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.