NB: THIS DISEASE IS NO LONGER LISTED IN CHAPTER 1.2.

3 OF THE AQUATIC CODE 1 2 3 4

CHAPTER 2.1.11. ENTERIC SEPTICAEMIA OF CATFISH (EDWARDSIELLA ICTALURI)

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1.

Scope

Enteric septicaemia of catfish (ESC) is considered to be an infection by the Gram-negative bacterium Edwardsiella ictaluri. There are three forms of this disease: acute haemorrhagic bacteraemia, chronic necrotising encephalitis and an unapparent carrier state. ESC has traditionally been a disease of North American freshwater catfish and is one of the most important infectious disease problems in the commercial catfish industry in the United States of America (USA). Recently, epizootics have been reported in native and North American species of freshwater catfish in commercial production systems in South-East Asia.

2.

Disease information
2.1. Agent factors
2.1.1. Aetiological agent, agent strains

Edwardsiella ictaluri is a Gram-negative motile rod that is oxidase negative, facultatively anaerobic, grows poorly at 37C and produces no hydrogen sulphide or indole. The agent is remarkably homogenous in biochemical profiles but plasmid and antigenic characteristics suggest that the non-channel catfish isolates differ somewhat from channel catfish isolates. The genome of this pathogen has been sequenced (http://micro-gen.ouhsc.edu/e_ictal/e_ictal_home.htm). 2.1.2. Survival outside the host

Although the agent appears to be primarily a pathogen of fish, it can survive in pond sediment and water for up to 4 months (26). 2.1.3. Stability of the agent (effective inactivation methods)

This pathogen is sensitive to heat, desiccation, UV exposure, detergents and most disinfectants. 2.1.4. Life cycle

The life cycle of Edwardsiella ictaluri is direct. Fish from a population that has recovered from the disease are considered carriers. Edwardsiella ictaluri has been detected in the kidney of such fish well over 4 months after exposure (2, 19), suggesting that carrier fish act as the natural reservoir for the organism. Researchers have found the bacterium in the gut of fish-eating birds using fluorescent antibody tests on ingesta but no E. ictaluri could be cultured, indicating that the bacteria were not viable (32, 37). This suggests that birds are not an important means of dissemination of this pathogen. However, there are many types of scavengers and opportunistic predators at the site of an ESC outbreak and physical spread of the bacterium to neighbouring ponds in infected tissues or on the surface of animals is likely.

2.2. Host factors

2.2.1. Susceptible host species

Edwardsiella ictaluri is one of the most important pathogens of channel catfish (Ictalurus punctatus), the primary species for catfish

aquaculture in USA. It can be an infectious bacterial pathogen of several other species of North American catfish, bullheads and madtoms (members of the family Ictaluridae: I. furcatus, Ameiurus nebulosis, A. catus, Noturus gyrinus) (13). This pathogen has also been associated with disease outbreaks in a variety of other members of the superorder Ostariophysi, including Asian freshwater catfish (Pangasiidae: Pangasius hypothammus [7], and Clariidae: Clarias batrachus), knifefish (Sternopygidae: Eigemannia virescens [16]) and cyprinids (Cyprinidae: Begal Danio Davario davario [35] and Rosy Barbs Puntius conchonius [14]). Experimental infections suggest that Pacific salmon are susceptible (4) and that zebra danios (Danio rerio) (25) and European catfish (Silurus glanis) are moderately susceptible (27). 2.2.2. Susceptible stages of the host

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2

Juveniles and adults are susceptible to the pathogen. The highest losses are in young fish (less than 1 year of age) during their first exposure to the pathogen in the optimal temperature range of 1828C. 2.2.3. Species or sub-population predilection (probability of detection)

In mixed populations, members of the Ictaluridae are most susceptible and among this group channel catfish are more susceptible than blue catfish. 2.2.4. Target organs and infected tissue

During acute ESC, the bacterium can be isolated from most tissues, but the target organ is the posterior kidney. In the chronic form, the target tissue is the brain, or occasional abscesses at the base of the pectoral or dorsal fin spine; often other tissues will give negative results by culture for E. ictaluri. In the unapparent carrier state, posterior kidney and gastrointestinal tract may give positive results by culture for the pathogen, but the use of enrichment and selective media may be needed. 2.2.5. Persistent infection with lifelong carriers

Edwardsiella ictaluri establishes a persistent lifelong infection in exposed fish.

2.2.6. Vectors

There are no known biotic vectors for E. ictaluri. 2.2.7. Known or suspected wild aquatic animal carriers

Wild and feral catfish may be reservoirs of E. ictaluri. Channel catfish in rivers and lakes throughout California were shown to have antibodies to E. ictaluri (6). An outbreak of ESC was confirmed in wild tadpole madtoms (Noturus gyrinus) (21), and an outbreak of ESC occurred in wild caught brown bullheads (Ameiurus nebulosis) when brought into a laboratory (15)

2.3. Disease pattern

2.3.1. Transmission mechanisms

During overt ESC, the bacterium can be transmitted from dead and dying fish to susceptible fish by cannibalism (18). It is believed that shedding with faeces is the main means of dissemination into the pond environment. Infection has been shown to occur by intestinal mucosa (oral uptake), and the olfactory mucosa (nasally). Recent evidence suggests that epidermal and branchial mucosa may be additionally important routes. During the natural course of ESC, the incubation period from first exposure to first deaths is approximately 8 days (18, 39). 2.3.2. Prevalence

ESC is a commonly reported disease problem in commercial catfish operations in the USA (24, 33). Most populations of cultured catfish in the endemic regions of the USA are carriers. The cryptic nature of the pathogen during the unapparent carrier state makes it difficult to determine the actual carrier rate within an endemic population. 2.3.3. Geographical distribution

Edwardsiella ictaluri is widely distributed in the USA and South-East Asia.

2.3.4. Mortality and morbidity

Acute outbreaks of ESC can cause losses of over 50% in a population. 2.3.5. Environmental factors

The highest losses occur in heavily stocked ponds that have experienced an environmental stressor within the 1828C temperature range.

2.4. Control and prevention

2.4.1. Vaccination

An attenuated mutant of E. ictaluri (RE-33) induced by passage in the presence of rifampicin (22) has been licensed for use as a vaccine (Aquavac-ESC, Intervet) and has been shown to provide protection when given at 7 days of age and in ovo (30). The use of vaccination has been shown to be helpful for fingerling production to reduce, but not eliminate, ESC.

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2.4.2.

Chemotherapy

Antibiotic-medicated feeds are generally effective against ESC. However, the disease must be identified during early stages of infection before the population experiences a suppressed appetite. Antibiotics that have proven effective include oxytetracycline HCL (Terramycin, Pfizer) 16 mg active ingredient per kg of fish for 10 days, a combination of sulfadimethoxine and ormetoprim (Romet 30, HofmannLaRoche) at 50 mg/kg fish for 5 days and florfenicol (Aquaflor, Schering Plough) at 10 mg/kg fish for 10 days. The latter two are specifically labelled for use on ESC in the USA. Withdrawal times before medicated channel catfish can go to market in the USA are 21 days for Terramycin, 3 days for Romet 30 and 12 days for Aquaflor. The use of antibiotics must be discriminate, and the full dose and application time period should be used once started. These measures are important to avoid development of antibiotic resistance, as E. ictaluri can acquire plasmids that provide multiple drug resistance. 2.4.3. Immunostimulation

Research on the use of immunostimulants in the feed is promising, but there is no widespread commercial application for any of these additives in the control of ESC. 2.4.4. Resistance breeding

Biological resistance to ESC has been demonstrated among genetic lines, but the use of genetics for controlling ESC has not gained widespread commercial application (38). 2.4.5. Restocking with resistant species

Biological resistance to ESC has been demonstrated in a similar species (blue catfish, I. furcatus) and channel blue hybrids are more resistant than channel catfish (41). There is some commercial production of the hybrid but difficulty in producing fry has hampered widespread adoption of the hybrid for commercial catfish aquaculture. 2.4.6. Blocking agents

None identified. 2.4.7. Disinfection of eggs and larvae

The establishment of ESC-free fish from an endemic population can be accomplished by disinfecting eggs using 100 ppm (parts per million) iodine (betadine) and producing fry using pathogen-free water. 2.4.8. General husbandry practices

Management to reduce ESC-associated losses is the best option when the pathogen becomes established on a facility or is endemic in a region. This is done by maintaining quality nutrition and feeding levels throughout the year and avoiding stressful events during the ESC temperature range. One practice that should especially be avoided is the stocking of potentially nave fish into an endemic population during or shortly before the temperatures are in the 1828C range. Also, the use of chemical treatments should be avoided in this temperature range. One management procedure that has proven effective in reducing losses is the modification of the feeding schedule to feeding on alternate days when ESC is occurring in the area (40). This apparently reduces faecaloral transmission of the pathogen.

Sampling
3.1. Selection of individual specimens
Samples for inspections/surveillance must be representative of all groups of a population derived from multiple independent stockings. Sampling from ponds should include any moribund fish and fish that school behind aerators when oxygen levels are not stressful. Sampling should not include the use of feed to attract fish or the use of baited hooks. Diseased fish with the chronic form of ESC often display sporadic or uncoordinated swimming and may occasionally be seen splashing at the surface.

3.2. Preservation of samples for submission

Samples for bacterial culture may be iced or refrigerated for up to 48 hours.

3.3. Pooling of samples

Pooling of samples is not recommended.
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3.4. Best organs or tissues

Posterior kidney and brain (also, any small abscesses on otherwise clinically normal fish).

3.5. Samples/tissues that are not suitable

Frozen samples are unreliable for culture. Often tissues other than brain or occasional abscesses at the base of the pectoral or dorsal fin spine will give negative results by culture for E. ictaluri.

Diagnostic methods
4.1. Field diagnostic methods
4.1.1. Clinical signs

ESC occurs predominantly at temperatures between 18C and 28C and is most severe in crowded production systems that contain many nave fish. In the USA, this results primarily in autumn- or spring-associated losses. The disease is substantially exacerbated by stressful events such as nitrite toxicity or low dissolved oxygen. Mixed infection with Flavobacterium columnare (columnaris disease) and/or channel catfish virus (channel catfish virus disease) are common. An affected population may show rapidly progressing mortality with affected fish being lethargic and resting at the bank. Affected fish will also demonstrate distressed uncoordinated swimming. As the outbreak worsens in the pond, the population will substantially reduce feeding activity. During the acute outbreaks, diseased fish will often demonstrate protruding eyes, abdominal distension and petechial haemorrhages on the face, body and fins. The gills may appear inflamed. Fish may also demonstrate raised 12 mm diameter red spots on the skin that can progress to shallow ulcers that do not penetrate the dermis. During the latter stages of an outbreak or in endemic populations at temperatures outside the optimum range, a chronic form of the disease is common. Fish with chronic ESC often display uncoordinated, spastic swimming indicating neurological dysfunction. Affected fish may display rigor or uncoordinated muscle twitching, resulting from the infection progressing to the brain. These fish may demonstrate protruding eyes and/or a raised lesion or ulcer on the top of the head. This form of the disease is sometimes referred to as hole-in-the-head disease. A less common chronic form of the disease is the formation of a small (23 mm diameter) abscess on the body of the fish, especially in the joint connecting the pectoral or dorsal spine to the body. 4.1.2. Behavioural changes

4.2. Clinical methods

4.2.1. Gross pathology

During acute outbreaks, diseased fish will often have protruding eyes, abdominal distension and petechial haemorrhages on the face, body and fins. The gills may be moderately inflamed. The skin may also demonstrate raised 12 mm diameter red spots or shallow ulcers that do not penetrate the dermis. Internally, acutely affects fish often demonstrate yellow or blood-tinged ascites. The liver may be mottled or have 23 mm diameter red spots. The spleen and posterior kidney may be swollen and the GI tract may display extensive diffuse reddening. Focal haemorrhages may also be evident in adipose tissue, mesenteries and musculature. 4.2.2. Clinical chemistry

Not evaluated. 4.2.3. Microscopic pathology

Extensive necrosis and inflammation of the interstitial tissue trunk kidney tissues is the most common histopathological sign associated with acute infections. Spleen, liver and intestines also generally display necrosis and inflammation. In the chronic form, meningoenphalitis and erosion of the cranial cartilage are commonly observed. 4.2.4. Wet mounts

Gill wet mounts often demonstrate some telangiectasis and some epithelial proliferation. Small bacterial rods may be seen on wet mounts of tissue squashes at 4001000.

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4.2.5.

Smears

Gram-negative rods are often evident in blood smears and tissue imprints. Often the bacteria are seen within macrophages. 4.2.6. NA 4.2.7. NA Electron microscopy/cytopathology Fixed sections

4.3. Agent detection and identification methods

4.3.1. Direct detection methods Microscopic methods

4.3.1.1.

4.3.1.1.1. Wet mounts

Wet mounts of blood or tissue imprints may be taken and the presence of bacterial rods may indicate a bacteraemic condition but this is of marginal diagnostic value.

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6

4.3.1.1.2. Smears Gram staining: tissue imprints from the posterior kidney or brain from diseased fish can be Gram stained. Edwardsiella ictaluri
will be seen as 12 m, Gram-negative rods, often within macrophages. This finding would indicate a bacterial infection. imprint is then allowed to dry, lightly heat fixed and Gram stained according to standard methods.

Method: a tissue sample is blotted with a laboratory wipe to remove blood, and then imprinted onto an alcohol-cleaned slide. The Antibody based detection: tissue imprints are made and heat fixed as described above and then stained using enzyme linked immunostaining or the fluorescent antibody technique using E. ictaluri-specific monoclonal antibody Ed9 (1), as described below. Negative controls using a monoclonal antibody that is not specific for E. ictaluri should be run simultaneously. Positive staining of

bacterial rods in the Ed9 sample, with negative staining of a matched negative control, is a confirmatory diagnosis. Cryostat sections from a known ESC-affected fish can be used as positive controls. This method is used primarily as a rapid confirmatory diagnostic test for ESC. It has also been used to detect E. ictaluri in decomposing fish that had ESC (10). It is not routinely used for inspections because E. ictaluri is easily cultured, culture protocols can be more sensitive and isolation allows for antibioticsensitivity testing.

4.3.1.1.3. Fixed sections

Formalin-fixed paraffin-embedded sections can be stained using immunohistochemical methods. Negative control slides as described above should be included. Fixed sections are used primarily for research (3) or retrospective evaluation (23). Positive staining is a confirmatory diagnosis and can be used to evaluate a disease condition, but should not be used for inspections/surveys. 4.3.1.2. Agent isolation and identification

4.3.1.2.1. Cell culture/artificial media Sampling and isolation of the agent

Bacteriological samples from freshly dead or moribund fish should be taken aseptically from the brain and kidney tissue. Brain sampling is required to detect the necrotising encephalitis form of ESC, since this form is not frequently associated with septicaemia. The samples should be streaked for isolation onto blood agar plates, brainheart infusion (BHI) agar or tryptic soy agar plates. The bacterium grows slowly but does not require special nutrients. In mixed cultures, E. ictaluri can be overgrown by more rapidly growing bacteria, but E. ictaluri is present in very high numbers in fish affected by ESC. Optimal temperature for incubation is 2830C. Culture methods are especially important because culture-based screening for antibiotic sensitivity will help in evaluating treatment options. A selective medium (Edwardsiella ictaluri medium: EIM) has been developed (31) that is useful when samples are taken from heavily contaminated environments, but it is not essential for diagnosing ESC under normal conditions. This medium can be used as a selective enrichment medium for identifying carriers (see below).

EIM formulation: 10g tryptone, 10 g yeast extract, 1.25 g phenylalanine, 1.20 g ferric ammonium citrate, 5 g sodium chloride,

0.03 g bromothymol blue, 17 g agar, and 990 ml distilled water. The components are dissolved, the pH adjusted to 7.0-7.2, then autoclaved at 121C for 15 minutes. A 10 ml solution containing 3.5 g mannitol, 1 g bile salts and 10 mg colistin is then filter-sterilised and added to the medium just prior to pouring the plates. EIM selective broth is made as described above without the use of agar. Also, 0.5 g/ml fungizone can be used to reduce fungal growth.

Evaluation: the medium allows the growth of E. ictaluri and inhibits the growth of most Gram-positive bacteria and most Gram-negative bacteria. Most of the bacteria that will grow can be differentiated by morphology. After 48 hours at 30C, E. ictaluri and E. tarda produce 0.51 mm translucent green colonies, Proteus spp. produce 23 mm brownish green colonies, Aeromonas spp. produce 2-5 mm yellowish-green opaque colonies, Yersinia ruckeri produce 12 mm yellowish-green colonies, Serratia marcescens produce 23 mm reddish colonies, and Enterocci produce 0.5 mm yellow colonies. Any
translucent green colonies should be evaluated using biochemical, specific antibody or DNA-based methods.

Culture for identifying carriers: for detecting low levels of the bacterium, a selective enrichment procedure can be used (19) in
which kidney tissue is homogenised, cultured overnight in liquid EIM and 100 l of this sample is plated onto BHI agar plates. Selective culture can be paired with filtration (8). For detecting a carrier state in a healthy population, kidney tissue has been homogenised in 0.5% triton-X 100, filtered onto 0.45 m nitrocellulose and grown on EIM agar medium. The membranes can then be immunostained using an E. ictaluri-specific antibody for rapid confirmatory diagnosis. Intraperitoneal administration of suspect carrier fish with 0.8 mg/g of Kenalog (triamcinolone acetonide) 2 weeks before attempted culture enhances the detection of the bacterium (2).

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Characteristics
Following incubation for 3648 hours at 2830C, E. ictaluri appears as smooth, circular (12 mm diameter), weakly haemolytic, slightly convex non-pigmented colonies with entire edges. It is a Gram-negative rod, measuring 0.752.5 m, is weakly motile by means of a peritrichous flagellation and is cytochrome oxidase negative. This bacterium grows slowly or not at all at 37C. After isolation, the bacterium should be identified by biochemical and serological characteristics (11, 13). Table 4.1 shows some of the characteristics of the species and biogroups of the genus Edwardsiella and similar bacteria that can be isolated from fish as given in Bergeys Manual of Determinative Bacteriology (9). The optimal growth temperature of 2830C should be used for evaluating biochemical characteristics. Edwardsiella ictaluri is biochemically less active than the other Edwardsiella species, but it appears to be homogeneous (28, 34). A clear-cut biotype variation is not detected. Edwardsiella ictaluri and E. tarda may be differentiated from each other biochemically by the production of indole and hydrogen sulphide (E. tarda produces both, while E. ictaluri does not). Also E. tarda, Yersinia ruckeri, Hafnia alvei and E. hoshinae grow well at 37C, whereas E. ictaluri does not. Miniature biochemical panels are commonly used at 30C, such as the API 20E system (bioMerieux Vitek, Inc.) that, with E. ictaluri, produces an identification code number of 4004000 (12). Table 4.1. Differentiation of the species and biogroups of the genus Edwardsiella and other Enterobacteriaceae found in fish (9) Characteristic Acid production from: D-Mannitol Sucrose Trehalose L-Arabinose Malonate utilisation Indole production Hydrogen sulphide in triple sugar iron (agar) Motility Citrate (Christensens) + + + + + + d + *Weakly motile at 28C (11).

Yersinia ruckeri Hafnia alvei

Wildtype + + + +

E. tarda
Biogroup 1 + + + + +

E. hoshinae
+ + + () + (+)

E. ictaluri
*

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259

4.3.1.2.2. Antibody-based antigen detection methods

Cultured bacteria and infected tissues can be evaluated by the indirect immunofluorescent antibody technique (IFAT) or enzyme linked immunostaining for confirmatory diagnosis (29).

4.3.1.2.2.1. Indirect immunofluorescent antibody technique (IFAT)

Smears are air-dried and heated for 2 minutes at 60C before being flooded and incubated for 5 minutes with specific antibody (use undiluted cell culture supernatant when using MAb Ed9 produced from cell culture). They are washed in phosphate-buffered saline (PBS), pH 7.2, flooded for 5 minutes with a commercially available fluorescein isothiocyanate (FITC)-conjugated mouse-Igspecific secondary antibody at the suggested working concentration (1). After rinsing, the slides are mounted with cover-slips using phosphate-buffered mounting medium and observed microscopically for bright green fluorescence under blue epiillumination. Smears of bacterial suspensions must be very thin. Positive and negative controls (such as E. tarda) should be stained on separate slides.

4.3.1.2.2.2. Enzyme-linked immunostaining

Smears are prepared as for IFAT the first steps are similar, but the secondary antibody is conjugated to horseradish peroxidase. A third incubation step with a substrate (DMOB, Sigma) is performed for 10 minutes and, after washing and drying, the smears are mounted in buffered glycerine and observed microscopically. If smears are too thick, they may produce nonspecific retention of the stain. Rinsing the smear again for 1 or 2 minutes in 1 N HCl can solve this problem.

4.3.1.2.3. Molecular techniques 4.3.1.2.3.1. Sequencing

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Assays based on polymerase chain reaction (PCR) amplification of structural RNA sequences from bacterial colonies and direct sequencing of the products are being adapted by several diagnostic bacteriology laboratories and some of these assays are already commercially available (MicroSeq, Applied Biosystems). Species confirmation can be carried out by amplifying and sequencing the 16S portion of the ribosomal RNA operon and comparing the sequence with GenBank accession AF310622. However, the E. tarda 16s sequence is very similar and the use of the 23S portion of the operon has been advocated (Genbank Accession DQ211093). In particular, E. ictaluri has a 98 bp insert in the 23S fragment in at least six of the ribosomal RNA operons that is not present in E. tarda (43).

4.3.1.2.3.2. Real-time PCR

An E. ictaluri-specific real-time PCR has been developed (5). This assay can be applied to isolated bacteria or for ESC diagnosis on tissues from a suspect case. DNA from trunk kidney or brain can be isolated using a commercial kit such as DNeasy Tissue Kit (Qiagen, Maryland, Delaware USA). Bacteria (<1 l) from a colony can be suspended in 200 l of lysis buffer (20 mM Tris/HCl, pH 8.0, 2 mM EDTA (ethylene diamine tetra-acetic acid), pH 8.0 and 1.2% Triton X-100) and lysed in a boiling water bath for 10 minutes. Real-time PCR can be performed in 25 l reactions containing 2 l DNA (1050 ng), 12.5 l of Platimum Quantitative PCR Super Mix-UDG (Invitrogen, Carlsbad, California USA), 200 nM of each primer and 200 nM of probe (5 FAM flourophore and 3 Black Hole Quencher from Biosearch Technologies, Novato California, USA). Reactions are run for 2 minutes at 50C, 2 minutes at 95C and 40 cycles of 15 seconds at 95C and 1 minute at 60C on a real-time PCR machine. Positive results are indicated by a threshold cycle of less than that of 0.1 pg purified E. ictaluri DNA. All reactions should include negative controls consisting of water for bacteria samples or negative tissue that was extracted at the same time for tissue samples. Table 4.2. Primers and probe for E. ictaluri specific real-time PCR (5) Forward primer Reverse Probe ACT-TAT-CGC-CCT-CGC-AAC-TC CCT-CTG-ATA-AGT-GGT-TCT-CG CCT-CAC-ATA-TTG-CTT-CAG-CGT-CGA-C

279 280 281 282 283 284 285

4.3.1.2.3.3. Loop-mediated isothermal amplification method (LAMP)

Loop-mediated isothermal amplification method (LAMP) has been developed and evaluated for E. ictaluri from cultures and infected tissue (42). The method can use the same DNA preparation procedures described above. The LAMP assay is performed in a 25-l reaction mixture: 1 ThermoPol buffer with 8 U Bst DNA polymerase (New England Biolabs, Beverly, MA), 6 mM MgSO4, 0.8 M betaine, 1.0 mM each deoxynucleotide triphosphate, 0.2 M each of F3 and B3 primers, 1.6 M each of FI and BI primers and 1 l bacterial lysate or 0.5 g tissue DNA template. The amplification is carried out at 65C for 1 hour. The product is electrophoresed on 2% agarose gel and stained with ethidium bromide. Table 4.3. Primers for E. ictaluri specific LAMP (42) F3 B3 FI BI TAA-GAC-TCC-AGC-CCT-CGG TTC-CCT-CGC-TGG-AAG-TGG GCC-CGC-AGG-AAA-CCA-TTG-ATT-TTT-TTC-CGC-CTT-ACC-GCT-CTG-AT GAG-GCC-CCG-GAG-CAG-TCA-TAT-TTT-GCG-ATA-AGT-TCG-CCT-TCT-GT

286 287 288 289 290 291 292 293 294 295 296
8

4.3.1.2.4. Agent purification

NA 4.3.2. Serological methods

Enzyme-linked immunosorbent assay (ELISA) methods have been developed to detect catfish antibodies to E. ictaluri and are widely used in research (17, 36). The first method uses whole heat-killed bacteria at 4 108 cells/ml PBS, 50 l/well to coat poly-L-lysinetreated ELISA plates (36). The plates are washed with PBS and then blocked by the addition of 100 l of 100 mM glycine and 1% bovine serum albumin in PBS for 30 minutes. The plates are then incubated for 30 minutes with dilutions of the sera to be tested, washed, and incubated with anti-fish-species immunoglobulin serum (MAb 9E1 can be used for channel catfish), washed, and incubated with an antibody conjugate (either horseradish peroxidase or alkaline phosphatase) specific to the secondary antibody. Then, the plate is washed and the chromogenic enzyme substrate is added, the colour is allowed to develop and the plate is read. The second method (17) is similar but uses a soluble major antigen (20) obtained by sonicating the bacteria, or merely by dialysing the supernatants of 24Manual of Diagnostic Tests for Aquatic Animals 2009

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Method

hour broth cultures, then concentrating the sample to 25 g/ml protein content. Approximately 100 l is used to coat the wells of the microplates. Optimal working concentrations must first be determined for each reagent used in the test. These techniques have proven useful for investigating the immune response of channel catfish to E. ictaluri, and may be applicable for screening populations of fish for previous exposure. However, the delay in the immune response after exposure, as well as any seasonal and genetic variability in the immune responses makes these methods unreliable for inspection purposes.

5.

Rating of tests against purpose of use

The methods currently available for targeted surveillance and diagnosis of ESC are listed in Table 5.1. The designations used in the Table indicate: a = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity; b = the method is a standard method with good diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or other factors severely limits its application; and d = the method is presently not recommended for this purpose. These are somewhat subjective as suitability involves issues of reliability, sensitivity, specificity and utility. Not all of the tests listed as category a or b have undergone formal standardisation and validation. Table 5.1. Methods for targeted surveillance and diagnosis Targeted surveillance
Clinical ESC Subclinical ESC

Presumptive diagnosis

Confirmatory diagnosis

Gross signs Histopathology IFAT or immunostaining Culture biochemical Selective culture biochemical PCR sequencing Real-time PCR* LAMP* Antibody-specific ELISA

c c b a b d b b d

d d c b a d c c b

c c a a a d a a d

d d a b b a a a a

310 311 312 313 314 315 316 317 318 319 320

IFAT= indirect fluorescent antibody test; PCR = polymerase chain reaction; *= relatively new test with limited testing for routine diagnostic work; LAMP= loop-mediated isothermal amplification; ELISA = enzyme-linked immunosorbent assay.

6.

Test(s) recommended for targeted surveillance to declare freedom from enteric septicaemia of catfish (Edwardsiella ictaluri)

Regular health inspections in spring and fall when the temperature is 2026C using sampling methods described and a selective enrichment culture method on posterior kidney and brain (the entire organ up to 0.5 g). This should be combined with diagnostic analysis that includes routine bacteriological culture of posterior kidneys and brain on nutrient rich agar at 2830C for 72 hours any time moribund fish are identified.

7.

Corroborative diagnostic criteria

7.1. Definition of suspect case
ESC is suspected if any of the following occurs: i) Channel catfish are found displaying clinical signs of ESC at any temperature.

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

ii) iii) iv)

Any population that has been diagnosed with ESC in the past even if no clinical signs are present. Any population with detectable antibodies to E. ictaluri. Any population that produces bacterial colonies that are Gram negative, cytochrome oxidase negative, hydrogen sulphide negative, indole negative and grows well at 30C but poorly at 37C.

7.2. Definition of confirmed case

ESC is confirmed if any other following are found: i) ii) Fish show clinical signs of disease and tissue smears contain IFAT or positive immunostaining bacterial rods or tissues test positive by E. ictaluri-specific real-time PCR or LAMP. Bacteria are isolated by culture and test positive by specific antibody based detection or molecular methods.

References
AINSWORTH J., CAPLEY G., WATERSTREET P. & MUNSON D. (1986). Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri. J. Fish Dis., 9, 439444.

Edwardsiella ictaluri. J. Aquat. Anim. Health, 6, 4452.

ANTONIO-BAXTA D.B. & HEDRICK R.P. (1994). Effects of the corticosteroid kenalog on the carrier state of juvenile channel catfish exposed to

BALDWIN T.J. & NEWTON J.C. (1993). Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and light electron microscopy findings. J. Aquat. Anim. Health, 5, 189198.

ictaluri. Dis. Aquat. Org., 8, 113117.

BAXA D.V., GROFF J.M., WISHKOVSKY A. & HEDRICK R.P. (1990). Susceptibility of nonictalurid fishes to experimental infection with Edwardsiella

Edwardsiella ictaluri in channel catfish. J. Aquat. Anim. Health, 15, 8086. Anim. Health, 6, 234241.

BILODEAU A.L., WALDBIESER C.G., TERHUNE J.S., WISE D.J. & WOLTERS W.R. (2003). A real-time polymersae chain reaction assay of the bacterium CHEN M.F., HENRY-FORD D., KUMLIN M.E., KEY M.L., LIGHT T.S., COX W.T. & MODIN J.C. (1994). Distribution of Edwardsiella ictaluri in California. J. Aquat.

CRUMLISH M., DUNG T.T., TURNBULL J.F., NGOC N.T.N. & FERGUSON H.W. (2002). Identification of Edwardsiella ictaluri from diseased freshwater catfish, Pangasius hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam. J. Fish Dis., 25, 733736. EARLIX D., PLUMB J.A. & ROGERS W.A. (1996). Isolation of Edwardsiella ictaluri from channel catfish by tissue homogenisation, filtration and enzyme linked immuosorbant assay. Dis. Aquat. Org., 27, 1924. FARMER J.J. & MCWORTHER A.C. (1984). Genus Edwardsiella, Ewing & McWorther (1965). In: Bergeys Manual of Determinative Bacteriology, Krieg N.R. & Holt J.G., eds. William & Wilkins: Baltimore, Maryland, USA. pp 486491.

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HANSON L.A. & ROGERS W.A. (1989). Enzyme Immunoassay Identification of Edwardsiella ictaluri in Decomposing Channel Catfish. J. World

HAWKE J.P. (1979). A bacterium associated with disease of pond cultured channel catfish, Ictalurus punctatus. J Fish Res Board Can, 36, 15081512. HAWKE J.P., DURBOROW R.M., THUNE R.L. & CAMUS A.C., (1998). ESC-Enteric Septicemia of Catfish. USDA-CSREES, Southern Regional Aquaculture Center. publication 477. HAWKE J.P., MCWHORTER A.C., STEIGERWALT A.G. & BRENNER D.J. (1981). Edwardsiella ictaluri sp. nov., the causative agent of enteric septicemia of catfish. Int. J. Syst. Bacteriol., 31, 396400.

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14.

HUMPHREY J.D., LANCASTER C., GUDKOVS N. & MCDONALD W. (1986). Exotic bacterial pathogens Edwardsiella tarda and Edwardsiella ictaluri from imported ornamental fish Betta splendens and Puntius conchonius, respectively: isolation and quarantine significance. Aust Vet J, 63, 369 71. IWANOWICZ L.R., GRIFFIN A.R., CARTWRIGHT D.D. & BLAZER V.S. (2006). Mortality and pathology in brown bullheads Amieurus nebulosus associated with a spontaneous Edwardsiella ictaluri outbreak under tank culture conditions. Dis Aquat Org., 70, 21925. KENT M.L. & LYONS J.M. (1982). Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News, 2, ii.

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punctatus, using exoantigen. Vet. Immunol. Immunopathol., 36, 359368.

KLESIUS P. (1993). Rapid enzyme-linked immunosorbent tests for detecting antibodies to Edwardsiella ictaluri in channel catfish, Ictalurus

KLESIUS P. (1994). Transmission of Edwardsiella ictaluri from from infected, dead to uninfected channel catfish. J. Aquat. Anim. Health, 6, 180 182. KLESIUS P.H. (1992). Carrier state of channel catfish infected with Edwardsiella ictaluri. J. Aquat. Anim. Health, 4, 227230. KLESIUS P.H. & HORST M.N. (1991). Characterization of a major outer-membrane antigen of Edwardsiella ictaluri. J. Aquat. Anim. Health, 3, 181 187. KLESIUS P., LOVY J., EVANS J., WASHUTA E. & ARIAS C. (2003). Isolation of Edwardsiella ictaluri from tadpole madtom in a southwestern New Jersey river. J. Aquat. Anim. Health, 15, 295301. KLESIUS P.H. & SHOEMAKER C.A., Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In: Veterinary Vaccines and Diagnostics, Schultz R.D., Editor. 1999, Academic Press: San Diego, California USA, pp. 523537. MITCHELL A.J. & GOODWIN A.E. (1999). Evidence that Enteric Septicemia of Catfish (ESC) was Present in Arkansas by the Late 1960s: New Insights into the Epidemiology of ESC. J. Aquat. Anim. Health, 11, 175178. NAHMS, (2003). Highlights of NAHMS Catfish 2003: Part II. 2003, National Animal Health Monitoring System, Centers of Epidemiology and Animal Health, USDA:APHIS.: Ft. Collins, CO. PETRIE-HANSON L., ROMANO C.L., MACKEY R.B., KHOSRAVI P., HOHN C.M. & BOYLE C.R. (2007). Evaluation of zebrafish Danio rerio as a model for Enteric Septicemia of Catfish. J. Aquat. Anim. Health, 19, 151158. PLUMB J.A. & QUINLAN E.E. (1986). Survival of Edwardsiella ictaluri in pond water and bottom mud. Progress. Fish Cult., 48, 212214. PLUMB J.A. & SANCHEZ D.J. (1983). Susceptibility of five species of fish to Edwardsiella ictaluri. J. Fish Dis., 6, 261266. PLUMB J.A. & VINITNANTHARAT S. (1989). Biochemical, Biophysical, and Serological Homogeneity of Edwardsiella ictaluri. J. Aquat. Anim. Health, 1, 5156. ROGERS W.A. (1981). Serological detection of two species of Edwardsiella infecting catfish. In International symposium on fish biologics: serodiagnostics and vaccines. Dev. Biol. Stand., 49, 169172. SHOEMAKER C.A., KLESIUS P.H. & BRICKER J.M. (1999). Efficacy of a modified live Edwardsiella ictaluri vaccine in channel catfish as young as seven days post hatch. Aquaculture, 176. SHOTTS E.B. & WALTMAN W.D. (1990). A medium for the selective isolation of Edwardsiella ictaluri. J. Wildl. Dis., 26, 214218. TAYLOR P.W. (1992). Fish-eating birds as potential vectors of Edwardsiella ictaluri. J. Aquat. Anim. Health, 4, 240243.

Health, 14, 263272.

WAGNER B.A., WISE D.J., KHOO L.H. & TERHUNE J.S. (2002). The epidemiology of bacterial diseases in food-size channel catfish. J. Aquat. Anim.

WALTMAN W., SHOTTS E.B. & HSU T.C. (1986). Biochemical characteristics of Edwardsiella ictaluri. Appl. Environ. Microbiol., 51, 101104. WALTMAN W.D., SHOTTS E.B. & BLAZER V.S. (1985). Recovery of Edwardsiella ictaluri from Danio (Danio devario). Aquaculture, 46, 6366.
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36. 37. 38. 39. 40. 41. 42. 43.

WATERSTRAT P., AINSWORTH J. & CHAPLEY G. (1989). Use of an indirect enzyme-linked immunosorbent assay (ELISA) in the detection of channel catfish, Ictalurus punctatus (Rafinesque), antibodies to Edwardsiella ictaluri. J. Fish Dis., 12, 8794. WATERSTRAT P.R., DORR B., GLAHN J.F. & TOBIN M.E. (1999). Recovery and viability of Edwardsiella ictaluri from great blue herons Ardea herodias fed E. ictaluri-infected channel catfish Ictalurus punctatus fingerlings. J. World Aquacult. Soc., 30, 115122. WISE D., KLESIUS P., SHOEMAKER C. & WOLTERS W. (2000). Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33). J. World Aquacult. Soc., 31, 206212. WISE D.J. & JOHNSON M.R. (1998). Effect of feeding frequency and Romet-medicated feed on survival, antibody response and weight gain of fingerling channel catfish (Ictalurus punctatus) after natural exposure to Edwardsiella icaluri. J. World Aquacult. Soc., 29, 170176. WISE D.J. & JOHNSON M.R. (1998). Effect of feeding frequency and Romet-medicated feed on survival, antibody response, and weight gain of fingerling channel catfish Ictalurus punctatus after natural exposure to Edwardsiella ictaluri. J. World Aquacult. Soc., 29, 169175. WOLTERS W.R., WISE D.J. & KLESIUS P.H. (1996). Survival and antibody response of channel catfish, blue catfish and channel catfish female blue catfish male hybrids after exposure to Edwardsiella ictaluri. J. Aquat. Anim. Health, 8, 249254. YEH H.Y., SHOEMAKER C.A. & KLESIUS P.H. (2005). Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. J Microbiol Methods, 63, 3644.

Edwardsiella ictaluri. Syst. Appl. Microbiol., 30, 93101.

ZHANG Y. & ARIAS C.R. (2007). Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of * * *

NB: There is an OIE Reference Laboratory for Enteric septicaemia of catfish (Edwardsiella ictaluri) (see Table at the end of this Aquatic Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).

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