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

Highlights

• 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 hopAJ2 and fepE.

• 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

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.

Introduction

Carassius auratus has been widely loved by consumers because it is rich in nutrients such as vitamins, protein and unsaturated fatty acids. Currently, the artificial culture of crucian carp in the Dongting Lake area (Hunan, China) is increasingly widespread and has become an important economic pillar of the area (Zou et al., 2020). However, the deterioration of water quality has led to the growth of bacteria, especially pathogenic bacteria, which eventually causes mass death of farmed aquatic animals; for example, aquatic diseases caused by Citrobacter freundii (Baldissera et al., 2018; Liu et al., 2020).

C. freundii is an aerobic or facultative anaerobic Gram-negative bacterium belonging to the genus Citrobacter of Enterobacteriaceae, which is widely distributed in the environment and is condition-causing bacterium for human-animal-fish co-occurrence (Bai et al., 2012; Karunasagar and Pai, 1992; Liu et al., 2018; Sato et al., 1982). In recent years, diseases caused by C. freundii in the aquaculture industry have become increasingly frequent and can infect many types of aquatic products, such as fish, shrimp, crabs, and turtles (Xiong et al., 2020; Zou et al., 2020). The main clinical symptoms of diseased aquatic products are redness and swelling of the anus, skin ulcerations, and pathological changes to the liver, spleen, and kidney (Baldissera et al., 2018; Sun et al., 2018). Adhesion is a prerequisite for pathogenic bacteria to cause disease. There have been previous reports that C. freundii could adhere to the intestines of mice and cause attaching and effacing (A/E) lesions to colon epithelial cells (Schauer and Falkow, 1993). However, the pathogenicity of C. freundii to fish cells and the site of infection in fish are unknown. A large number of studies have indicated that we can monitor pathogens in the body in real-time by using fluorescent proteins to label pathogens and study the infection sites of pathogenic invasion (Cao et al., 2019; Ling et al., 2001). This provides an intuitive and convenient technique for studying the infection mechanism of C. freundii.

Despite the increase in aquatic diseases caused by C. freundii, the method of prevention and treatment is still traditional and involves the use of antibiotics. Long-term use of antibiotics triggers a series of negative effects, such as antibiotic residue and drug resistance, and previous reports have indicated that C. freundii contains multiple resistance genes (Zhou et al., 2019). In order to better prevent C. freundii infection within aquatic products, it is very important to develop an effective and environmentally friendly treatment, such as a vaccine. Vaccines have become one of the most effective means of preventing disease in fish and other farmed animals (Wang and Lu, 2009). As a new type of inactivated vaccine, bacterial ghost vaccines use the principle that the LysisE gene of the bacteriophage PhiX174 is accurately expressed in Gram-negative bacteria by the temperature control system of pL/pR-cI837 to form bacterial ghost (Yan et al., 2018). The bacterial ghost retain the original cell morphology, surface antigens and adhesion characteristics of the cell to the utmost extent. A large number of studies have shown that ghost vaccines produce stronger innate immunity, humoural and cellular immunity than traditional inactivated vaccines (Kwon et al., 2009; Tu et al., 2010; Wang et al., 2018; Zhang et al., 2019).

In the present study, we isolated a novel C. freundii strain from the intestines of crucian carp, which was highly pathogenic and caused a new disease in the water system of Dongting Lake. We firstly sequenced and analyzed its whole genome, and then analyzed its pathogenicity against crucian carp in vivo and in vitro, and constructed a fluorescent marker of C. freundii to determine the site of infection in crucian carp. Moreover, we prepared C. freundii ghost and evaluated the immune effects on crucian carp. Our research provides data support and a theoretical basis for the study of pathogenic mechanisms and the prevention of C. freundii.