Flavobacterium psychrophilum - Experimental challenge and immune response

Abstract

The disease rainbow trout fry syndrome (RTFS) is caused by the bacterial fish pathogen Flavobacterium psychrophilum. It has been the cause of great losses of rainbow trout in aquacultures both in Denmark and around the world. It was estimated that RTFS resulted in the death of 88 million fry in 1998, which amounted to a loss of 18 million DKK. Disease outbreaks are typically seen at water temperatures below 15 °C and typically with fry mortality rates from 50-60 %. Disease outbreaks are usually first observed shortly after the onset of feeding after the yolk sack is depleted. At this point the immune system of the fry is not fully developed. Theoretically, the infection pressure could be subdued by vaccinating larger fish, but no commercial vaccine is yet available. Diagnostic methods are well described and the disease is treated with antibiotics. To prevent disease outbreaks and subsequent use of antibiotics, further knowledge of the disease is needed. Previous studies focusing on various types of aquacultures demonstrated the presence of F. psychrophilum in all examined farms. The bacterium was demonstrated in gills, skin, internal organs and wounds both during RTFS outbreaks and periods without disease. The main purpose and focus of the present thesis was to increase knowledge of the immune response following infection with F. psychrophilum, which may contribute to the future development of vaccines and other preventive measures. The project consisted of three main tasks: 1) Establish an experimental infection model imitating natural infection, 2) examine the immune response in blood and selected organs, and 3) examine potential portals of entry for the bacterium. Previous experimental immersion-challenges involving F. psychrophilum have resulted in none or low mortality in rainbow trout fry, unless the fish are stressed or have their surface compromised through e.g. injuries to the skin. The effect of a range of hydrogen peroxide (H2O2) concentrations was tested on fry in order to assess mortality. An appropriate dose was subsequently combined with immersion in a diluted bacterial broth. The method was shown to increase the mortality of RTFS significantly and as a result, this model for infection was used for the planned studies. All subsequent investigations thus include two infected groups, one of which was pretreated with hydrogen peroxide, while the other was not. Real-time PCR (RT-PCR) was used to examine the immune response in the head kidney during the first eight days after infection, and enzyme-linked immunosorbent assay (ELISA) was used to evaluate the production of antibodies 50 days post-exposure. A pro-inflammatory response was observed in both groups infected with F. psychrophilum. However, only a weak pattern was observed in the regulation of the adaptive response, while numerous correlations between the regulation of genes and the amount of F. psychrophilum 16S rRNA were demonstrated. Exposure to H2O2 before immersion in F. psychrophilum also influenced correlation between regulation of genes and pathogen load in several cases. Pre-treatment with H2O2 e.g. delayed the positive correlation between IgM and pathogen load in the head kidney. Antibodies against F. psychrophilum were present in the blood of both infected groups 50 days after infection, but no significant difference could be observed at this time point. Morphological changes in the gill tissue after exposure to F. psychrophilum and H2O2 was examined on tissue sections stained with hematoxylin and eosin (H&E). Exposure to F. psychrophilum or H2O2 resulted in epithelial lifting and formation of edemas, but in both cases the tissue was regenerating after 192 hours. However, when the fish had been exposed to both H2O2 and F. psychrophilum, the damage was still evident at this time point. The relative pathogen load measured as 16S rRNA was highest at the first sampling and decreased steadily with no significant effect from pre-treatment with H2O2. No bacteria were observed on the H&E-stained tissue or visualized using a F. psychrophilum-specific probe with fluorescence in situ hybridization. Changes in gene expression in the gills after exposure to F. psychrophilum and H2O2 was also examined using RT-PCR. The observed immune response was limited, and neither a typical Th1 or Th2 response was observed. The results indicate that immersion in F. psychrophilum may suppress the immune response in its host and induce lesions in the gills. A single exposure to H2O2 influenced the immune response to subsequent exposure to F. psychrophilum in both head kidney and gills, and leads to increased mortality. The results show a need for further investigation regarding the potential connection between routine non-medical treatments like H2O2 in aquaculture and disease outbreaks.