Final Report Summary - AQUABAC (The use of potentially protective bacteria in aquaculture against fish pathogenic Flavobacterium spp.)
Project context and objectives
Salmonid aquaculture is a growing industry and significant employer in the EU and elsewhere (FAO Fisheries and Aquaculture Department, 2007). Rainbow trout fry syndrome (RTFS) and cold water disease (BCWD) have become major problems in salmonid aquaculture, causing significant mortalities especially in rainbow trout hatcheries. The causative agent of RTFS and BCWD is Flavobacterium psychrophilum. Antibiotics are the only treatment available for F. psychrophilum infections in aquaculture. However, several publications have already reported emergence of antibiotic resistance strains of F. psychrophilum on fish farms (e.g. Bruun et al. 2003. Aquaculture 215:11-20). Because of the emergence of antibiotic resistance of pathogens in aquaculture, it has been suggested that probiotics should be studied as alternatives to minimise the use of antibiotics (FAO Fisheries Technical Paper No.469). The objective in this project was to investigate the effectiveness of probiotic bacteria against F. psychrophilum infections in rainbow trout (Oncorhynchus mykiss). We were also interested in elucidating the mode of action of any useful probiotic bacteria.
Project work and results
1. Probiotics effective against F. psychrophilum: during this project, we conducted feeding experiments with two potential probiotics, which were originally isolated from the surface of rainbow trout eggs: Pseudomonas sp. M174 and M162. After feeding with these probiotics, fish were challenged with F. psychrophilum to see if they would prevent infection. Results showed that both probiotics had the capacity to decrease mortality in infected rainbow trout fry. Pseudomonas M174 fed fish had a cumulative mortality of 23 %, which was significantly less than control fish mortality 45 % (Korkea-aho et al. 2011. J. Appl. Microbiol. 111: 266-277). Mortality for Pseudomonas M162 fed fish was 35 % and was also significantly different from the control group, where mortality was 57 %. Both feeding experiments were repeated with similar results.
We have also conducted feeding experiments with two known probiotics: Aeromonas sobria GC2 and Bacillus sp JB-1. Both probiotics are known to be effective against several bacterial diseases of rainbow trout (Brunt et al. 2007. J. Fish Dis. 30: 573-579), but have not been tested against F. psychrophilum before. However, in our experiments statistically significant lower mortality was not achieved for these probiotic fed fish compared to controls, when fish were challenged with F. psychrophilum.
The ability of probiotics to adhere to and colonise the gut was also investigated. The results showed that viable cells of M174 and M162 were still found in the intestinal content of the fish two weeks after fish were fed with probiotics, although probiotics were most likely transient as the number of probiotic cells in fish intestine dropped after one week following the return to normal diet. The probiotics produced siderophores and interestingly, when inhibition properties against growth of F. psychrophilum were tested in iron scarce media, M174 inhibited the growth of F. psychrophilum but M162 did not have this property. Also other enzymatic activities of the extra cellular products (ECP) of the probiotics and pathogen were studied. The ECPs were extracted from probiotic bacterial strains: M162, M174, GC2 and JB-1, as well as F. psychrophilum JIP02/86 and their enzyme activity analysed on agar plates. Results showed that M174, M162 and GC2 had protease activity as revealed by clear zones in casein agar plates. Also, M174 cleared lysozyme agar plates. It is possible that one of the modes of action of M174 against F. psychrophilum is the production of inhibitory substances.
2. Immune properties of probiotics: innate immunological parameters of M174- and M162-fed fish were also investigated in vivo. After two weeks of probiotic feeding, fish samples were collected and immunological and haematological parameters analysed. Results showed that M174-fed fish had significantly higher respiratory burst activity than controls. However, no differences were shown in the numbers of erythrocytes or leucocytes in the blood, or in phagocytic activity or lysozyme activity between M174-fed and control fish. Furthermore, M162 stimulated peripheral blood leukocyte counts, serum lysozyme activity and total serum immunoglobulin levels after three weeks from the start of feeding.
Also, production of natural and specific antibodies were tested from the serum of M162-fed fish, but these were not significant when compared to control fish. Specific antigen production against F. psychrophilum of probiotic ECPs and whole cell products (WCP) were analysed to determine their capability of producing cross-reactions by western blotting using monoclonal and polyclonal anti F. psychrophilum, but no antigenic blots were produced by ECPs nor WCPs of M162 and M174 probiotics. These results suggest that one mode of action of probiotics is to stimulate the fish's innate immunity. One of the reasons for the high mortality seen in fry during RTFS outbreaks could be the lack of a developed specific immune response in these young fish. However, the young fish may be protected by innate immunity, and thus the effect of immunostimulants and probiotics may be beneficial in improving their innate disease resistance. Furthermore, we have preliminary results from microarray analysis indicating that gene expression of several innate immunologically related genes were up-regulated in the spleen of M162-fed fish. The gene expression data confirms our cellular level immunological response results, and furthermore gives a more specific picture of host interactions with probiotics.
Identification and comparison of probiotics: the probiotics M162 and M174 were identified as a Pseudomonas sp. by 16S rRNA gene sequencing comparing the data with the nucleotide database (BLASTN). The probiotics were simultaneously isolated from the eggs of healthy rainbow trout. To confirm that they are different organisms, the 16S rRNA partial gene sequences between the two organisms were compared and were found to have a 97 % identity. The 3 % difference found between the two isolates does confirms that M174 and M162 are indeed in different species, where as 97 % 16S rRNA gene sequence similarity confirms that they are part of the same operational taxonomic unit. Beside the genetic similarity of the probiotics, differences in phenotype and modes of actions against F. psychrophilum, also suggest that they are different species. Comparison of genotypic and phenotypic differences of the probiotics suggests that they have different modes of action against F. psychrophilum and further studies combining these probiotics could be useful.
Project outcomes and impact
Our results showed for the first time the potential of probiotics to control F. psychrophilum infections. Moreover, M174 and M162 have several modes of action including production of inhibitory substances, colonisation of intestine and stimulating the immunity of fish. This work has developed alternative disease control strategies to the use of chemotherapeutants and antibiotics. Indeed, probiotics could provide economical and safe treatment against F. psychrophilum infections in salmonid fish and could help strengthen Europe's position in the global aquaculture market. Probiotics used during the early life stages of fish can positively affect fish welfare, as probiotic bacterial treatments also enhance fish immunity and survival, as well as promoting the development of good indigenous microbial flora of fish. During this project, all the research has been designed to ensure that the utility of these probiotics are realised fully in an applied context and would benefit salmonid aquaculture in Europe. The project was carried out in Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK.
Salmonid aquaculture is a growing industry and significant employer in the EU and elsewhere (FAO Fisheries and Aquaculture Department, 2007). Rainbow trout fry syndrome (RTFS) and cold water disease (BCWD) have become major problems in salmonid aquaculture, causing significant mortalities especially in rainbow trout hatcheries. The causative agent of RTFS and BCWD is Flavobacterium psychrophilum. Antibiotics are the only treatment available for F. psychrophilum infections in aquaculture. However, several publications have already reported emergence of antibiotic resistance strains of F. psychrophilum on fish farms (e.g. Bruun et al. 2003. Aquaculture 215:11-20). Because of the emergence of antibiotic resistance of pathogens in aquaculture, it has been suggested that probiotics should be studied as alternatives to minimise the use of antibiotics (FAO Fisheries Technical Paper No.469). The objective in this project was to investigate the effectiveness of probiotic bacteria against F. psychrophilum infections in rainbow trout (Oncorhynchus mykiss). We were also interested in elucidating the mode of action of any useful probiotic bacteria.
Project work and results
1. Probiotics effective against F. psychrophilum: during this project, we conducted feeding experiments with two potential probiotics, which were originally isolated from the surface of rainbow trout eggs: Pseudomonas sp. M174 and M162. After feeding with these probiotics, fish were challenged with F. psychrophilum to see if they would prevent infection. Results showed that both probiotics had the capacity to decrease mortality in infected rainbow trout fry. Pseudomonas M174 fed fish had a cumulative mortality of 23 %, which was significantly less than control fish mortality 45 % (Korkea-aho et al. 2011. J. Appl. Microbiol. 111: 266-277). Mortality for Pseudomonas M162 fed fish was 35 % and was also significantly different from the control group, where mortality was 57 %. Both feeding experiments were repeated with similar results.
We have also conducted feeding experiments with two known probiotics: Aeromonas sobria GC2 and Bacillus sp JB-1. Both probiotics are known to be effective against several bacterial diseases of rainbow trout (Brunt et al. 2007. J. Fish Dis. 30: 573-579), but have not been tested against F. psychrophilum before. However, in our experiments statistically significant lower mortality was not achieved for these probiotic fed fish compared to controls, when fish were challenged with F. psychrophilum.
The ability of probiotics to adhere to and colonise the gut was also investigated. The results showed that viable cells of M174 and M162 were still found in the intestinal content of the fish two weeks after fish were fed with probiotics, although probiotics were most likely transient as the number of probiotic cells in fish intestine dropped after one week following the return to normal diet. The probiotics produced siderophores and interestingly, when inhibition properties against growth of F. psychrophilum were tested in iron scarce media, M174 inhibited the growth of F. psychrophilum but M162 did not have this property. Also other enzymatic activities of the extra cellular products (ECP) of the probiotics and pathogen were studied. The ECPs were extracted from probiotic bacterial strains: M162, M174, GC2 and JB-1, as well as F. psychrophilum JIP02/86 and their enzyme activity analysed on agar plates. Results showed that M174, M162 and GC2 had protease activity as revealed by clear zones in casein agar plates. Also, M174 cleared lysozyme agar plates. It is possible that one of the modes of action of M174 against F. psychrophilum is the production of inhibitory substances.
2. Immune properties of probiotics: innate immunological parameters of M174- and M162-fed fish were also investigated in vivo. After two weeks of probiotic feeding, fish samples were collected and immunological and haematological parameters analysed. Results showed that M174-fed fish had significantly higher respiratory burst activity than controls. However, no differences were shown in the numbers of erythrocytes or leucocytes in the blood, or in phagocytic activity or lysozyme activity between M174-fed and control fish. Furthermore, M162 stimulated peripheral blood leukocyte counts, serum lysozyme activity and total serum immunoglobulin levels after three weeks from the start of feeding.
Also, production of natural and specific antibodies were tested from the serum of M162-fed fish, but these were not significant when compared to control fish. Specific antigen production against F. psychrophilum of probiotic ECPs and whole cell products (WCP) were analysed to determine their capability of producing cross-reactions by western blotting using monoclonal and polyclonal anti F. psychrophilum, but no antigenic blots were produced by ECPs nor WCPs of M162 and M174 probiotics. These results suggest that one mode of action of probiotics is to stimulate the fish's innate immunity. One of the reasons for the high mortality seen in fry during RTFS outbreaks could be the lack of a developed specific immune response in these young fish. However, the young fish may be protected by innate immunity, and thus the effect of immunostimulants and probiotics may be beneficial in improving their innate disease resistance. Furthermore, we have preliminary results from microarray analysis indicating that gene expression of several innate immunologically related genes were up-regulated in the spleen of M162-fed fish. The gene expression data confirms our cellular level immunological response results, and furthermore gives a more specific picture of host interactions with probiotics.
Identification and comparison of probiotics: the probiotics M162 and M174 were identified as a Pseudomonas sp. by 16S rRNA gene sequencing comparing the data with the nucleotide database (BLASTN). The probiotics were simultaneously isolated from the eggs of healthy rainbow trout. To confirm that they are different organisms, the 16S rRNA partial gene sequences between the two organisms were compared and were found to have a 97 % identity. The 3 % difference found between the two isolates does confirms that M174 and M162 are indeed in different species, where as 97 % 16S rRNA gene sequence similarity confirms that they are part of the same operational taxonomic unit. Beside the genetic similarity of the probiotics, differences in phenotype and modes of actions against F. psychrophilum, also suggest that they are different species. Comparison of genotypic and phenotypic differences of the probiotics suggests that they have different modes of action against F. psychrophilum and further studies combining these probiotics could be useful.
Project outcomes and impact
Our results showed for the first time the potential of probiotics to control F. psychrophilum infections. Moreover, M174 and M162 have several modes of action including production of inhibitory substances, colonisation of intestine and stimulating the immunity of fish. This work has developed alternative disease control strategies to the use of chemotherapeutants and antibiotics. Indeed, probiotics could provide economical and safe treatment against F. psychrophilum infections in salmonid fish and could help strengthen Europe's position in the global aquaculture market. Probiotics used during the early life stages of fish can positively affect fish welfare, as probiotic bacterial treatments also enhance fish immunity and survival, as well as promoting the development of good indigenous microbial flora of fish. During this project, all the research has been designed to ensure that the utility of these probiotics are realised fully in an applied context and would benefit salmonid aquaculture in Europe. The project was carried out in Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK.