| 论文摘要： |
Flavobacterium columnare is a gram-negative pathogen that causes columnaris disease, which causes economic losses mainly in carp and catfish aquaculture. In this work, the strain Ci2323 was isolated from diseased grass carp (Ctenopharyngodon idella). It exhibited characteristic yellow pigmentation and a rhizoid spreading colony on modified Shiehs agar. Genome sequencing identified Ci2323 as F. columnare, a species well matched to F. columnare F2S17 with an average nucleotide identity (ANI) of 99.29 %. F. columnare Ci2323 is susceptible to florfenicol and tetracycline but resistant to oxacillin. Subsequently, F. columnare Ci2323 was attenuated by rifampicin passaging and mutant strains with shorter rhizoids were acquired. Of these, mutant strains that form immature biofilm are more severely attenuated in grass carp. The mutant strain CiR6 is hyper-adhesive to zebrafish larvae, forms an immature biofilm and secretes reduced extracellular proteins (ECP), in particular, the type VI secretion system cargo protein TssD and a similar to 13 kDa hypothetical protein. Genome comparison between the wild-type Ci2323 and the mutant CiR6 revealed the absence of gldJ, porU and the gliding motility-associated C-terminal domain-containing protein. Grass carp vaccinated by bath immersion with F. columnare mutant strain CiR6 for 2 and 4 weeks developed a relative percent survival (RPS) of 83.66 % and 71.76 %, respectively, when being challenged by bath immersion with 5-fold LD50 of the F. columnare wild-type Ci2323. Transcript levels of IgM, IgZ and IL-10 peaked 4 weeks after immunization. In conclusion, rifampicin passaging of the clinical isolate F. columnare Ci2323 yielded an attenuated vaccine candidate that may aid in the management of columnaris disease. |
