2.5. Pathogenicity
Based on Koch’s rule, challenge tests were performed to test the
pathogenicity of the isolated strains. The isolated bacteria SZ01 was
cultured in liquid medium at 28 °C for 18h and then collected at 4 ° C
for 5 minutes at 4000 rpm. Contents were resuspended in sterile PBS
buffer. The concentration of the bacterial suspension was adjusted to
106,107,108 and
109 CFU/mL using sterile PBS. A total of 150 normalH. nipponia were randomly divided into 5 groups termed ABCDE,
where ABCD were the experimental groups and group E was the control
group. Each group contained 30 leeches. In groups A-D, 0.1mL of
bacterial solution was intraperitoneally injected into each H.
nipponia . The bacterial concentrations were 109,
108, 107 and 106CFU/mL respectively, which was equivalent to 108,
107, 106 and 105CFU for each H. nipponia . The same volume of PBS was injected
into the same area of the H. nipponia for the control group.
During the experiment, dissolved oxygen was kept at 7.5-8.5, water
temperature was 24-26 ℃ and fully aerated tap water was used for
breeding until death. Clinical symptoms and mortality were both recorded
daily. At the same time, dead H. nipponia were dissected and
bacteria were isolated and purified. Purified bacteria were identified
using 16S rRNA sequencing.