFU/mL of E. piscicida for 60-minutes (3 tanks/treatment; 15 fish/tank; n = 45 fish per treatment). In this trial, challenged trout experienced 40% mortality while challenged salmon demonstrated 13.3% mortality. Histologically, infected fish demonstrated foci of necrosis with associated bacteria in renal interstitium, the spleen, and less frequently within the liver. Lesions were more severe and intralesional bacteria more prevalent in the 5-mo trout than the 5-mo salmon. These findings indicate that removal of the mucous barrier or creation of a wound was crucial to facilitate bacterial entry and pathogenicity via immersion challenge. Additionally, the age and/or size of the fish appeared to significantly impact the virulence of E. piscicida in the fish. Overall, this challenge method was able to effectively and reproducibly produce significant mortality in salmonids ≤5 g.
Yoandy Coca1, Zeinab Yazdi1, Eileen Henderson2, Haitham H. Mohammed3, Taylor I. Heckman4,5, Matt J. Griffin5,6, Esteban Soto1
1Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, CA; 2California Animal Health & Food Safety Laboratory, San Bernardino, CA, 92408; 3Department of Rangeland, Wildlife and Fisheries Management, Texas A&M University, College Station, TX; 4Department of Wildlife, Fisheries and Aquaculture, College of Forest Resources, Mississippi State University, Starkville, MS; 5Mississippi Agriculture and Forestry Experiment Station, Delta Research and Extension Center, Mississippi State University, Stoneville, MS; 6Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS
Piscine lactococcosis is an emerging infectious disease caused by Lactococcus petauri, L. garvieae, and L. formosensis. In North America, outbreaks of piscine lactococcosis have been mainly diagnosed in rainbow trout (Oncorhynchus mykiss); however, isolates of L. petauri have also been recovered from other major cultured fish species, including Nile tilapia (Oreochromis niloticus) and catfish (Ictalurus spp.). The virulence of L. petauri in these non-salmonid species has not been fully explored under laboratory-controlled conditions. In this study, the pathogenicity of two L. petauri strains, one isolated from catfish in Alabama, USA, and one from rainbow trout in California, USA, was evaluated in experimental challenges using Nile tilapia fingerling infection model. The isolates represent two different genotypes of L. petauri present in the USA. Since tilapia can be cultured at a wide range of temperatures, in vivo experiments were conducted at 25°C and 30°C (±2°C). Fish were challenged via intra-coelomic injection of ~10⁷ CFU/fish, or by immersion in 5 L of water containing ~10⁷ CFU/mL for 1hr. Morbidity and mortality were monitored twice daily for three weeks. Water samples from each tank were collected 6 hours, 1, 9 and 10 days post-challenge to evaluate L. petauri DNA load using a species-specific quantitative PCR. No significant morbidity or mortality was observed in any of the treatment groups during the challenge. At the end of the three weeks, all fish were euthanized and brain samples from ten fish per group were cultured on nutrient agar to evaluate bacterial persistence in survivors. Additionally, three fish from each group were preserved in 10% buffered formalin for histopathological examination. None of the surviving fish yielded positive isolation for L. petauri. Bacterial DNA in the water was detected only 6h and 1 d post-challenge in some treatments. All together, these findings suggest that the tested L. petauri strains pose minimal risk to tilapia.
Mia L. Reed, Esteban Soto
Department of Veterinary Medicine and Epidemiology, University of California, Davis, CA 95616, USA
Biofilms are bacterial communities that can form on inert materials, such as nets, tanks, or pipes, as well as in vivo in various tissues. In vivo biofilms can contribute to antimicrobial resistance and microbial persistence. Skin lesions on fish are often contaminated with opportunistic and/or obligate pathogens that can form biofilms. Disruption of these biofilms is essential to improving outcomes for fish that sustain damage to their skin. Honey has been shown to aid in wound healing and inhibit many species of bacteria, yet the efficacy of medical-grade honey against biofilm-forming fish pathogens, such as Aeromonas spp., has yet to be explored. In this study, we investigated the in-vitro efficacy and necessary exposure time (1 or 10 minutes) of medical-grade honey against A. veronii (n = 3) and A. caviae (n = 1) isolates previously recovered from koi, Australian lungfish, or white sturgeon. Initially, the capacity of Aeromonas spp. to form biofilm was assessed using the Minimal Biofilm Eradication Concentration (MBEC) method for 24 hours at different temperatures (15°C, 20°C, or 25°C). After 24 hours, Aeromonas spp. formed biofilms at concentrations between 106.31 and 107.49 CFU/mL. The data also suggest that peak biofilm formation occurred at 20°C for each isolate, with biofilm concentrations ranging from 106.94 to 107.49 CFU/mL. There were significant temperature-dependent differences for all isolates except A. veronii from the lungfish. Exposing the biofilms to honey caused a significant (p <0.0001 – 0.02) reduction in bacterial growth in broth, effectively decreasing the area under the growth curve (28 – 75% reduction) regardless of isolate, biofilm growth temperature, or honey exposure time with the exception of a single isolate grown at 25°C with a 1-minute honey exposure. However, there appeared to be a temperature-dependent difference between isolates and the effects of honey exposure time on viable bacteria from the biofilm directly following exposure. With a 1-minute exposure, all isolates grown at 15°C exhibited a log colony forming unit (CFU) reduction (0.7 to 2.3-fold), whereas no isolates grown at 25°C exhibited a log CFU reduction. With a 10-minute exposure, all isolates grown at 15°C exhibited a 1.5 to 2.7-fold log reduction in CFU, all isolates grown at 20°C experienced a 1 to 1.5-fold log reduction, and two isolates exhibited a 0.4 and 0.8-fold log reduction when grown at 25°C. Taken together, these data suggest that medical-grade honey is effective at reducing the growth of biofilm-enclosed Aeromonas spp. There is variation in the efficacy of honey on viable bacteria concentration based on bacterial isolate, biofilm-formation temperature, and honey exposure time. This study suggests that medical-grade honey may be an effective topical treatment for biofilm-forming fish pathogens by restricting bacterial growth, allowing the fish immune system time to respond or providing more time for medical interventions.