title: The Animal Model Determines the Results ofAeromonasVirulence Factors.
abstract: The selection of an experimental animal model is of great importance in the study of bacterial virulence factors. Here, a bath infection of zebrafish larvae is proposed as an alternative model to study the virulence factors ofAeromonas hydrophila. Intraperitoneal infections in mice and trout were compared with bath infections in zebrafish larvae using specific mutants. The great advantage of this model is that bath immersion mimics the natural route of infection, and injury to the tail also provides a natural portal of entry for the bacteria. The implication of T3SS in the virulence ofA. hydrophilawas analyzed using the AH-1::aopBmutant. This mutant was less virulent than the wild-type strain when inoculated into zebrafish larvae, as described in other vertebrates. However, the zebrafish model exhibited slight differences in mortality kinetics only observed using invertebrate models. Infections using the mutant AH-1ΔvapA lacking the gene coding for the surface S-layer suggested that this protein was not totally necessary to the bacteria once it was inside the host, but it contributed to the inflammatory response. Only when healthy zebrafish larvae were infected did the mutant produce less mortality than the wild-type. Variations between models were evidenced using the AH-1ΔrmlB, which lacks the O-antigen lipopolysaccharide (LPS), and the AH-1ΔwahD, which lacks the O-antigen LPS and part of the LPS outer-core. Both mutants showed decreased mortality in all of the animal models, but the differences between them were only observed in injured zebrafish larvae, suggesting that residues from the LPS outer core must be important for virulence. The greatest differences were observed using the AH-1ΔFlaB-J (lacking polar flagella and unable to swim) and the AH-1::motX (non-motile but producing flagella). They were as pathogenic as the wild-type strain when injected into mice and trout, but no mortalities were registered in zebrafish larvae. This study demonstrates that zebrafish larvae can be used as a host model to assess the virulence factors ofA. hydrophila. This model revealed more differences in pathogenicity than thein vitromodels and enabled the detection of slight variations in pathogenesis not observed using intraperitoneal injections of mice or fish.
authors.affiliation: Department of Immunology and Genomics, Marine Research Institute-Consejo Superior de Investigaciones Científicas, Vigo Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Romero
authors.affiliation: Department of Immunology and Genomics, Marine Research Institute-Consejo Superior de Investigaciones Científicas, Vigo Spain.
authors.forename: Paolo R
authors.initials: PR
authors.lastname: Saraceni
authors.affiliation: Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Merino
authors.affiliation: Department of Immunology and Genomics, Marine Research Institute-Consejo Superior de Investigaciones Científicas, Vigo Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Figueras
authors.affiliation: Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Tomás
authors.affiliation: Department of Immunology and Genomics, Marine Research Institute-Consejo Superior de Investigaciones Científicas, Vigo Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Novoa
keywords: Aeromonas; animal model; in vivo infection; mice; mutant Aeromonas; rainbow trout; virulence factors; zebrafish larvae