We collected Chinese sturgeons displaying clinical signs and transported them to our laboratory (Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China) for disease diagnosis and isolation of pathogens. For infection experiments, we obtained healthy hybrid sturgeons (Acipenser baeri [male] × Acipenser schrenckii [female]) from farms without any history of such diseases. The hybrid sturgeons selected were energetic, showed no visible scars, and weighed 100 ±10 g. The hybrid sturgeons were temporarily housed in buckets for 7 days to confirm their health before infection testing.

We investigated the breeding environment and water source of the Chinese sturgeon farm to determine the temperature and water quality conditions during the onset of disease as well as the disease history and drug use at the farm. To identify the key characteristics of the disease, we examined the body surface and anatomy of Chinese sturgeons displaying typical signs and those in critical condition. Fish showing typical signs were brought back to the laboratory, where we examined the internal organs and gills under an optical microscope to look for parasites and fungi (9).

Initially, we anesthetized the Chinese sturgeons and placed them on ice and disinfected their entire body with 75% ethanol. Using an inoculation ring, we sampled blood, kidneys, and intestines of each fish and inoculated the samples onto brain–heart infusion agar plates, incubated at 28°C for 24 hours. We selected the dominant strain on the plate for further purification, resulting in a dominant strain temporarily named zhx1. To preserve the purified strain, we added 15% glycerol, mixed well, and then stored it at –80°C for future use (10,11).

We fixed intestines, spleen, liver, kidneys, and gills of the Chinese sturgeons that had displayed clinical signs with 10% neutral formalin fixative. We then prepared slides of the tissues and performed histologic examination according to standard methods (12,13). We inoculated the isolated strain zhx1 onto brain–heart infusion agar plates and cultured them at 28°C for 18 hours. We then washed the bacterial moss with sterile phosphate-buffered saline (PBS) and adjusted the bacterial suspension to different concentrations.

For the experiment with healthy fish, we randomly divided 150 healthy hybrid sturgeons into 5 group of 30 each: A, B, C, D, and E. Each sturgeon in groups A–D was injected with 0.1 mL of bacterial suspension at the base of the ventral fin, at concentrations of 10⁹ CFU/mL for group A, 10⁸ CFU/mL for group B, 10⁷ CFU/mL for group C, and 10⁶ CFU/mL for group D. The sturgeons in the control group (group E) were injected with an equal dose of sterile PBS at the same site. During the experiment, the fish were not fed, dissolved oxygen was maintained between 7.5 and 8.5 mg/L, the water temperature was controlled at 21°C to 22°C, and the fully aerated tap water was changed daily. We collected visceral tissues of dying sturgeons for bacterial isolation and purification and monitored the condition of the experimental hybrid sturgeons until deaths ceased. We recorded our observations of the experimental sturgeons daily and calculated the mortality rate.

We analyzed drug sensitivity of the zhx1 isolate by using the disk-diffusion method according to the Clinical and Laboratory Standards Institute antimicrobial drug sensitivity experimental standard (14). We inoculated the zhx1 isolate into brain–heart infusion and maintained the culture at 28°C with constant temperature oscillation (200 r/min) for 24 hours. Subsequently, we diluted the bacterial suspension with PBS to a concentration of 10⁷ CFU/mL. Next, we spread 100 μL of the bacterial suspension onto Mueller-Hinton agar plates, placed the drug-sensitive disks on the plates, incubated them at 28°C for 24 hours, and measured the diameter of the inhibition rings.

We extracted genomic DNA from bacterial cultures of zhx1 in brain–heart infusion by using a genomic DNA extraction kit (TaKaRa, https://www.takarabiomed.com). We used the PacBio Sequel platform (https://www.pacb.com) to sequence the entire genome. We used the hierarchical genome-assembly process version 2.3.0, single-molecular real-time analysis, for read assembly (15). We conducted predictions of coding DNA sequences by using Glimmer 3.02 (16). We generated the circular map of the genome by using Circos version 0.64 (17) and predicted genome islands by using the IslandPath-DIOMB genomic island prediction method (18). We predicted transfer RNA by using tRNAscan-Sev1.3.1 and ribosomal RNA by using Barrnap 0.7 software (19). We identified clusters of regularly interspaced short palindromic repeats by using MinCED (20). We performed functional annotation through a BLASTP search (BLAST 2.2.28+, https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the National Center for Biotechnology Information nonredundant database, gene database, string database, and gene ontology (GO) database. Protein function classifications were based on clustering of protein homology group (clusters of orthologous genes) annotations by using the string database and BLASTP comparisons (21). We compared predicted genes with the Kyoto Encyclopedia of Genes and Genomes database by using the BLAST algorithm to identify corresponding genes involved in specific biologic pathways, based on the Kyoto Encyclopedia of Genes and Genomes orthogonal number obtained from the comparison (22). GO annotations were performed by using Blast2GO.

A phylogenetic tree based on bacterial whole-genome sequencing results (Figure 3, panel A) revealed clustering of zhx1 with Y. ruckeri, confirming that the isolated strain zhx1 is Y. ruckeri. The whole genome of zhx1 consists of a circular chromosome spanning 3,772,850 bp with an average guanine-cytosine content of 47.61% (Figure 3, panel B). Genome DNA sequencing generated 855,183 reads totaling 8,513,300,630 bp; sequencing depth was 1990×, and coverage rate was 100%. We identified 3,475 coding sequence genes, 22 ribosomal RNA genes, and 80 transfer RNA genes. Using the genomic island prediction method, we found 8 putative genomic islands in zhx1. In zhx1, we also identified 2 clustered regularly interspaced short palindromic repeats containing multiple short and repetitive sequences, 21-47–bp long. The sequencing reads are available in the National Center for Biotechnology Information Sequence Read Archive database (accession no. PRJNA1007872).

The zhx1 genome consisted of 443 virulence factor-related genes (e.g., which affect hemolysin, flagella, enterobactin, and outer membrane protein A), 135 drug-resistance-related genes (e.g., which affect macrolide, fluoroquinolones, aminoglycosides, cephalosporins, tetracyclines, and phenicol), 109 carbohydrate enzyme-associated genes, and 514 host–pathogen interaction–associated genes.

We determined the sensitivity of the zhx1 isolate to 20 antimicrobial agents. zhx1 was highly sensitive to chloramphenicol and florfenicol and exhibited varying degrees of resistance to other drugs, especially those commonly used in aquaculture (e.g., doxycycline, neomycin) (Table).