Significance of Flavobacterium diseases on salmonid farming in Chile Ruben Avendao-Herrera1, Pedro Ilardi2, Jorge Fernndez1

Universidad Andrs Bello, Facultad de Ciencias Bsicas, Departamento de Ciencias Bsica. Santiago, Chile. 2 Laboratorio de Patologa de Peces, Veterqumica, Santiago Chile
Correspondence: R. Avendao-Herrera (reavendano@yahoo.com)

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 (35 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 12C. However, most farm water temperatures, with exception in summer, are between 8 to 14C 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 20052008. 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 nonChilean 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, with only taking a look to the cell morphology it would have been simple to know that the samples were not F. psychrophilum because they were all rod (4). In addition, growth at 28C and with 3% NaCl as well as the activity observed on aesculin and growth on Columbia blood agar (a-haemolytic) and trypticase soy agar are in disagreement with the biochemical description made by Bernardet & Grimont (11) and Bernardet et al. (12) for F. psychrophilum. Recently, the eight isolates have been formally recognized as a novel species, for which the name Chryseobacterium piscicola sp. nov. was proposed (13). With respect to these isolates they were biochemical, serological and genetically homogeneous. 6. Virulence of the Chilean isolates Experimental infection studies have been carried out with F. psychrophilum, C. piscicola and bacterial mixtures of the both microorganisms using healthy Atlantic salmon (4, 9). The pathogenicity assays showed that the F. psychrophilum isolates tested, regardless of their host origin (salmon or trout), were able to cause mortality or to induce disease when fish were intraperitoneally injected. In fact, the Atlantic salmon isolate was less virulent than the rainbow trout isolate, with cumulative mortalities of 30% and 50%, respectively. In the case of the results of the virulence testing with C. piscicola alone gave no mortalities and did not induce the disease when fish were infected with the bacterium by bath. However, mortalities reached 100% in fish, 23 days after the exposure to simultaneously the mixture of both bacterial species. We speculate that the degree of pathogenicity or expression of the pathogenic potential of disease-producing microorganisms is increased when F. psychrophilum and Chryseobacterium isolates are mixed, since the isolates alone did not kill fish even when high concentrations were applied. Based on the course of the experimental challenge studies; C. piscicola can be considered as an opportunistic pathogen for Atlantic salmon.

7. Antimicrobial treatment In Chile, the list of fully authorized licensed antimicrobial for aquaculture is quite large. At present, 6 antibiotics are officially registered for salmonids disease treatments, including flumequine, amoxicillin, oxytetracycline, erythromycin, florfenicol and oxolinic acid (www.sag.cl). Although, the mortality can be controlled using some of these drugs, the flavobacterium disease has economic significance due to lowered quality of fish as well as high costs of antimicrobial agents, mainly oxytetracycline and/or florfenicol which also contributes greatly to a rapid appearance of multiresistance and further diminish the effect of chemotherapy (14, 15). In Chilean aquaculture, the total antibiotic used for salmonids production has been estimated to be between 385,635 and 325,616 kg in the year 2007 and 2008, respectively (16). Official data indicates that trout and salmon production uses approximately 8% of drugs per annum to treat flavobacteriosis (17) (Fig. 4). In practice, the treatment commonly used against external disease is bath with oxytetracycline (100125 ppm x 1 h at closed flow for 3 days), while to internal flavobacteriosis the most effective compounds is oral treatment with florfenicol at 20 mg/kg x 15 days. As such, all antibiotics require veterinary prescription. However, the last year, potentiated sulphonamide, a drug not registered for the use in fish, and that was not routinely used for the treatment of flavobacteriosis in Chilean farms, has increased to 21.87 kg (0.01% of total antimicrobial used in 2008) (16). Apparently studies do not exist that guarantee the administration of this antimicrobial agent. In fact, Valdebenito and Avendao (9) reported that all F. psychrophylum strains were resistant to trimethoprim-sulfamethoxazole and highly susceptible to amoxicillin and florfenicol with mean inhibition zones of c. 63 and 43 mm, respectively. Interestingly, Soltani et al. (18) indicated that potentiated sulphonamide is unsuitable to control F. psychrophilum because of intrinsic resistance. A similar situation has been observed with trimethoprim, one of the components of the potentiated sulphonamide combinations (11, 18, 19). Consequently, good diagnosis that includes an antibiotic sensitivity test plays an important role in the use of the correct early treatment. In the case of C. piscicola, additional studies are necessary to evaluate the risk for salmonid cultures, particularly because our isolates exhibit multiresistance to antibiotics that may impair the treatments (4, 20). 8. Prevention methods and vaccination The low efficacy of the chemotherapy in controlling these ulcerative diseases in salmonids as well as the no registered commercial vaccines against F. psychrophilum and other Flavobacterium-like has led to an effort in developing preventive measures, including the use of surface acting disinfectants (i.e. formalin, sea salts or sodium chloride, hydrogen peroxide, etc) administered by immersion and the use of auto-vaccines which together with maintenance of fish at low densities, cleaning of the tanks, adequate feed composition, movement restrictions, etc., helped to decrease the impact of flavobacteriosis in the cultured fish. The recent application of recirculation system have been suggested as a possibility for keeping the amount of F. psychrophilum low; but further studies are needed to confirm this hypothesis. Although no commercial vaccine against flavobacteriosis caused by F. psychrophilum are available, in Chile, during years a Flavobacterium columnare vaccine or bivalent fish vaccine (against F. columnare infection and Yersinia ruckeri) were used to prevent the disease. However, no antigenic correlation exists between both freshwater pathogens. Therefore, the farmers will have to analyse the true benefit to cost ratio to determine if these vaccines, particularly the monovalent, is feasible. From 1999, authorization to produce and sell autogenous immersion bacterins made from single farm isolates against infections with F. psychrophilum was issued to prevent this disease (21). A good example of the successful results of the vaccines used in Chile is the amount of the liters sold for the years 2000 to 2002 increasing from 76 to 281 liters, respectively (21). More recent antecedents about the autogenous vaccine commercialization are scarce. However, the regulatory requirements for the development and formulation of autogenous vaccines are strictly regulated;

causing time consuming (more than 6 months) to obtain the authorization and is the reason why the farmers have less conviction on replacing the use of antibiotics by these immunological products. Needs for the future 1. In the near future it will be necessary to strengthen research for the creation of vaccine to combat flavobacteriosis. On the basis of the facts presented, it is important to consider the inclusion of all serogroups of F. psychrophilum occurring in Chile in flavobacteriosis vaccine as recommended (22). 2. Another need is to reduce the use of drugs employed in the treatment against F. psychrophylum and/or other pigmented yellow bacteria, in order to avoid the damage to the environment. 3. Finally, remember farmers that the vaccine do not let solve their husbandry problems nor eradicates the disease completely, reason why the performance of the vaccine is very dependent on the health status of the fish at the time of vaccination. References 1- Godoy M.G., Aedo A., Kibenge M.J., Groman D.B., Yason C.V., Grothusen H., Lisperguer A., Calbucura M., Avendao F., Imiln M., Jarpa M. & Kibenge F.S., 2008. First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile. BMC Veterinary Research, 4:428 2- Nematollahi A., Decostere A., Pasmans F. & Haesebrouck F., 2003. Flavobacterium psychrophilum infections in salmonid fish. Journal of Fish Diseases, 26:563574. 3- Bustos P.A., Calbuyahue J., Montaa J., Opazo B., Entrala P. & Solervisenc R., 1995. First isolation of Flexibacter psychrophilus, as causative agent of rainbow trout fry syndrome (RTFS), producing rainbow trout mortality in Chile. Bulletin of the European Association of Fish Pathologists, 15:162164. 4- Ilardi P. & Avendao-Herrera R., 2008. Isolation of Flavobacterium-like bacteria from diseased salmonids cultured in Chile. Bulletin of the European Association of Fish Pathologists, 28:176185. 5- Bader J.A. & Shotts E.B., 1998. Identification of Flavobacterium and Flexibacter species by species-specific polymerase chain reaction primers to the 16S ribosomal RNA gene. Journal of Aquatic Animal Health, 10:311319. 6- Urdaci M.C., Chakroun C., Faure D. & Bernardet J.-F., 1998. Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum. Research in Microbiology, 149:519530. 7- Izumi S. & Wakabayashi H., 2000. Sequencing of GyrB and their application in the identification of Flavobacterium psychrophilum by PCR. Fish Pathology, 35:9394. 8- Wiklund T., Madsen L., Bruun M.S. & Dalsgaard I., 2000. Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification. Journal of Applied Microbiology, 88:299397. 9- Valdebenito S. & Avendao-Herrera R., 2009. Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in Chile. Journal of Fish Diseases 32:321333. 10- Avendao-Herrera R., Araya P. & Fernndez J., 2009. Molecular analysis of Flavobacterium psychrophilum isolates from salmonid farms in Chile. Bulletin of the European Association of Fish Pathologists, (in press). 11- Bernardet J.-F. & Grimont P.A.D., 1989. Deoxyribonucleic acid relatedness and phenotypic characterization of Flexibacter columnaris sp. nov., rev., Flexibacter psychrophilus sp. nov., and Flexibacter maritimus Wakabayashi, Hikida and Masumura 1986. International Journal of Systematic Bacteriology, 39:346354. 12- Bernardet J.-F., Nakagawa Y. & Holmes B., 2002. Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. International Journal of Systematic and Evolutionary Microbiology, 52:10491070.

13- Ilardi P., Fernndez J. & Avendao-Herrera R., 2009.Chryseobacterium piscicola sp. nov., isolated from diseased salmonid fish. International Journal of Systematic and Evolutionary Microbiology, (in press). 14- Cabello F., 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environmental Microbiology, 8:11371144. 15- Miranda C.D. & Rojas R., 2007. Ocurrence of florfenicol resistance in bacteria associated with two Chilean salmon farms with different history of antibacterial usage. Aquaculture, 266:3946. 16- Ministerio de Economa Fomento y Reconstruccin Ord. N 3411. 17- Servicio Nacional de Pesca ORD. N 520112109. 18- Soltani M., Shanker S. & Munday B.L., 1995. Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected antimicrobials. Journal of Fish Diseases, 18:555565. 19- Lorenzen E., Dalsgaard I. & Bernardet J.-F., 1997. Characterization of isolates of Flavobacterium psychrophilum associated with coldwater disease or rainbow trout fry syndrome I: phenotypic and genomic studies. Diseases of Aquatic Organisms, 31:197208. 20- Ilardi P., Abad J., Rintamki P., Bernardet J.-F. & Avendao-Herrera R., 2009. Phenotypic, serological and molecular evidence of Chryseobacterium piscicola in farmed Atlantic salmon, Salmo salar L., in Finland. Journal of Fish Diseases, (in press). 21- Bravo S. & Midtlyng P., 2007. The use of fish vaccines in the Chilean salmon industry 19992003. Aquaculture, 270:3642. 22- Toranzo A.E., Romalde J.L., Magarios B. & Barja J.L., 2009. Present and future of aquaculture vaccines against fish bacterial diseases. In: The use of veterinary drugs and vaccines in Mediterranean aquaculture (Ed: Rogers C. & Basurco B.), 155176 pp.

2000

1638

1588

Number of diagnostics

1600 1200 800 400 0 2005 2006 Year 2008 867

Figure 1 Total number of samples analyzed within the Program of Passive Monitoring of salmon.

40 35 25

Diagnostics (%)

30 25 20 15 10 5 0

23

9,9

2005

2006 Year

2008

SRS BKD Atypical furunculosis

IPNv Vibriosis Others

Flavobacteriosis Streptococcal infections

Figure 2 Distribution of the diagnosis informed by the laboratories according to disease.

300 250

264

N diagnostics

200 127 150 84 100 50 0 2005 2006 Year Oncorhynchus tshawytscha Oncorhynchus mykiss Oncorhynchus kisutch Salmo salar 2008 2009 13 34 22 5 116 59 1 14 9

Figure 3 Diagnosis of flavobacteriosis informed by the laboratories according to salmon species cultured in Chile.

Figure 4 Relative distributions of antimicrobials, according to diagnosis, in species salmonids during year 2007(A) and 2008 (B).