2.1. Sampling
Diseased H. nipponia were collected in the Jingzhou area of Hubei Province in China. These leeches contained a body length of 3-8 cm and showed typical pathological symptoms. Dying leeches were stored in oxygen containing bags and immediately transported to the laboratory for diagnosis and pathogen isolation. Meanwhile, normalH. nipponia purchased from the farm were 3-5 cm in length and cultured in an inflated water tank at 28 ℃ for one week for the pathogenicity test.
2.2. Pathogen isolation
Typical symptoms of skin mucus and visceral tissues in H. nipponia were examined for parasites. Bacteria were isolated in a secondary biosafety cabinet (ESCO Singapore). Dying, diseased leeches were placed on ice and sterilized using 75% ethanol. Visceral tissue of each H. nipponia was added to an inoculation ring and placed on brain heart infusion agar (BHIA; Difco, USA) for 24 hours at 28 ℃. The dominant strain was selected and purified with the purified strain temporarily named SZ01. Next, 15% glycerin was added into the purified strain, mixed and frozen at - 80 ℃.
2.3. Biochemical characterization of bacterial isolates
The SZ01 strain was inoculated on brain heart infusion agar and kept at a constant temperature of 28 ℃ for 24 hours before being stained by gram. The physical and chemical indexes of the strain were determined using the micro biochemical identification tube of bacteria based on the manual of common bacterial system identification (Dong and Cai, 2001).
2.4. 16S ribosomal DNA sequencing analysis
The isolated strain SZ01 was inoculated on brain heart infusion agar, cultured at 28 ℃ for 24 hours and a single colony was dissolved in 10 μL sterile water, which was used as a PCR template.
The primer used to detect the 16S rRNA sequence was 27F: 5’-AGAGTTTGATC(C/A)TGGCTCAG-3’, 1492R: 5’-GGTTACCTTGTTACGACTT-3’(Polz and Cavanaugh, 1998). The PCR reaction system included 2×Taq PCR mix, 50 μL; ddH2O, 47 μL; 1 μL for each primer and 1 μL for each template. Reaction conditions included 95 ℃, 1 min; 98 ℃, 15s, 55 ℃, 30s; 72 ℃, 2min; 35 cycles in this stage, 72 ℃, 15min. The amplified product was verified using 1% agarose gel electrophoresis as the target fragment size and then sent to Shanghai bioengineering for purification and sequencing. The 16S rRNA gene sequence of the SZ01 strain was put into the NCBI for comparison. The 16S rRNA sequences of Klebsiella and important aquatic pathogens were selected and analyzed by cluster x software. The phylogenetic tree was constructed by the Neighbor-Joining method using the mega 6.0 software and the confidence of the bootstrap test was 10000 times.