Francisella orientalis is a facultative intracellular pathogenic bacterium that triggers the disease known as francisellosis and can swiftly spread within tilapia populations, resulting in considerable losses in aquaculture. Thus, implementing preventive and control strategies, including good aquaculture practices, rigorous biosecurity control, and vaccination are crucial to curbing the disease’s dissemination. Francisella spp. inactivated vaccine poses numerous challenges owing to the bacterium’s knotty cultivation process in the laboratory, characterized by slow growth under a temperature range of 22 °C to 24 °C and specific nutritional demands.
The inactivation process aims to render cells incapable of causing diseases, contaminating, or proliferating in the environment. However, this process must preserve the bacterial structure to ensure that the cells remain immunogenic and antigenic, thereby retaining the main epitopes of the cellular structure. Therefore, this study aimed to produce an inactivated vaccine against francisellosis. The vaccine was developed using the pathogenic strain, isolated from fish in commercial fish farming. It was cultured in a Schott® bottle containing 700 mL of BHI broth added with 0.2% L-cysteine hydrochloride and 1% glucose to enhance growth. The culture was incubated in an orbital shaker at 150 rpm at 22 °C for 72 hours to promote optimal growth conditions. Gram staining, catalase, and oxidase tests, along with PCR analysis, were employed to confirm the identity and purity of the bacterial culture. Results revealed Gram-negative coccobacilli with negative catalase and oxidase reactions. Additionally, PCR testing yielded a positive result, with the presence of a compatible fragment at 286bp. The bacterial culture underwent inactivation using formalin, followed by washes with 0.9% saline solution three times. Inactivation was validated by inoculating 0.1mL of the culture onto chocolate agar (CHOC) and BHI broth supplemented with glucose and cysteine followed by incubation at 22°C for 14 days. After 7 days 0.1mL of BHI broth was inoculated onto CHOC, with another 14 days of incubation. Upon this process, no colonies or turbidity were observed in either the agar plates or the broth, confirming successful inactivation.
The bacterial suspension was emulsified with Montanide® adjuvant in the recommended proportion by the manufacturer. This is a promising approach for the development of inactivated vaccines, as it aims to maintain the bacterial structure, thereby preserving its immunogenicity and antigenic properties. Such a vaccine holds significant potential for application in Brazilian tilapia farming, offering a viable solution for francisellosis prevention in aquaculture.