Identification of twelve Aeromonas spp. species with monoclonal antibody-based immunoassay in water and fish samples

Aeromonas spp. are the causative agents of motile Aeromonas septicemia (MAS) in fish, as well as gastroenteritis and infections in humans. The antigen diversity between Aeromonas spp. and serotypes greatly challenges the identification of this zoonotic pathogen in environmental, food, and clinical samples. In the present study, six conserved peptides in the outer membrane of Aeromonas were selected and used as immunogens after conjugation to produce monoclonal antibodies (mAbs). Peptides 1 and 4 with the amino sequences of “VYDKDGTTFD” and “GGFKGKLSYQTND”, effectively elicited cross-reactive antibodies against Aeromonas in mice. Based on the mAbs 1B1 and 14H5, the developed sandwich immunoassay detected twelve Aeromonas spp. species, including A. hydrophila (8/8), A. veronii (7/7), A. caviae, A. sobria, A. dhakensis, A. media, A. popoffii, A. punctata subsp. caviae, A. bivalvium, A. lacus, A. jandaei, A. bestiarum and A. salmonicida (1/2). The detection limit of these strains primarily ranged from 1.69 × 104 to 4.57 × 105 CFU mL−1. No cross-reactivity was observed with the other tested strains. The sensitivity and specificity of this method was 96% (26/27) and 100% (15/15), respectively. Furthermore, real sample analyses detected river water samples containing 105 CFU mL−1 of A. hydrophila or A. caviae, and detected Carassius auratus samples showing hemorrhagic septicemia after enrichment in buffered peptone water. The established immunoassay may be promising as an effective screening method for monitoring Aeromonas spp. in environmental and food samples. [Display omitted] Broad-spectrum mAbs are the cornerstone for an accurate immune-diagnosis of versatile environmental pathogens. Many species in Aeromonas spp. (e.g., A. hydrophila, A. veronii, A. sobria, A. caviae, and A. salmonicida) cause severe infection in fish, as well as gastroenteritis diseases in humans. The antigen diversity between Aeromonas spp. and serotypes greatly challenges the identification of this zoonotic pathogen. So far mAbs against Aeromonas are usually prepared with a whole-cell antigen or inclusion body of recombinant proteins, the high affinity and broad cross-reactivity against Aeromonas spp. are difficult to balance. In this study, conserved and surface-exposed peptides of Aeromonas spp. were bioinformatically selected for antigen preparation and producing broad-spectrum mAbs with controlled specificity. The established immunoassay for Aeromonas spp. sensitively detected twelve