Detection, genotyping and pathogenicity of virulence genes in Aeromonas species isolated from diseased freshwater fish

Aeromonas spp. are gram-negative bacteria that can infect a variety of aquatic and terrestrial animals, including humans and cause motile aeromonad septicemia. The prevalence of Aeromonas spp. in various water and food sources poses a significant public health threat. Aeromonas spp. encompass a diversity of strains or genotypes with varying pathogenic potentials. Many strains are pathogenic, but others are avirulent and produce little harm to the host. The availability of methods to assess the pathogenic potential of Aeromonas spp. would contribute to the improved control and prevention strategies for this bacterial infection. An approach to the direct detection of potentially pathogenic Aeromonas spp. is the use of virulence determinants as genetic markers. To investigate the correlation between virulence genes and pathogenicity of Aeromonas spp., 173 strains of Aeromonas were isolated from different diseased freshwater fish from 2009 to 2018 as the research object. Virulence genes including aer, act, fla, lip, gcaT, exu, ast, alt, eprCAI and ahyB were detected and the bacterial pathogenicity with different virulence genotypes was assessed through intraperitoneal (IP) injection challenged Carassius auratus gibelio (gibel carp). Molecular identification revealed that A.veronii (119/173, 68.8%) and A.hydrophila (50/173, 28.9%) were the prevalent species based on gyrB gene sequencing. The distribution of 10 virulence genes including aer (162/173, 93.64%), act (131/173, 75.72%), ast (55/173, 31.79%), alt (58/173, 33.53%), lip (152/173, 87.86%), exu (154/173, 89.02%), fla (143/173, 82.66%), gcaT (148/173, 85.55%), eprCAI(41/173, 23.70%) and ahyB (51/173, 29.48%) were determined and these strains were sorted into 7 clusters (Ⅰ-Ⅶ) and formed 53 genotypes according to the virulence distribution profile. 8-10 virulence genes were present in 24.86% (43/173) Aeromonas strains, among which 38 strains were identified as A. hydrophila and 4 strains A.veronii. The eprCAI (0/119, 0%), ahyB (19/119, 15.13%), ast (7/119, 5.88%) and alt (24/119, 20.17%) genes were detected from the A.veronii isolates. Most of the Aeromonas strains (94.22%, 163/173) had hemolytic activity. At the challenge dose of 3.0×10⁶CFU per fish, 3 strains of A.veronii (Ⅳ-5, Ⅴ-1 and Ⅵ genotype) and 16 strains of A.hydrophila (Ⅰ, Ⅱ-1, Ⅱ-3, Ⅱ-5, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅲ-5, Ⅳ-1 and Ⅳ-2 genotype) were highly pathogenic and the mortality reached 80%-100%.The study suggested that A.veronii was the most prevalent species in the diseased fish. A.veronii isolates had fewer virulence genes and lower pathogenicity than A.hydrophila ones. The result may provide reference for epidemiological investigation of motile aeromonad septicemia and vaccine research.