A probiotic Enterobacter sp. provides significant protection against Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) after injection by two different routes

• Probiotic Enterobacter sp. enhance disease protection in fish
• Injection of probiotics stimulates anti-Flavobacterium psychrophilum antibodies
• Innate and adaptive immunity is enhanced following injection of probiotic bacteria

To determine the potential for protection against Flavobacterium psychrophilum infection, a study was conducted where a probiotic, Enterobacter sp. strain C6-6, was delivered to rainbow trout, Oncorhynchus mykiss, via injection. Two separate studies were conducted. In a preliminary study groups of rainbow trout (mean weight, 2 g) were either left unhandled or intramuscularly (IM) injected with a standardized concentration of either a 48 or 72 h culture of C6-6 and subsequently challenged with two different doses of F. psychrophilum 7, 28 and 56 days post-injection (PI). The relative survival ranged from 66 to 87%, 42 to 53% and 0 to 18% at 7, 28 and 56 days PI, respectively. In the second study groups of rainbow trout (mean weight, 1.3 g) received either an intraperitoneal injection (IP) of phosphate buffered saline (PBS; negative control), supernatant from a C6-6 culture, formalin killed C6-6, or live C6-6 and were subsequently challenged with F. psychrophilum 7 or 28 days PI. Log rank survival analysis showed a significant (P < 0.05) reduction in mortality for fish receiving these treatments at both 7 and 28 days PI. Injection of live C6-6 resulted in the lowest mortality at either time point and cumulative mortality at 7 days PI was significantly reduced (P < 0.05) in fish receiving formalin killed (62.7 ± 7.4%) or live Enterobacter C6-6 (48 ± 6.9%) when compared to the negative controls (92 ± 2.3%). Additionally, at 28 days PI, fish receiving the formalin killed or live C6-6 had significantly increased antibody titers against F. psychrophilum. This was not expected and suggests that protection observed at 28 days could be in part due to a cross protective adaptive immune response. Antibody titers were not detected at 7 days PI but significant protection was observed and indicates that innate immunity was responsible for this. Taken together, results from this study indicate that protection against F. psychrophilum after either IM or IP injection of this naturally occurring bacterium, either alive or dead, is at least in part dependent on the enhanced immune function(s) of the treated fish. This may shed light on protective mechanisms associated with the use of the Enterobacter sp. and may be useful as a potential alternate strategy for reducing the impacts from F. psychrophilum infection through non-specific immune-enhancement during times of increased fish stress or as a possible adjuvant.