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¹ US Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, 11649 Leetown Road, Kearneysville, West Virginia 25430, USA
² Corresponding author (email: starliper{at}usgs.gov)

	ABSTRACT

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
ABSTRACT:   There are a number of significant diseases among cultured and free-ranging freshwater fishes that have a bacterial etiology; these represent a variety of gram-negative and gram-positive genera. Confirmatory diagnosis of these diseases involves primary isolation of the causative bacterium on bacteriologic media. Frequently used "general" bacteriologic media simply provide the essential nutrients for growth. For most of the major pathogens, however, there are differential and/or selective media that facilitate primary recovery. Some specialized media are available as "ready-to-use" from suppliers, while others must be prepared. Differential media employ various types of indicator systems, such as pH indicators, that allow diagnosticians to observe assimilation of selected substrates. An advantage to the use of differential media for primary isolation is that they hasten bacterial characterization by yielding the appropriate positive or negative result for a particular substrate, often leading to a presumptive identification. Selective media also incorporate agent(s) that inhibit the growth of contaminants typically encountered with samples from aquatic environments. Media that incorporate differential and/or selective components are ideally based on characters that are unique to the targeted bacterium, and their use can reduce the time associated with diagnosis and facilitate early intervention in affected fish populations. In this review, the concepts of general and differential/selective bacteriologic media and their use and development for fish pathogens are discussed. The media routinely employed for primary isolation of the significant bacterial pathogens of fishes are presented.
  Key words:  Bacteria, bacteriologic media, culture, fish, freshwater, primary isolation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
Diseases are a significant threat to the health of free-ranging and hatchery-reared populations of fishes, including a suite of important sport and restoration species. Prevention of pathogen introductions and disease transmissions is the most prudent and cost-effective means for management of fishery resources (Piper et al., 1982). Because fish reside in, or are reared in open-environment waters, and pathogens are transmitted horizontally via the water column, diseases are a recurring concern. Health management is a primary objective for fishery resource agencies, particularly the U.S. Fish and Wildlife Service’s (FWS) regional Fish Health Centers and State Fishery Offices. The foundations for a program in fish health management include maintaining optimal husbandry, observing fish for development of disease signs or gross pathology, and providing periodic health/pathogen inspections and diagnostics. In the event of a disease outbreak, quick intervention with control and treatment measures is essential to preclude or minimize mortality and disease spread to other populations. With a bacterial etiologic agent, effective disease control can be dependent upon identification of the pathogen by primary bacterial culture. This approach offers a definitive diagnosis and allows for subsequent antimicrobial sensitivity testing against candidate therapeutic agents, which minimizes the development of antibiotic-resistant strains. It also provides a source of isolates from various hosts or geographic areas that can be archived and used in research.

Health and pathogen inspections at fish hatcheries conducted by FWS Fish Health Centers and State Agencies involve the evaluation of tissues from a defined number of individuals per lot (i.e., species and year-class) of fish (American Fisheries Society–Fish Health Section, 2004). During inspections of facilities that have no historic record of diseases and that have current resident fish that are apparently healthy, two or three general bacteriologic media that can be used to culture a broad spectrum of potential pathogens, are typically used. On the other hand, specialized media (selective and/or differential) that target specific pathogens are often used at those facilities where a specific bacterium or disease has previously occurred, or where a bacterium is enzootic or deemed exotic. Specialized media have also been developed for fastidious bacterial pathogens and have been used to support surveillance programs, such as the FWS Wild Fish Health Survey (www.fws.gov/wildfishsurvey/), related to the detection of reportable pathogens in free-ranging fish.

General bacteriologic growth media are used to culture all bacteria present in tissues, including pathogenic and non-pathogenic organisms. Many of the fish pathogens do not require additional steps during sample processing for isolation, such as a pre-enrichment. However, mincing or homogenization of tissues facilitates recovery of some pathogens, including Mycobacterium spp. and Renibacterium salmoninarum. Aseptic techniques are employed when tissues are collected. Media in petri plates are typically streak-plate inoculated, or dilutions of tissues are made and volumes of each dilution are drop-plate inoculated. Two types of general media are used, which can be distinguished by their nutrient richness. Rich-nutrient general media that are frequently used include brain-heart infusion agar (BHIA), tryptic soy agar (TSA), and blood agar base with 5% sheep blood; these media are available as "ready-to-prepare" from suppliers (e.g., BD Difco [Becton, Dickinson and Co.], Sparks, Maryland, USA). Growth of some fish pathogens, such as Flavobacterium psychrophilum and Flavobacterium columnare, however, can be inhibited by high-nutrient media. For this reason, reduced-nutrient media also are used; the most common is cytophaga medium (Anacker and Ordal, 1959); R2A (BD Difco) is a good medium for isolation of bacteria from aquatic environments. Cytophaga medium is composed of 0.05% tryptone, 0.05% yeast extract, 0.02% beef extract, and 0.02% sodium acetate. Other pathogens have unique requirements for general growth. Media for R. salmoninarum (Sanders and Fryer, 1980) contain l-cysteine, marine source bacteria require salt supplementation (typically 1–1.5% NaCl), and Lowenstein or Middlebrook media are necessary for primary culture of Mycobacterium spp. A major difference in primary isolation of fish bacterial pathogens compared to isolation of bacteria in human or veterinary medicine is incubation temperature. With few exceptions, fish bacteria are inhibited by incubation temperatures above 30 C. Many fish disease diagnostic laboratories use packaged microbiologic identification systems as a tool for bacterial identifications. Caution should be exercised when using such systems because they are typically used for mammalian isolates, which have optimal incubation temperatures of 37 C. Because aquatic-origin isolates might not be included in manufacturers’ databases, and assimilation of various substrates is often temperature dependant, fish diagnostic laboratories that rely on packaged systems need to develop their own databases and expected biocodes for the bacteria that they routinely encounter.

The recipes of differential media include at least one substrate that targets a particular characteristic (i.e., phenotype) that is unique to the bacterium. This character, or substrate, needs to be identified through extensive biochemical testing with a significant number of isolates of that species originating from a variety of geographic locations. Furthermore, the substrate must lend itself to inexpensive incorporation into a medium, and the bacterial action toward it must be observable with the aid of an indicator system that clearly differentiates between positive and negative reactions. It is not often that a bacterial species possesses a unique phenotype, so the goal is to select a character that a minimal number of bacteria will share. An example of this is Coomassie brilliant blue (CBB) medium (Udey, 1982; Cipriano and Bertolini, 1988), which is an effective differential medium for field and laboratory work with Aeromonas salmonicida. Coomassie brilliant blue in the medium stains the A-layer protein on the cell surface of A. salmonicida, resulting in blue colonies. Occasionally, blue colonies are encountered on CBB that are not A. salmonicida, and this presumptive identification of A. salmonicida can be easily confirmed with additional biochemical tests. This approach reduces work as compared to systems using a nondifferential medium, where numerous colonies would have to be characterized. More important, a confirmatory disease diagnosis can be made more quickly. Sensitivity (i.e., reducing the number of presumptive colonies) can be enhanced by incorporating more than one differential substrate and/or by making the medium selective as well as differential; however, such media are more complicated to prepare. Various indicator systems are used in media to identify contrasting substrate assimilation results. One of the more frequently used is an acidic/basic response that utilizes one of numerous pH indicators (Difco Manual, 1998). For example, changes in pH are used to demonstrate assimilation of carbon source, such as carbohydrates, citrate, or malonate, or the decarboxylation/deamination of amino acids (i.e., arginine, lysine, ornithine). Depending on the medium, a pH change may be visualized as a colored bacterial colony against the contrasting color of the medium or a change to the color of the medium adjacent to colonial growth. Often, pH indicator systems are used in the same recipe along with other substrates and indicator systems. For example, triple sugar iron agar (BD Difco) has lactose, sucrose, and glucose along with phenol red as the pH indicator, and a hydrogen sulfide system, which uses thiosulfate as the sulfur source and a ferrous compound to form a visible (black) precipitate with hydrogen sulfide. Differential media may be prepared to detect enzymes that degrade a variety of substrates, including proteins (e.g., gelatin, casein, tyrosine, or elastin), a complex carbohydrate (starch), or a glycosaminoglycan (chondroitin). Media that contain these substrates are usually poured in petri plates where visible zones of clearing under or surrounding the bacterial growth indicate positive reactions. It should be noted that the percentage of the substrate used can affect the result. A high concentration of substrate may overwhelm weak positive enzymatic action, and care must be taken to use an appropriate (i.e., minimal) concentration to allow visualization of a zone of clearing, but not too high an amount as to yield false negative results. Differential media also can utilize protein stains, which can interact with bacterial cells during growth, resulting in colony staining.

Numerous environmental bacteria grow on both general and differential media used for work with fish. The flora varies with each locale, and it is generally believed that many of these bacterial species have not been characterized. Typically, there are a number of colonies that grow on differential media plates that may appear phenotypically similar to the target organism. A selective medium is developed in a similar manner to a differential medium, in that the selective agent allows for unimpeded growth by the target bacterium, while inhibiting as many non-target organisms as possible. The advantage of a selective bacteriologic medium is that the growth of many of those confounding, presumptive colonies is inhibited. This effect is obvious when comparing results of paired plates with and without the selective agent(s). Selective media are particularly useful for isolation of relatively slow-growing fish pathogens such as R. salmoninarum and F. columnare. Examples of selective agents incorporated into media include antibacterial agents, antifungal agents, bile salts, crystal violet, sodium azide, phenlyethanol, selenite, and tellurite. For some of the pathogens, it is useful to take advantage of their unique characteristics and use non-specific selective techniques, such as increased salt (NaCl), pH tolerance, nutrient richness of the medium, and incubation temperature. The goal for primary isolation of a specific bacterial etiology is to employ a combination or multifaceted differential and selective approach to maximize the sensitivity of culture and promote a quick diagnosis and treatment.

	Yersinia ruckeri

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
Yersinia ruckeri is the cause of enteric redmouth disease in rainbow trout (Oncorhynchus mykiss), and BHIA and TSA are routinely used for field isolations and for laboratory studies. A differential medium (SW; Waltman and Shotts, 1984) has been developed for primary isolation of Y. ruckeri. It relies on two characters to increase selectivity, hydrolysis of Tween 80 and negative assimilation of mannitol. Colonies will be green (negative mannitol is indicated with the pH indicator bromthymol blue) with a halo of precipitate (Tween 80 hydrolysis with calcium chloride) adjacent to and surrounding each colony. Since the publication of SW medium, there have been important observations on its preparation and use. The original paper gave a percentage of 0.0003% for bromthymol blue, which was subsequently confirmed to be an error and should be 0.003% (Shotts, 1991). Even with the correct percentage, in most instances, the medium is pale yellow at the specified pH of 7.4. If sleeved plates stored in the refrigerator are greenish in color, they will quench toward yellow within a few days. This is caused by a chemical change in the Tween 80. A solution to this problem is to adjust the pH according to the color of the medium (green), which will be in the range of pH 8.0 to 9.5, and prepare and use the medium fresh. The increased pH is advantageous for primary isolation of Y. ruckeri, which grows well, while many environmental bacteria will not. However, the isolation of Tween 80 hydrolysis-negative strains from England has affected its usefulness for field isolations (Hastings and Bruno, 1985; Rodgers and Hudson, 1985; Davies and Frerichs, 1989). Rodgers (1992) developed a differential and selective medium (ROD) for Y. ruckeri. Colonies on ROD are yellow (maltose and ribose assimilation) on a red medium (pH 7.4) with yellow deposits in the medium (sodium desoxycholate) surrounding colonies. ROD also contains a hydrogen sulfide indicator system (Y. ruckeri does not produce hydrogen sulfide) and sodium dodecyl sulfate and sodium desoxycholate as antibacterials.

	Renibacterium salmoninarum

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
The cause of bacterial kidney disease in salmonid fish species, this gram-positive rod-shaped bacterium grows slowly, and media that support its growth are prepared containing l-cysteine HCl (typically 0.1%). Laboratory broth cultures take 10–14 days for log growth; a similar time is required for colonies to develop on primary plates to 2–3 mm. Because this bacterium is so slow to grow, field primary plates are very susceptible to contamination, which greatly confounds recovery and diagnosis. To eliminate this problem, selective kidney disease medium (SKDM; Austin et al., 1983) is an excellent choice for primary culture; this medium incorporates three antibacterials (cycloserine, polymyxin B, oxolinic acid) and an anti-fungal (cycloheximide). KDM2, the medium of Evelyn (1977), is used extensively for laboratory growth cultures by laboratories in geographic regions with endemic disease. Both SKDM and KDM2 include serum in their recipes: fetal bovine serum, fetal calf serum, or normal calf serum may be used. It was determined that sera from various sources suffice for bacterial growth, but some differences may be encountered (e.g., fetal bovine sera result in a clearer medium in petri plates). Daly and Stevenson (1985) reported that activated charcoal may be used to substitute for serum. They found that serum was not providing growth nutrients but was acting as a detoxifier, and charcoal would perform this same function. Evelyn et al. (1989) observed satellitism in R. salmoninarum, whereby secreted growth products enhanced its own subsequent growth, and from this, metabolite supplementation was described (Evelyn et al., 1990). Metabolite is made simply by filter sterilizing a spent R. salmoninarum broth culture, then including this (usually at 1–5%) in future preparations of either broth or solid media. For primary isolation, it can be used to supplement KDM2 or SKDM. Metabolite does not result in an increase in colony-forming units/g (cfu/g) isolated, but it yields a more luxurious growth (i.e., larger, quicker) of colonies, and this allows for quicker diagnostic confirmation of disease.

	Aeromonas spp.

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
The most significant pathogen of the genus Aeromonas to salmonid fish species is A. salmonicida subspecies salmonicida, the cause of furunculosis. Although it may be readily isolated from infected fish or grown in laboratory cultures using TSA, BHIA, or other general media, TSA supplemented with 0.01% CBB (Udey, 1982; Cipriano and Bertolini, 1988) offers an excellent presumptive identification. Suspect blue colonies are visible within 48 hr, and single colonies can be selected for easy and quick characterization with a few simple additional tests. Tryptic soy agar also aids in production of the brown water-soluble pigment, another presumptive character, in addition to the blue colony. Congo red agar (Ishiguro et al., 1985), a differential medium, is analogous to CBB in function, except that the A-layer cell protein binds congo red and colonies are red.

Motile Aeromonas spp. (e.g., A. hydrophila) are ubiquitous in untreated water supplies and are generally considered as secondary or opportunistic pathogens to cool- and cold-water fishes. These are not considered a significant primary cause of disease because diseases of salmonids are related to water temperature; salmonid fishes are reared at water temperatures between 10 C and 16 C, whereas the optimal temperatures for diseases from this group of bacteria are 22 C and above. Motile Aeromonas spp. are significant causes of diseases in warm-water fish species. These bacteria may be readily isolated from fish and are easily cultured in the laboratory on any of the routine general media, including TSA and BHIA. They grow to 5-mm-diameter, and larger, colony sizes within 24 hr. Selective and differential media are available to aid with primary isolation of Aeromonas spp. from fish, including Rimler-Shotts (RS; Shotts and Rimler, 1973) and SGAP-10C (starch-glutamate-ampicillin-penicillin, 10 µg/L C from glucose; Huguet and Ribas, 1991). SGAP-10C may also be used for isolation and quantification of the organisms from water (Jenkins and Taylor, 1995).

	Edwardsiella spp.

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
Edwardsiella ictaluri, the cause of enteric septicemia of catfish (ESC; Hawke et al., 1981), is one of the leading causes of mortality to channel catfish aquaculture. This enteric bacterium can be isolated on general media including TSA blood agar or BHIA, or the Edwardsiella isolation medium of Shotts and Waltman (1990). Colistin is the selective agent in EIM, and, if necessary, the authors state that 0.5 µg/mL Amphotericin B (Fungizone®) could be included in the recipe for selection against fungal contamination.

Edwardsiella tarda is an enteric bacterium that has been reported as a cause of disease to channel catfish (Meyer and Bullock, 1973). Although it is not unusual to isolate E. tarda, it is not thought to be as significant as E. ictaluri. This bacterium is common in reptiles, grows on routinely employed general media, and may also be isolated from fish on EIM. Edwardsiella tarda colonies grow quicker than E. ictaluri colonies and are easily differentiated by subsequent biochemical testing, such as the ability of E. tarda to produce hydrogen sulfide. Specific primary isolation of E. tarda is best accomplished using an enrichment step in either double-strength Salmonella-Shigella broth or selenite-cystine broth followed by direct plating onto Salmonella-Shigella agar (BD Difco; Wyatt et al., 1979; Muratori et al., 2000).

	Flavobacterium spp.

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
Bacteria of this genus can be difficult to isolate from fish because they are often associated with skin lesions that can harbor heavy loads of other nonpathogenic bacteria. Two to three days of incubation are necessary for most Flavobacterium spp., and in general, a longer incubation time increases the chance for overgrowth by contaminants. Fish pathogenic Flavo-bacteria are long (up to about 5 µM), thin, gram-negative rods that are cultured on reduced-nutrient media, and most researchers and diagnosticians prepare and use media according to published recipes. A general medium that is often used is cytophaga medium (Anacker and Ordal, 1959). Flavobacterium columnare (formerly Flexibacter columnaris and Cytophaga columnaris), which causes columnaris disease in many cool- and warm-water free-ranging and cultured fish species, is the most frequently isolated pathogen. Hawke and Thune (1992) described a selective primary isolation medium (SCA; selective cytophaga agar) for F. columnare; it is prepared by supplementing cytophaga medium with 5 µg neomycin/ml and 200 IU polymyxin B/ml of medium. A differential and selective medium for F. columnare was described by Bullock et al. (1986) that incorporated 4 µg neomycin sulfate/ml media as a selective antibacterial and gelatin for a differential character. Flavobacterium columnare is gelatinase positive, and on SCA, a clear zone surrounding the colonies is visible after incubation. Shieh medium supplemented with 1 µg tobramycin/ml is another selective medium that has been reported for primary isolation from diseased fish (Decostere et al., 1997).

Flavobacterium psychrophilum (formerly Flexibacter psychrophilus and Cytophaga psychrophila), is the cause of bacterial cold-water disease and fry syndrome to cultured and wild rainbow trout and salmon species. Like F. columnare, the most commonly used medium for primary isolation and laboratory growth cultures is cytophaga medium (Anacker and Ordal, 1959). Other media have been described in the literature but have yet to be frequently used by fish diagnosticians. For the most part, all of the described media for F. psychrophilum are general growth media and are not differential or selective. Optimal growth temperature for this bacterium is 15–16 C, which is cool enough to retard some but not all contaminant growth during primary isolation, especially from lesions. Kidney tissue can afford a more aseptic sample for primary isolation from clinically diseased fish. Several of the other reported media (Anderson and Conroy, 1969; Holt, 1987; Rangdale et al., 1997) are variations to the recipe of cytophaga medium, while others contrast with the generally accepted requirement for low nutrient concentration. For example, serum (up to 5%) has been incorporated (Lorenzen, 1993; Brown et al., 1997). Daskalov et al. (1999) supplemented cytophaga medium with galactose, glucose, rhamnose, and skimmed milk. A significant improvement in growth of F. psychrophilum on cytophaga medium was reported by Rangdale et al. (1997) by increasing tryptone tenfold (to 0.5%) and increasing beef extract from 0.02% to 0.05%.

	Less common fish bacterial pathogens

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
Many diseases of fishes with a bacterial etiology are currently limited to certain hosts and geographic origins but may result in high mortality given optimal circumstances. Like other animals, stressed or immunocompromised fish often are more susceptible to opportunistic pathogens. Low dissolved oxygen and temperatures, for example, in addition to representing common stressors, may serve to enhance pathogen growth. Such environmental stressors along with opportunistic pathogens can result in mortality events with a combined and related noninfectious and infectious etiology. These types of problems are more common to cultured fishes and are sometimes a consequence of rearing conditions.

Carnobacterium maltaromicus (formerly C. piscicola and Lactobacillus piscicola; Mora et al., 2003), a gram-positive rod, is the causal agent of a disease that has been primarily associated with hatchery-reared rainbow trout brood stock at spawning or postspawning (Herman et al., 1985; Starliper et al., 1992). During spawning season, fish can be stressed by gamete development and through handling as part of the husbandry of spawning. Primary isolation of C. maltaromicus can be accomplished using general bacteriologic media, such as TSA, BHIA, or blood agar. Two selective media for gram-positives are Rogosa SL (for Lactobacillus spp.) and azide blood agar (Difco Manual, 1998). Although neither of these has been extensively field tested for primary isolation from fish, they could be useful for C. maltaromicus. Starliper and Morrison (2000) used Rogosa SL agar and azide blood agar (Difco Manual, 1998) during a general surveillance for fish pathogens, including Lactobacillus and related gram-positive genera from freshwater mussels; however, C. maltaromicus was not isolated.

Streptococcus iniae is a gram-positive cocci that causes diseases in both freshwater and marine water fishes. Disease in tilapia (Oreochromomis niloticus) often is associated with fish reared under intensive culture, recycle systems. Water quality has been noted as a predisposing factor, and improving water quality has been demonstrated to be pivotal in alleviating disease and mortality. General media, BHIA, or blood agar, may be used for primary isolation; however, increased sensitivity is reported with media that have a selective capability for S. iniae. Thallium acetate-oxolinic acid (TAOA) agar medium and TAOA with 3% horse blood, which allows for the detection of hemolysis, have been reported to greatly improve primary isolations of S. iniae from Japanese flounder (Paralichthys olivaceus), and from water and sediment from culture systems having infected fish (Nguyen and Kanai, 1999; Nguyen et al., 2002). In the United States, Columbia CNA agar (BD Difco) is routinely employed in diagnostic cases where S. iniae is the suspected cause (J. Hawke, Louisiana State University, pers. comm.). This medium is prepared with 5% blood to detect hemolysin production, and it contains colistin sulfate and nalidixic acid as selective antibacterial agents.

There are many non-glucose-fermenting bacteria that are ubiquitous in freshwater aquatic environments and are frequently isolated from healthy and diseased fishes. Among the common genera isolated are Pseudomonas, Acintobacter, Moraxella, Shewanella, Alcaligenes, and Branhamella. These bacteria grow on any of the previously described high- and reduced-nutrient general bacteriologic media. On streak plating from external sites such as mucus and gills or from internal sites such as kidney tissues, it is common to culture up to 10–20 total colonies per plate from apparently healthy fish. Members of these genera are only occasionally identified as causes of diseases and are generally considered opportunistic; disease problems associated with these agents probably relate to underlying stress-induced problems in culture conditions and one or more of these bacteria might be present in relatively higher numbers as secondary infections. Pseudomonas fluorescens and Shewanella putrifaciens are two examples of species that have been identified as occasional causes of disease and mortality. Both are readily culturable from fish external lesions or kidneys using general media. Selective media have been described for Pseudomonas spp., including Pseudomonas isolation agar (BD Difco) and the media of Gould et al. (1985) for primary isolation of fluorescent Pseudomonads (e.g., P. fluorescens).

Mycobacterium spp. cause chronic diseases in both freshwater and marine fishes, and infected fish typically demonstrate classical signs such as lethargy, wasting, and poor growth; external lesions may be present as well as granulomas in internal tissues. Primary isolation is commonly made using Lowenstein and Middlebrook media (BD Difco). Rhodes et al. (2004) developed a tissue preparation and media inoculation procedure that enhances the recovery of Mycobacterium spp. from fish (i.e., striped bass M. saxatilis) using Middlebrook 7H9 medium supplemented with antimicrobials (Teska et al., 1997) to reduce overgrowth by contaminants and thereby increase recovery of the pathogen.

Photobacterium damselae subspecies piscicida (formerly Pasturella piscicida), which causes fish pasturellosis, has been isolated from freshwater and saltwater fishes. Primary isolation of the bacterium is accomplished on TSA, TSA with 5% sheep blood, BHIA, or blood agar base containing 5% sheep blood. This bacterium produces a systemic infection and can be readily isolated from kidney tissues. Primary isolation from fish from marine environments may be enhanced by supplementation of any of the aforementioned media with 1% NaCl.

Intracellular bacterial pathogens cause significant diseases and mortality to a variety of fish species. Piscirickettsia salmonis, cause of piscirickettsiosis (salmonid rickettsial septicemia), and Francisella sp. (francisellosis) are two gram-negative pathogens that can be difficult to recover from diseased specimens. For both of these diseases, various authors report that tissue processing or homogenization prior to inoculation of media or cell lines greatly enhances culture success.

At present, there are no reports of bacteriologic media that allow for successful primary isolation or laboratory cultures of P. salmonis. However, this bacterium has been isolated using cell-culture techniques, usually with Chinook salmon embryo (CHSE-214) cells (Fryer et al., 1990, 1992; Gaggero et al., 1995; Palmer et al., 1996; Skarmeta et al., 2000; Fryer and Hedrick, 2003; Birkbeck et al., 2004a), epithelioma papulosum ciprini cells (Fryer and Hedrick, 2003), or brown bullhead catfish (Ictalurus nebulosus) cells, (Almendras et al., 1997). In the brown bullhead catfish cells, cytopathic effect (CPE) associated with the type strain of P. salmonis (ATCC VR 1361; Fryer et al., 1992) was not observed until 45 days following inoculation. Birkbeck et al. (2004b) showed approximately 100-times greater cell yield of P. salmonis grown in Sf21 insect cells (from the fall armyworm [Spodoptera frugiperda]), compared to CHSE-214 cells. Fryer et al. (1990) isolated P. salmonis using the CHSE-214 cell line; although the isolate could be subcultured in five of seven cell lines on passage, it could not be subsequently grown on media. These reports indicate an optimum incubation temperature ranging from 15 C to 18 C, and antimicrobials should not be used in cell-culture media or any tissue transport or processing buffers or media.

Nyland et al. (2006) isolated Francisella sp. from Atlantic cod (Gadus morhua L.) on blood agar plates supplemented with 0.1% cysteine and 1% glucose, and the primary culture demonstrated excellent growth in salmon head kidney cells with medium B1817. This media consists of marine broth, fetal bovine serum, yeast-olate, l-cysteine HCl, and glucose (Ottem et al., 2007). Olsen et al. (2006) were not successful at primary isolation of Francisella sp. from Atlantic cod kidney, heart, and spleen on blood agar (Oxoid; Basing-stoke, UK) with 5% bovine blood or blood agar with 1.5% NaCl at 22 C and 15 C, respectively. However, they recovered isolates through cell cultures, and at an incubation temperature of 22 C, heavy growth was demonstrated on cystine heart agar (BD Difco). Growth was limited to the initial streak lines; single colonies did not develop. On cystine heart agar with 5% sheep blood, small colonies were noted at 3 days and enlarged to 2–3 mm in diameter with an extended incubation time. Isolates did not grow on buffered charcoal yeast or Bacto chocolate agar enriched (BD Difco) media. Kamaishi et al. (2005) isolated Francisella sp. from three-line grunt (Parapristipoma trilineatum) on cystine-heart agar (BD Difco) supplemented with 1% hemoglobin after two weeks at 25 C. Eugon agar (BD BBL, Franklin Lakes, New Jersey, USA) with 1% hemoglobin and Eugon broth (BD BBL) with 2 mM FeCl₃ also supported bacterial culture growth; the latter was also good for large-scale cultivation (Kamaishi et al., 2005). Hsieh et al. (2006) isolated Francisella sp. from tilapia (Oreochromis spp.) on Thayer-Martin medium in a 5% CO₂ incubator at 23 C; colonies took 3–6 days to develop. They also recovered the bacterium in CHSE-214 cells, noting CPE at 7 days. However, they did not recover the bacterium from fish using blood agar, MacConkey, BHIA with 5% sheep red blood cells, blood cystine-glucose agar, or in 6% NaCl broth. Francisella sp. causing disease in hybrid striped bass (Morone chrysops x M. saxatilis) was identified through phylogenetic analyses of bacterial DNA from kidney tissues (Ostland et al., 2006). Ostland et al. (2006) were unsuccessful in their attempts at primary isolation using the following bacteriologic media: TSA with 5% sheep blood, BHIA, BHIA with 5% sheep blood, BHIA with 1% NaCl, Mac-Conkey, Mueller-Hinton with 5% sheep blood, Middlebrook 7H10, Lowenstein-Jensen (LJ), LJ–Gruft modification, and chocolate and SP4 agars. In addition, they did not culture the causative bacterium in CHSE-214 incubated at 15 C.

Suggested media for the fish bacterial pathogens discussed in this paper and guidelines for incubation temperatures are summarized in Table 1.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION Yersinia ruckeri Renibacterium salmoninarum Aeromonas spp. Edwardsiella spp. Flavobacterium spp. Less common fish bacterial... LITERATURE CITED

 
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AMERICAN FISHERIES SOCIETY (AFS)–FISH HEALTH SECTION (FHS). 2004. Fish Health Section, blue book: Suggested procedures for the detection and identification of certain finfish and shellfish pathogens. 2004 Edition. AFS-FHS, Bethesda, Maryland, USA, CD-ROM.

ANACKER, R. L., AND E. J. ORDAL. 1959. Studies on the Myxobacterium Chondrococcus columnaris. I. Serological typing. Journal of Bacteriology 78: 25–32.[Free Full Text]

ANDERSON, J. I. W., AND D. A. CONROY. 1969. The pathogenic myxobacteria with special reference to fish diseases. Journal of Applied Bacteriology 32: 30–39.[Medline]

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Received for publication 27 September 2006.

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