The novel pathogenic Citrobacter freundii (CFC202) isolated from diseased crucian carp (Carassius auratus) and its ghost vaccine as a new prophylactic strategy against infection.

Citrobacter freundii , a common pathogen of freshwater fish, severely restricts the sustainable development of the aquaculture industry. In this study, the novel C. freundii strain CFC202 was isolated from the intestines of crucian carp, which was annotated with three virulence factors related to promoting bacterial colonization and causing anemia by the whole genome analysis. We found that the strain adhered to the surface of hepatic L8824 cells, and actin aggregation occurred at the adhesion site of the strain. Then, C. freundii was fluorescently labelled and imaged the distribution of crucian carp under an In Vivo Imaging System (IVIS) during natural infection. The results showed that this strain mainly gathered in the intestines of crucian carp, and the intestinal villi were severely damaged by TEM. From the results of histopathological examination, the intestinal mucosa was obviously shed and various organs were damaged. Hence, we speculated that C. freundii could cause attaching and effacing (A/E) lesions to fish cells, which in turn caused pathological changes to various organs. Moreover, we constructed C. freundii ghost (CFG) and evaluated their immune effects by oral and injection immunization in crucian carp. Compared with the non-immunized group, non-specific immune indicators (ACP, AKP, SOD, and LZM), agglutination antibody titres of the serum, and the expression of immune-related genes (TNF-α and IL-6) of the head kidney and spleen were significantly increased in the immunized group (P < 0.01), indicating that innate and specific immune responses were induced by the CFG. In addition, the immunized fish showed higher protection than the non-immunized fish after challenge: oral control (20.00% survival); oral test (80.21% survival); injected control (6.67% survival); injected test (87.50% survival). Taken together, our research was the first to demonstrate that the constructed CFG might be developed as either oral or injected vaccines against C. freundii in aquaculture. • Citrobacter freundii CFC202 was firstly isolated from the diseased crucian carp in the Dongting Lake area of China. • Genome analysis of CFC202 showed it could mediate adhesion to cell surface through virulence factors such as hop AJ2 and fep E. • Strain CFC202 could cause attaching and effacing lesions to fish cells in vivo and in vitro. • This study was the first to construct Citrobacter freundii ghost and evaluate their immune effects in crucian carp. • Immunization with CFG vaccine in advance in crucian carp could improve disease resistance against Citrobacter freundii. [ABSTRACT FROM AUTHOR]