Persistence of Flavobacterium psychrophilum in the aquatic environment.

Abstract

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD). Despite RTFS causing significant economic losses in salmonid aquaculture, there is limited information on the epidemiology of the disease and the pathogenicity of the bacterium. An important step for the development of successful control strategies for the disease is the development of rapid, sensitive and specific diagnostic tools for the isolation and identification of the pathogen in environmental and fish samples. This study examined the potential of using a number of different methods to identify the bacterium in environmental samples. In addition the ability of the bacterium to survive under conditions of prolonged starvation and the morphological as well as functional changes that occur, were examined. The potential of using a polymerase chain reaction (PCR) to detect and monitor F. psychrophilum in farming systems was investigated in this study. After validating the PCR assay in terms of sensitivity and specificity, two different surveys were performed on two fish farms with similar tank layouts, both of which had an endemic problem with RTFS. The results of both surveys indicated that the majority of areas of both farms sampled (egg incubators, hatchery, fry tanks, broodstock, farm outlet) were positive for the pathogen, while both inlets were negative. An inappropriate farm layout and possibly ineffective disinfection procedures may have led to the outbreaks of RTFS with high mortalities on these farms. Morphological and functional changes in F. psychrophilum, observed under conditions of starvation were examined. Bacteria maintained in stream water stopped multiplying and became small and rounded. Their culturability declined until it was no longer possible to obtain colonies on agar plates 19 weeks after setting up of the experiment. However, even after 36 weeks, it was still possible to obtain growth of the bacterium by a resuscitation step in Cytophaga broth. The culturability of the bacterium did not correspond with its viability as tested with a Live/Dead kit. Bacteria maintained in distilled water or treated with a disinfectant, appeared non-viable and non-culturable 1h after setting up the experiment. No morphological changes were observed in the bacteria maintained under these conditions. Bacteria maintained in iv broth were present as long, slim rods, some of which developed into 'ring' formations. Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria. There was also a marked decline in the sensitivity of a PCR method used to detect bacteria 16 weeks from the onset of the study, with differences also observed in the sensitivity of the PCR between bacteria maintained under the different treatments. The ability of F. psychrophilum to attach to unfertilised rainbow trout eggs and to hydrocarbon n-hexadecane was examined, whereby five different isolates of F. psychrophilum obtained from a variety of origins were compared. The effect of the age of the bacterium and conditions of starvation on the ability of the bacterium to adhere, were also evaluated. The different isolates were found to exhibit a similar ability to attach to both substrates. Increased surface hydrophobicity and a greater ability to attach to the surface of the eggs were observed with bacteria aged for one month, compared to bacteria cultured in Cytophaga agar for only three days. The potential of four different methods to identify and enumerate F. psychrophilum in water samples was also examined. Semi-quantitative PCR and quantitative PCR using an internal standard appeared to be specific but lacked reproducibility. The in situ hybridisation technique detected bacteria from three-day old culture but no signal was observed when one-month old bacteria were tested. Further investigation is needed to validate the method in terms of specificity. The IFAT method provided positive results with aged bacteria however, there is concern over the specificity and sensitivity of the antibodies used. The ability of F. psychrophilum to colonise the surface of rainbow trout eggs after a bath challenge and the effects of this colonisation on the egg was also investigated. The bacterium appeared not to penetrate within the eggs after colonisation of the surface of the eggs. Further studies are needed to confirm this, however, especially under adverse environmental conditions as often observed in commercial hatcheries during incubation (e.g. high egg densities, reduced water flow, higher water temperature). The techniques used in this study show great potential for the investigation of the epidemiology of the disease and once the problems discussed are resolved, their application will undoubtedly assist in the successful development of control strategies for RTFS.