Significance of Flavobacterium diseases on salmonid farming in Chile Ruben Avendaño-Herrera1, Pedro Ilardi2, Jorge Fernández1 1 Universidad Andrés Bello, Facultad de Ciencias Básicas, Departamento de Ciencias Básica. Santiago, Chile. 2 Laboratorio de Patología de Peces, Veterquímica, Santiago – Chile Correspondence: R. Avendaño-Herrera ([email protected]) Abstract Chile is considered the second largest producer of farmed salmon in the world. This intensive fish farming has resulted in growing problems as bacterial diseases, and infections by Flavobacterium psychrophilum, Chryseobacterium species and other yellow-pigmented strains are considered to be one of the major problems in the freshwater stage in Chilean aquaculture where infections can result in 5 to 90% mortality rate of fingerlings, particularly in Atlantic salmon. The infections are frequently found in association with infectious pancreatic necrosis virus (IPNV), saprolegnia or parasitic fish pathogens as Ichthyophthirius multifilis, but the significance of these combinations is unclear. According to the distribution of the diagnosis informed by the laboratories showed that flavobacteriosis is the second pathology affecting salmon cultures, after Piscirickettsia salmonis. In the present article we review current knowledge on these bacterial pathogens, focusing on important aspects such as biochemical, serological and genetic characterization of the microorganisms. The diagnostic procedures employed in Chile are also discussed. We include the current status of prevention and control strategies, studies of virulence, and we attempt to highlight fruitful areas for continued research. 1. Introduction The intensive exploitation of salmonids is an activity of high economical importance in Chile, being this country considered the second largest producer of farmed salmon in the world with an estimated production of approximately 655 ton during the year 2007 (www.salmonchile.cl). The production mainly takes place in the South of the country and is dominated by the marine culture of Atlantic salmon (Salmo salar). However, the involvement of infectious salmon anaemia virus (ISAV) in a disease outbreak was officially verified in Atlantic salmon in mid-June 2007 (1), and to date 62.3% of Chilean marine sites is affected by the disease. Therefore, this intensification of fish farming has resulted in growing problems of infectious and/or non-infectious diseases, which cause significant economic losses due to mortalities of Atlantic salmon eggs, younger fish and post-smolt in seawater. Flavobacterium psychrophilum (formerly Cytophaga psychrophila and Flexibacter psychrophilus) is the causative agent of bacterial cold-water disease (BCWD) in a large number of freshwater fish species worldwide (2) and is of considerable economic significance to aquaculture producers. In Chile, F. psychrophilum was first isolated from a rainbow trout (Oncorhynchus mykiss) farm in 1993 (3); subsequently, this pathogen has been detected in other host species, such as Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch) in different regions of the South of the country. It seems clear that movement of fish, in particularly fry, between farms located in different geographical areas has played an important role in the spreading of the disease across Chile. 2. Pathogenesis of infection Actually, the intensive fish farming has resulted in growing problems as bacterial diseases, and infections by F. psychrophilum, Chryseobacterium species and other yellow-pigmented strains are considered to be one of the major problems in the freshwater stage in Chilean aquaculture where infections can result in 5 to 70% mortality rate of fingerlings. In mid-2008, the disease was observed with a high level mortality (40 to 90%) following rain events particularly in Atlantic salmon (3–5 g). The infections are also frequently found in association with infectious pancreatic necrosis virus (IPNV), saprolegnia or parasitic fish pathogens as Ichthyophthirius multifilis, but the significance of these combinations is unclear. Outbreaks occurred repeatedly during winter months at temperatures lower than 12ºC. However, most farm water temperatures, with exception in summer, are between 8 to 14ºC throughout the year, causing chronic flavobacteriosis outbreaks. To avoid confusion with other fish diseases, the Chilean fish farmers use to designate this ulcerative condition as flavobacteriosis due to the aetiological agent of the disease. In general, the main signs of flavobacteriosis are similar to the signs reported in other geographic areas. The affected fish show gross external lesions such as ulcers on skin and muscles, on the flank or in the peduncle area. In the case of smaller fish, exophthalmia and haemorrhagic on abdominal areas, as well as, frayed fins and tail rots are also observed. 3. Impact of the disease In spite of the impact of flavobacteriosis as well as other fish diseases the compilation of numerical antecedents is not simple. In fact, in Chile there is no knowledge of the total cases of flavobacteriosis. The main causes are: • The Chilean authority does not force to report the outbreak, only those diseases that are included in List 1. • Therefore, the data are taken from the cases that arrive at the laboratories and that do not represent mortality or prevalence of flavobacteriosis in the country. • The fish affected by flavobacteriosis are taken by salmon farmers after at least a drug treatment. The laboratory diagnoses are included within the Program of Passive Monitoring of salmon, which obligates the diagnostic laboratories to report the results obtained to authorities. According to the Servicio Nacional de Pesca (www.sernapesca.cl), total of samples analyzed within the Program of Passive Monitoring of salmon between 2005 and 2008 showed values among 1638 to 867 cases reported, but no data of the year 2007 is available (Fig. 1). The principal cause appeared to be due to the ISAV discovery in Chile in the same period. In general, the distribution of the diagnosis informed by the laboratories according to disease showed that flavobacteriosis is the second pathology affecting salmon cultures, after Piscirickettsia salmonis. The percentage of total flavovacteriosis cases are given in Figure 2 for the period 2005–2008. It is important to denote that not all flavobacteriosis outbreaks samples are taken to diagnosis as well as that most analysis are made in Atlantic salmon (Fig. 3). 4. Diagnostic techniques for flavobacteriosis From a diagnosis point of view, one of the most critical steps is the lack of methods to identify F. psychrophilum from others that are phenotypically similar. The majority of the Chilean laboratories employ for the F. psychrophilum diagnosis the API ZYM test. Therefore, the probability of false positives to this pathogen is high. Moreover, misidentification of F. psychrophilum strains using miniaturized system API ZYM has been reported recently by our group (4). On the other hand, the precise diagnosis of the disease requires the isolation of the microorganism(s) from fish tissue, and then the study of biochemical traits of the isolated bacteria, a task that in some cases can be very difficult to perform due to overgrowth produced by several other bacterial species present within the lesions. Economic damage for fish farmers is severe and can be augmented due to delay or misinterpretation of the disease diagnosis. To overcome this difficulty, different species-specific polymerase chain reactions (PCR) have been development in recent years as tools for the diagnosis (5, 6, 7, 8), proving to be very useful include in diagnosing sub-clinical infection in Atlantic salmon. However, until now the application of these molecular techniques are limited to research laboratories. 5. Biochemical, serological and genetic characterization of Chilean isolates Biochemical, antigenic and genetic characteristics of 20 Chilean F. psychrophilum strains derived from farmed Atlantic salmon and rainbow trout have been detailed recently (9, 10). In these studies, biochemical and physiological analyses showed that Chilean F. psychrophilum strains, regardless of the host species, constitute a phenotypically very homogeneous group matching with previous descriptions of this pathogen. However, serological assays indicated the existence of antigenic heterogeneity with four patterns of serological reactions. Using different molecular typing methods, such as pulsed-field gel electrophoresis, 16S rRNA alleles, randomly amplified polymorphic DNA and repetitive extragenic palindromic PCR a marked homogeneity was also revealed, indicating that the disease outbreaks in Chilean farms are dominated by a closely related cluster of the F. psychrophilum strains, while significant genetic differences between these isolates and the non- Chilean strains were observed. Restriction fragment length polymorphism of PCR analysis showed that gyrase genotypes B-S or B-R were found in Chilean isolates from rainbow trout and Atlantic salmon, whereas genotype A was not found. In parallel, we received eight yellow pigmented bacteria associated with diseases in the fingerling stages of Atlantic salmon cultured in Puerto Montt, while the other cases were detected in rainbow trout cultured in Osorno. The distance between both outbreaks is of approximately 109 km. These microorganisms were often isolated from mixed cultures of F. psychrophilum, being all of them initially diagnosed as member of the F. psychrophilum species. However,