University of California-Davis - Aquatic Animal Health Lab
University of California-Davis - Aquatic Animal Health Lab
Dr. Esteban Soto
Best presentation award winner
Kim L. Jacobsen1, Matt Griffin2, Brett S. Phinney3, Michelle Salemi3, Zeinab Yazdi1, Sujita Balami2, Caitlin Older4, Tania Rodriguez-Ramos5, Brian Dixon5, and Esteban Soto1
1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616; 2Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS 38776; 3Proteomics Core Facility, University of California Davis, CA 95616; 4Warmwater Aquaculture Research Unit, Agricultural Research Service, U.S. Department of Aquaculture, Stoneville, MS 38776; 5Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
Edwardsiellosis is a bacterial disease caused by Edwardsiella spp. estimated to cause annual economic losses of over $50 million/year to the US aquaculture industry. Emergence of Edwardsiella piscicida among economically important cultured fish species, as well as evidence of antimicrobial resistance among some bacterial populations, has fueled a strong interest in the development of vaccines against piscine Edwardsiellosis. With the advent of novel protocols for enhanced antigen uptake and recently developed commercial adjuvants, investigation into the development of recombinant protein vaccines against major fish pathogens has gained increased attention. Protein vaccines offer a potential safe and cost-effective method of targeting the host immune response against select immunogenic antigens that may be conserved across diverse bacterial clades. This last feature is particularly important for protection against E. piscicida which is known to have at least six distinct genetic clades in North America. In this study, we aimed to identify immunogenic E. piscicida antigens in salmonids and catfish and evaluate their ability to stimulate protective immunity against subsequent challenge. Rainbow trout (O. mykiss), channel catfish (I. punctatus), and channel catfish (♀) × blue catfish (I. furcatus) (♂) hybrids (n = 10-13 fish/treatment) maintained in flow-through fresh water at 18±1°C (trout) and 25±1°C (catfish), were inoculated with formalin-killed whole-bacteria preparations containing a representative isolate from each E. piscicida clade suspended in Montanide ISA 763 A adjuvant to generate anti-E. piscicida IgM. Harvested serum was then used in a shotgun immunoproteomic approach for discovery of immunogenic E. piscicida peptides via LC/MS-MS that were consistently identified from multiple bacterial clades. Identified proteins were further evaluated based on their amino acid sequence homology, predicted subcellular localization, potential virulence function, and reported protective effect against other Gram-negative pathogens. From this analysis we selected three proteins for further investigation as vaccine candidates: GroEL, the glycine 2TM zipper domain-containing protein, and coproporphyrinogen-III oxidase. To evaluate the protective effect of the selected proteins, chinook salmon (O. tshawytscha) (n = 24/treatment) maintained in flow-through freshwater at 18±1°C were either orally (PO) or intra-coelomically (IC) immunized with vaccine preparations containing purified recombinant protein (10 µg protein/fish) suspended in the appropriate adjuvant (Montanide ISA 763 A for IC; Montanide GR1 for PO) and subsequently challenged via IC injection of ~106 CFU/fish of E. piscicida isolate S11-285, a known virulent strain to salmonids. Humoral response in surviving fish 56 days post-challenge was evaluated via IgM serum ELISA. The IC GroEL vaccine preparation induced a significant anti-protective effect (63.16% mortality) compared to sham vaccinated fish (22.3% mortality) (p=0.0071). Indirect ELISA revealed that fish vaccinated with the IC GroEL vaccine generated a significantly higher specific anti-GroEL antibody response (p=0.0153) compared to sham vaccinated fish regardless of exposure status. ELISA results also showed that sham vaccinated fish challenged with E. piscicida S11-285 had significantly higher (p=0.0238) specific anti-GroEL antibody response than non-exposed sham vaccinated fish, suggesting the viability of our shotgun immunoproteomic workflow as a screening method for identification of immunogenic bacterial antigens for fish vaccinology.
Best presentation award winner
1,2Renata C. Egger, 1Henrique César P. Figueiredo, 2Esteban Soto
1Department Preventive Veterinary Medicine, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616
The genus Edwardsiella comprises gram-negative bacteria in the family Hafniaceae. Four species in the genus, namely, E. tarda, E. piscicida, E. anguillarum and E. ictaluri have been associated with disease outbreaks in cultured tilapia in Latin-America. Still, little is known on the diversity of members in the genus, and until recently, E. tarda was considered to be the only pathogenic agent for tilapia. Therefore, this study aimed to determine the taxonomic classification and genetic diversity of Edwardsiella spp. affecting Nile tilapia in Brazil, the largest producer of tilapia in the Americas, and associate it to bacterial virulence. Isolates of Edwardsiella spp. were collected from diseased tilapia farmed in different regions of the country (Southeast=25, Northeast=7, Central-West=5 and South=3) between 2015 and 2022. Following bacterial isolation, the taxonomic classification of the isolates was determined through phylogenetic analysis based on the sequence of the dnaJ gene. Multi locus sequence typing (MLST) using the adk (adenylate kinase), atpD (β subunit of ATPase), dnaJ (heat shock protein 40), gapA (glyceraldehyde-3-phosphodehydrogenase), glnA (glutamine synthetase), hsp60 (60-KDs heat shock protein), phoR (phosphate regulon sensor protein), pyrG (cytidine triphosphate synthase), rpoA (α subunit of RNA polymerase) and tuf (elongation factor Tu) genes was also evaluated. Representative isolates from each genetic cluster identified in the MLST analysis were selected for in vitro challenges using immortalized Mozambique tilapia (Oreochromis mossambicus) brain cell line (OmB). The isolates were individually inoculated onto OmBs at a multiplicity of infection (MOI) of 10:1 (OmBs:bacteria), incubated at 28°C for 24h (E. tarda) or 48h (E. anguillarum and E. piscicida), and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) was used to quantify lactate dehydrogenase in the cell media. Among the bacterial isolates analyzed, 20 E. tarda, 19 E. anguillarum and one E. piscicida were identified. The MLST analysis identified 23 new sequence types (STs) for the isolates in this study (ST27-49), 19 STs for E. tarda, three for E. anguillarum and one for E. piscicida. The phylogenetic analysis based on the MLST revealed three main genetic clusters for the E. tarda isolates. All E. anguillarum and E. piscicida isolates presented Types 1, 3 and 6 secretion systems genes, and one E. tarda isolate had type 4 secretion system genes. All isolates had virulence genes related to hemolysis, adhesion, pili, fimbria, catalase and superoxide enzymes. The cytotoxicity of the three species to Omb cells varied. Edwardsiella anguillarum ST32 and ST28 were the two most cytotoxic species (28.28% and 23.61%, respectively). Among E. tarda isolates, genetic cluster two was the most cytotoxic (14.45% cytotoxicity). Edwardsiella piscicida isolates presented low cytotoxicity to OmBs.
1Claudio Murgia, 1Zeinab Yazdi, 1Esteban Soto Martinez
1Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, CA, 95616
Mycobacteriosis is a bacterial disease commonly found in captive fish. This disease is caused by various species of Mycobacterium, all of which have zoonotic potential. Currently, no vaccines or treatments are available for fish mycobacteriosis, and the primary control measures are eradication of infected stocks and facility disinfection. While research has been conducted to establish the disinfectant susceptibility of some Mycobacterium spp., most studies focus on planktonic forms, and only few studies address biofilms. Thus, this study aimed to compare biofilm formation among different Mycobacterium spp. previously recovered from outbreaks of mycobacteriosis in fish, and investigate their resistance to common disinfectants used in aquaculture systems. Initially, the minimum biofilm eradication concentration (MBEC) assay® system was used to determine biofilm formation of M. chelonae, M. salmoniphilum, M. arcueilense, and M. marinum at 28°C. Mature biofilms of each isolate were then used to evaluate the susceptibility of the different species to povidone-iodine, sodium hypochlorite (bleach), hydrogen peroxide, Virkon® Aquatic, and 70% ethanol. Different susceptibility was found between the different mycobacterial species. Mycobacterium marinum was susceptible to all disinfectants tested, while M. arcueilense was susceptible to bleach, povidone-iodine, and 70% ethanol (30 min treatment). However, at the currently recommended doses for aquaculture, none of the disinfectants were effective in eliminating M. chelonae or M. salmoniphilum biofilms. These preliminary results suggest that biofilm formation can vary among different Mycobacterium spp. and highlight the need for effective disinfectant strategies in aquaculture facilities to prevent the spread of mycobacteriosis. Further research is necessary to optimize disinfection protocols and ensure the health and safety of aquaculture systems.