Among bacterial diseases affecting cultured salmonid fish, Bacterial Cold-Water Disease, caused by Flavobacterium psychrophilum, produces high mortality and morbidity and consequently, economical losses worldwide.
Attempts to control this situation through the use of antimicrobials have been limited due to the rapid appearance of antibiotic-resistant bacteria. Moreover, the determinants of antibiotic resistance that have emerged and selected in this aquatic environment have the potential of being transmitted by horizontal gene transfer to bacteria of the terrestrial environment, including human and animal pathogens.
Evidence indicating that antibiotic-resistant bacteria and antibiotic resistance determinants pass from the aquatic to the terrestrial environment has resulted in a drastic restriction of the use of antibiotics in aquaculture in many countries. For this reason, it is important to find new products with antimicrobial activity without the aforementioned undesirable effects of antibiotics.
In this sense, natural food additives or phytobiotics, which combined different mechanisms of action against pathogenic bacterial species (bactericidal/ bacteriostatic activities, Quorum Sensing inhibition), are potential candidates for the development of prevention strategies in aquaculture. This work describes the effect of the dietary administration to rainbow trout of the phytobiotic Liptofry (Liptosa S.A, Spain) in the improvement of disease resistance against Flavobacterium psychrophilum.
In this study 4000 healthy rainbow trout, distributed in two experimental groups (2000 individuals/each) were maintained in an aquaculture facility of the north of Spain, until its transfer to the aquarium of the Faculty of Biology of the University of Santiago de Compostela. Rate of food supply of both groups was established based on the requirements of the fish, taking as a reference the parameters of size and weight of the specimens and the temperature of the water.
- Group 1: Fish fed with feed supplemented with the phytobiotic Liptofry manufactured by Lípidos Toledo SA (Liptosa SA, Spain).
- Group 2: Fish fed with no supplemented feed (control fish).
The usefulness of feeding the fish with diet supplemented with phytobiotics in the prevention of Bacterial cold-water disease (BCWD) in rainbow trout was determined by experimental infection, using a virulent strain RBT4.1.04 of the species F. psychrophilum. With this aim, 120 fish of each experimental group (group 1 and group 2) were anaesthetized by immersion in tricaine methane sulfonate (MS-222, Sigma) (60mg/L) and infected by intra-peritoneal injection of a bacterial suspension containing 109 CFU/mL. Differences in survival between fish fed with phytobiotics (Group 1) and control fish (Group 2) after challenge with Flavobacterium psychrophilum were analysed by Chi-square test (P<0.05) (Figure 1).
Histopathological analysis was performed in order to evaluate the effect of the administration of the phytobiotic Liptofry in fish tissues. Tissue samples from control and Liptofry-feeded fish were taken at 24, 48, 72 y 96 h after experimental infection. Bouin’s fixed samples were embedded in paraffin wax, sectioned at 3 μm in thickness, and stained with hematoxylin-eosin (H-E), Phosphotungstic acid-haematoxylin stain (PTAH) and Periodic acid–Schiff stain (PAS) for light microscopy observation.
In our study, fish fed with the Liptofry supplemented diet (group 1) showed an average cumulative mortality of 27 following challenge experiment, compared to 62 percent mortality observed in control fish (group 2) (Table 1). The survival of fish fed with the phytobiotic Liptofry was significantly higher than those of the control group (p<0.05).
Table 1. Experimental infection with F. psychrophilum to evaluate the effect of the Liptofry supplemented diet in the prevention of BCWD
Histopathological analysis showed that fish of both experimental groups showed similar lesions, characterised by a moderate inflammatory response at the coelomic cavity, necrotic areas affecting the spleen, liver, and pancreatic lobes, and degenerative lesions of renal tubules, showing pyknosis and dequamation of epitelial cells (Figure 2),
Results of this study, suggest that the use of feed supplemented with the phytobiotic Liptofry in the diet of rainbow trout may contribute to the prevention of diseases caused by the bacterial pathogen Flavobacterium psychrophilum. Thus, these diet supplements could be used as an alternative to antibiotics or vaccines in the control and/or prevention of infectious diseases. Although distribution of the inflammatory response in fish of groups 1 and 2 infected with Flavobacterium psychrophilum was similar, the severity and extent of lesions, and the number of animals affected were higher in the control group. These findings might correlate with significant differences of mortality in groups one and two.
Acknowledgements
This work was supported by Grant CDTI-IDI-20140147 from the Centro para el Desarrollo Tecnológico e Industrial (CDTI), Ministerio de Economía y Competitividad, Spain
