TK- Edwardsielia Ictaluri _Chapter 1.2.3 of the Aquatic Code _Manual of Diagnostic Tests for Aquatic...

NB : TH IS D ISEASE IS NO LONGER L ISTED IN CHAPTER 1 .2 .3 OF THE AQUAT IC CODE

Manual of Diagnostic Tests for Aquatic Animals 2009 1

CHAPTER 2.1.11. 1 2

ENTERIC SEPTICAEMIA OF CATFISH 3 (EDWARDSIELLA ICTALURI) 4

1. Scope 5

Enteric septicaemia of catfish (ESC) is considered to be an infection by the Gram-negative bacterium Edwardsiella ictaluri. There are three forms 6 of this disease: acute haemorrhagic bacteraemia, chronic necrotising encephalitis and an unapparent carrier state. ESC has traditionally been a 7 disease of North American freshwater catfish and is one of the most important infectious disease problems in the commercial catfish industry in 8 the United States of America (USA). Recently, epizootics have been reported in native and North American species of freshwater catfish in 9 commercial production systems in South-East Asia. 10

2. Disease information 11

2.1. Agent factors 12

2.1.1. Aetiological agent, agent strains 13

Edwardsiella ictaluri is a Gram-negative motile rod that is oxidase negative, facultatively anaerobic, grows poorly at 37°C and produces no 14 hydrogen sulphide or indole. The agent is remarkably homogenous in biochemical profiles but plasmid and antigenic characteristics 15 suggest that the non-channel catfish isolates differ somewhat from channel catfish isolates. The genome of this pathogen has been 16 sequenced (http://micro-gen.ouhsc.edu/e_ictal/e_ictal_home.htm). 17

2.1.2. Survival outside the host 18

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). 19

2.1.3. Stability of the agent (effective inactivation methods) 20

This pathogen is sensitive to heat, desiccation, UV exposure, detergents and most disinfectants. 21

2.1.4. Life cycle 22

The life cycle of Edwardsiella ictaluri is direct. Fish from a population that has recovered from the disease are considered carriers. 23 Edwardsiella ictaluri has been detected in the kidney of such fish well over 4 months after exposure (2, 19), suggesting that carrier 24 fish act as the natural reservoir for the organism. Researchers have found the bacterium in the gut of fish-eating birds using 25 fluorescent antibody tests on ingesta but no E. ictaluri could be cultured, indicating that the bacteria were not viable (32, 37). This 26 suggests that birds are not an important means of dissemination of this pathogen. However, there are many types of scavengers and 27 opportunistic predators at the site of an ESC outbreak and physical spread of the bacterium to neighbouring ponds in infected tissues 28 or on the surface of animals is likely. 29

2.2. Host factors 30

2.2.1. Susceptible host species 31

Edwardsiella ictaluri is one of the most important pathogens of channel catfish (Ictalurus punctatus), the primary species for catfish 32 aquaculture in USA. It can be an infectious bacterial pathogen of several other species of North American catfish, bullheads and 33 madtoms (members of the family Ictaluridae: I. furcatus, Ameiurus nebulosis, A. catus, Noturus gyrinus) (13). This pathogen has also 34 been associated with disease outbreaks in a variety of other members of the superorder Ostariophysi, including Asian freshwater 35 catfish (Pangasiidae: Pangasius hypothammus [7], and Clariidae: Clarias batrachus), knifefish (Sternopygidae: Eigemannia virescens 36 [16]) and cyprinids (Cyprinidae: Begal Danio Davario davario [35] and Rosy Barbs Puntius conchonius [14]). Experimental infections 37 suggest that Pacific salmon are susceptible (4) and that zebra danios (Danio rerio) (25) and European catfish (Silurus glanis) are 38 moderately susceptible (27). 39

2.2.2. Susceptible stages of the host 40

Chapter 2.1.11. – Enteric septicaemia of catfish (Edwardsiella ictaluri)

2 Manual of Diagnostic Tests for Aquatic Animals 2009

Juveniles and adults are susceptible to the pathogen. The highest losses are in young fish (less than 1 year of age) during their first 41 exposure to the pathogen in the optimal temperature range of 18–28°C. 42

2.2.3. Species or sub-population predilection (probability of detection) 43

In mixed populations, members of the Ictaluridae are most susceptible and among this group channel catfish are more susceptible 44 than blue catfish. 45

2.2.4. Target organs and infected tissue 46

During acute ESC, the bacterium can be isolated from most tissues, but the target organ is the posterior kidney. In the chronic form, 47 the target tissue is the brain, or occasional abscesses at the base of the pectoral or dorsal fin spine; often other tissues will give 48 negative results by culture for E. ictaluri. In the unapparent carrier state, posterior kidney and gastrointestinal tract may give 49 positive results by culture for the pathogen, but the use of enrichment and selective media may be needed. 50

2.2.5. Persistent infection with lifelong carriers 51

Edwardsiella ictaluri establishes a persistent lifelong infection in exposed fish. 52

2.2.6. Vectors 53

There are no known biotic vectors for E. ictaluri. 54

2.2.7. Known or suspected wild aquatic animal carriers 55

Wild and feral catfish may be reservoirs of E. ictaluri. Channel catfish in rivers and lakes throughout California were shown to have 56 antibodies to E. ictaluri (6). An outbreak of ESC was confirmed in wild tadpole madtoms (Noturus gyrinus) (21), and an outbreak of ESC 57 occurred in wild caught brown bullheads (Ameiurus nebulosis) when brought into a laboratory (15) 58

2.3. Disease pattern 59

2.3.1. Transmission mechanisms 60

During overt ESC, the bacterium can be transmitted from dead and dying fish to susceptible fish by cannibalism (18). It is believed that 61 shedding with faeces is the main means of dissemination into the pond environment. Infection has been shown to occur by intestinal 62 mucosa (oral uptake), and the olfactory mucosa (nasally). Recent evidence suggests that epidermal and branchial mucosa may be 63 additionally important routes. During the natural course of ESC, the incubation period from first exposure to first deaths is 64 approximately 8 days (18, 39). 65

2.3.2. Prevalence 66

ESC is a commonly reported disease problem in commercial catfish operations in the USA (24, 33). Most populations of cultured 67 catfish in the endemic regions of the USA are carriers. The cryptic nature of the pathogen during the unapparent carrier state makes 68 it difficult to determine the actual carrier rate within an endemic population. 69

2.3.3. Geographical distribution 70

Edwardsiella ictaluri is widely distributed in the USA and South-East Asia. 71

2.3.4. Mortality and morbidity 72

Acute outbreaks of ESC can cause losses of over 50% in a population. 73

2.3.5. Environmental factors 74

The highest losses occur in heavily stocked ponds that have experienced an environmental stressor within the 18–28°C temperature 75 range. 76

2.4. Control and prevention 77

2.4.1. Vaccination 78

An attenuated mutant of E. ictaluri (RE-33) induced by passage in the presence of rifampicin (22) has been licensed for use as a 79 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 80 vaccination has been shown to be helpful for fingerling production to reduce, but not eliminate, ESC. 81



2.4.2. Chemotherapy 82

Antibiotic-medicated feeds are generally effective against ESC. However, the disease must be identified during early stages of 83 infection before the population experiences a suppressed appetite. Antibiotics that have proven effective include oxytetracycline HCL 84 (Terramycin, Pfizer) 16 mg active ingredient per kg of fish for 10 days, a combination of sulfadimethoxine and ormetoprim (Romet 30, 85 Hofmann–LaRoche) at 50 mg/kg fish for 5 days and florfenicol (Aquaflor, Schering Plough) at 10 mg/kg fish for 10 days. The latter 86 two are specifically labelled for use on ESC in the USA. Withdrawal times before medicated channel catfish can go to market in the 87 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 88 full dose and application time period should be used once started. These measures are important to avoid development of antibiotic 89 resistance, as E. ictaluri can acquire plasmids that provide multiple drug resistance. 90

2.4.3. Immunostimulation 91

Research on the use of immunostimulants in the feed is promising, but there is no widespread commercial application for any of 92 these additives in the control of ESC. 93

2.4.4. Resistance breeding 94

Biological resistance to ESC has been demonstrated among genetic lines, but the use of genetics for controlling ESC has not gained 95 widespread commercial application (38). 96

2.4.5. Restocking with resistant species 97

Biological resistance to ESC has been demonstrated in a similar species (blue catfish, I. furcatus) and channel × blue hybrids are 98 more resistant than channel catfish (41). There is some commercial production of the hybrid but difficulty in producing fry has 99 hampered widespread adoption of the hybrid for commercial catfish aquaculture. 100

2.4.6. Blocking agents 101

None identified. 102

2.4.7. Disinfection of eggs and larvae 103

The establishment of ESC-free fish from an endemic population can be accomplished by disinfecting eggs using 100 ppm (parts per 104 million) iodine (betadine) and producing fry using pathogen-free water. 105

2.4.8. General husbandry practices 106

Management to reduce ESC-associated losses is the best option when the pathogen becomes established on a facility or is endemic in 107 a region. This is done by maintaining quality nutrition and feeding levels throughout the year and avoiding stressful events during the 108 ESC temperature range. One practice that should especially be avoided is the stocking of potentially naïve fish into an endemic 109 population during or shortly before the temperatures are in the 18–28°C range. Also, the use of chemical treatments should be 110 avoided in this temperature range. One management procedure that has proven effective in reducing losses is the modification of the 111 feeding schedule to feeding on alternate days when ESC is occurring in the area (40). This apparently reduces faecal–oral 112 transmission of the pathogen. 113

3. Sampling 114

3.1. Selection of individual specimens 115

Samples for inspections/surveillance must be representative of all groups of a population derived from multiple independent stockings. 116 Sampling from ponds should include any moribund fish and fish that school behind aerators when oxygen levels are not stressful. Sampling 117 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 118 sporadic or uncoordinated swimming and may occasionally be seen splashing at the surface. 119

3.2. Preservation of samples for submission 120

Samples for bacterial culture may be iced or refrigerated for up to 48 hours. 121

3.3. Pooling of samples 122

Pooling of samples is not recommended. 123



3.4. Best organs or tissues 124

Posterior kidney and brain (also, any small abscesses on otherwise clinically normal fish). 125

3.5. Samples/tissues that are not suitable 126

Frozen samples are unreliable for culture. Often tissues other than brain or occasional abscesses at the base of the pectoral or dorsal fin 127 spine will give negative results by culture for E. ictaluri. 128

4. Diagnostic methods 129

4.1. Field diagnostic methods 130

4.1.1. Clinical signs 131

ESC occurs predominantly at temperatures between 18°C and 28°C and is most severe in crowded production systems that contain 132 many naïve fish. In the USA, this results primarily in autumn- or spring-associated losses. The disease is substantially exacerbated by 133 stressful events such as nitrite toxicity or low dissolved oxygen. Mixed infection with Flavobacterium columnare (columnaris disease) 134 and/or channel catfish virus (channel catfish virus disease) are common. An affected population may show rapidly progressing 135 mortality with affected fish being lethargic and resting at the bank. Affected fish will also demonstrate distressed uncoordinated 136 swimming. As the outbreak worsens in the pond, the population will substantially reduce feeding activity. During the acute outbreaks, 137 diseased fish will often demonstrate protruding eyes, abdominal distension and petechial haemorrhages on the face, body and fins. 138 The gills may appear inflamed. Fish may also demonstrate raised 1–2 mm diameter red spots on the skin that can progress to shallow 139 ulcers that do not penetrate the dermis. During the latter stages of an outbreak or in endemic populations at temperatures outside 140 the optimum range, a chronic form of the disease is common. Fish with chronic ESC often display uncoordinated, spastic swimming 141 indicating neurological dysfunction. Affected fish may display rigor or uncoordinated muscle twitching, resulting from the infection 142 progressing to the brain. These fish may demonstrate protruding eyes and/or a raised lesion or ulcer on the top of the head. This 143 form of the disease is sometimes referred to as hole-in-the-head disease. A less common chronic form of the disease is the 144 formation of a small (2–3 mm diameter) abscess on the body of the fish, especially in the joint connecting the pectoral or dorsal 145 spine to the body. 146

4.1.2. Behavioural changes 147

– 148

4.2. Clinical methods 149

4.2.1. Gross pathology 150

During acute outbreaks, diseased fish will often have protruding eyes, abdominal distension and petechial haemorrhages on the face, 151 body and fins. The gills may be moderately inflamed. The skin may also demonstrate raised 1–2 mm diameter red spots or shallow 152 ulcers that do not penetrate the dermis. Internally, acutely affects fish often demonstrate yellow or blood-tinged ascites. The liver 153 may be mottled or have 2–3 mm diameter red spots. The spleen and posterior kidney may be swollen and the GI tract may display 154 extensive diffuse reddening. Focal haemorrhages may also be evident in adipose tissue, mesenteries and musculature. 155

4.2.2. Clinical chemistry 156

Not evaluated. 157

4.2.3. Microscopic pathology 158

Extensive necrosis and inflammation of the interstitial tissue trunk kidney tissues is the most common histopathological sign 159 associated with acute infections. Spleen, liver and intestines also generally display necrosis and inflammation. 160

In the chronic form, meningoenphalitis and erosion of the cranial cartilage are commonly observed. 161

4.2.4. Wet mounts 162

Gill wet mounts often demonstrate some telangiectasis and some epithelial proliferation. Small bacterial rods may be seen on wet 163 mounts of tissue squashes at ×400–1000. 164



4.2.5. Smears 165

Gram-negative rods are often evident in blood smears and tissue imprints. Often the bacteria are seen within macrophages. 166

4.2.6. Fixed sections 167

NA 168

4.2.7. Electron microscopy/cytopathology 169

NA 170

4.3. Agent detection and identification methods 171

4.3.1. Direct detection methods 172

4.3.1.1. Microscopic methods 173

4.3.1.1.1. Wet mounts 174

Wet mounts of blood or tissue imprints may be taken and the presence of bacterial rods may indicate a bacteraemic condition 175 but this is of marginal diagnostic value. 176



4.3.1.1.2. Smears 177

Gram staining: tissue imprints from the posterior kidney or brain from diseased fish can be Gram stained. Edwardsiella ictaluri 178 will be seen as 1–2 µm, Gram-negative rods, often within macrophages. This finding would indicate a bacterial infection. 179

Method: a tissue sample is blotted with a laboratory wipe to remove blood, and then imprinted onto an alcohol-cleaned slide. The 180 imprint is then allowed to dry, lightly heat fixed and Gram stained according to standard methods. 181

Antibody based detection: tissue imprints are made and heat fixed as described above and then stained using enzyme linked 182 immunostaining or the fluorescent antibody technique using E. ictaluri-specific monoclonal antibody Ed9 (1), as described below. 183 Negative controls using a monoclonal antibody that is not specific for E. ictaluri should be run simultaneously. Positive staining of 184 bacterial rods in the Ed9 sample, with negative staining of a matched negative control, is a confirmatory diagnosis. Cryostat 185 sections from a known ESC-affected fish can be used as positive controls. This method is used primarily as a rapid confirmatory 186 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 187 for inspections because E. ictaluri is easily cultured, culture protocols can be more sensitive and isolation allows for antibiotic-188 sensitivity testing. 189

4.3.1.1.3. Fixed sections 190

Formalin-fixed paraffin-embedded sections can be stained using immunohistochemical methods. Negative control slides as 191 described above should be included. Fixed sections are used primarily for research (3) or retrospective evaluation (23). Positive 192 staining is a confirmatory diagnosis and can be used to evaluate a disease condition, but should not be used for 193 inspections/surveys. 194

4.3.1.2. Agent isolation and identification 195

4.3.1.2.1. Cell culture/artificial media 196

Sampling and isolation of the agent 197

Bacteriological samples from freshly dead or moribund fish should be taken aseptically from the brain and kidney tissue. Brain 198 sampling is required to detect the necrotising encephalitis form of ESC, since this form is not frequently associated with 199 septicaemia. The samples should be streaked for isolation onto blood agar plates, brain–heart infusion (BHI) agar or tryptic soy 200 agar plates. The bacterium grows slowly but does not require special nutrients. In mixed cultures, E. ictaluri can be overgrown by 201 more rapidly growing bacteria, but E. ictaluri is present in very high numbers in fish affected by ESC. Optimal temperature for 202 incubation is 28–30°C. Culture methods are especially important because culture-based screening for antibiotic sensitivity will 203 help in evaluating treatment options. 204

A selective medium (Edwardsiella ictaluri medium: EIM) has been developed (31) that is useful when samples are taken from 205 heavily contaminated environments, but it is not essential for diagnosing ESC under normal conditions. This medium can be used 206 as a selective enrichment medium for identifying carriers (see below). 207

EIM formulation: 10g tryptone, 10 g yeast extract, 1.25 g phenylalanine, 1.20 g ferric ammonium citrate, 5 g sodium chloride, 208 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, 209 then autoclaved at 121°C for 15 minutes. A 10 ml solution containing 3.5 g mannitol, 1 g bile salts and 10 mg colistin is then 210 filter-sterilised and added to the medium just prior to pouring the plates. EIM selective broth is made as described above 211 without the use of agar. Also, 0.5 µg/ml fungizone can be used to reduce fungal growth. 212

Evaluation: the medium allows the growth of E. ictaluri and inhibits the growth of most Gram-positive bacteria and most 213 Gram-negative bacteria. Most of the bacteria that will grow can be differentiated by morphology. After 48 hours at 30°C, E. 214 ictaluri and E. tarda produce 0.5–1 mm translucent green colonies, Proteus spp. produce 2–3 mm brownish green colonies, 215 Aeromonas spp. produce 2-5 mm yellowish-green opaque colonies, Yersinia ruckeri produce 1–2 mm yellowish-green 216 colonies, Serratia marcescens produce 2–3 mm reddish colonies, and Enterocci produce 0.5 mm yellow colonies. Any 217 translucent green colonies should be evaluated using biochemical, specific antibody or DNA-based methods. 218

Culture for identifying carriers: for detecting low levels of the bacterium, a selective enrichment procedure can be used (19) in 219 which kidney tissue is homogenised, cultured overnight in liquid EIM and 100 µl of this sample is plated onto BHI agar plates. 220 Selective culture can be paired with filtration (8). For detecting a carrier state in a healthy population, kidney tissue has been 221 homogenised in 0.5% triton-X 100, filtered onto 0.45 µm nitrocellulose and grown on EIM agar medium. The membranes can then 222 be immunostained using an E. ictaluri-specific antibody for rapid confirmatory diagnosis. Intraperitoneal administration of 223 suspect carrier fish with 0.8 mg/g of Kenalog (triamcinolone acetonide) 2 weeks before attempted culture enhances the 224 detection of the bacterium (2). 225



Characteristics 226

Following incubation for 36–48 hours at 28–30°C, E. ictaluri appears as smooth, circular (1–2 mm diameter), weakly haemolytic, 227 slightly convex non-pigmented colonies with entire edges. It is a Gram-negative rod, measuring 0.75–2.5 µm, is weakly motile by 228 means of a peritrichous flagellation and is cytochrome oxidase negative. This bacterium grows slowly or not at all at 37°C. After 229 isolation, the bacterium should be identified by biochemical and serological characteristics (11, 13). Table 4.1 shows some of the 230 characteristics of the species and biogroups of the genus Edwardsiella and similar bacteria that can be isolated from fish as 231 given in Bergey’s Manual of Determinative Bacteriology (9). The optimal growth temperature of 28–30°C should be used for 232 evaluating biochemical characteristics. Edwardsiella ictaluri is biochemically less active than the other Edwardsiella species, but 233 it appears to be homogeneous (28, 34). A clear-cut biotype variation is not detected. Edwardsiella ictaluri and E. tarda may be 234 differentiated from each other biochemically by the production of indole and hydrogen sulphide (E. tarda produces both, while E. 235 ictaluri does not). Also E. tarda, Yersinia ruckeri, Hafnia alvei and E. hoshinae grow well at 37°C, whereas E. ictaluri does not. 236 Miniature biochemical panels are commonly used at 30°C, such as the API 20E system (bioMerieux Vitek, Inc.) that, with E. ictaluri, 237 produces an identification code number of 4004000 (12). 238

Table 4.1. Differentiation of the species and biogroups of the genus 239 Edwardsiella and other Enterobacteriaceae found in fish (9) 240

Characteristic Yersinia ruckeri Hafnia alvei E. tarda E. hoshinae E. ictaluri Acid production from: Wildtype Biogroup 1

D-Mannitol + + – + + –

Sucrose – – – + –

Trehalose + + – – + –

L-Arabinose – + – + (–) –

Malonate utilisation – d – – –

Indole production – – + + – –

Hydrogen sulphide in triple sugar iron (agar)

– – + – – –

Motility – + + + + –*

Citrate (Christensen’s) + – + + (+) –

*Weakly motile at 28°C (11). 241

4.3.1.2.2. Antibody-based antigen detection methods 242

Cultured bacteria and infected tissues can be evaluated by the indirect immunofluorescent antibody technique (IFAT) or enzyme 243 linked immunostaining for confirmatory diagnosis (29). 244

4.3.1.2.2.1. Indirect immunofluorescent antibody technique (IFAT) 245

Smears are air-dried and heated for 2 minutes at 60°C before being flooded and incubated for 5 minutes with specific antibody 246 (use undiluted cell culture supernatant when using MAb Ed9 produced from cell culture). They are washed in phosphate-buffered 247 saline (PBS), pH 7.2, flooded for 5 minutes with a commercially available fluorescein isothiocyanate (FITC)-conjugated mouse-Ig-248 specific secondary antibody at the suggested working concentration (1). After rinsing, the slides are mounted with cover-slips 249 using phosphate-buffered mounting medium and observed microscopically for bright green fluorescence under blue epi-250 illumination. Smears of bacterial suspensions must be very thin. Positive and negative controls (such as E. tarda) should be 251 stained on separate slides. 252

4.3.1.2.2.2. Enzyme-linked immunostaining 253

Smears are prepared as for IFAT – the first steps are similar, but the secondary antibody is conjugated to horseradish 254 peroxidase. A third incubation step with a substrate (DMOB, Sigma) is performed for 10 minutes and, after washing and drying, 255 the smears are mounted in buffered glycerine and observed microscopically. If smears are too thick, they may produce non-256 specific retention of the stain. Rinsing the smear again for 1 or 2 minutes in 1 N HCl can solve this problem. 257

4.3.1.2.3. Molecular techniques 258

4.3.1.2.3.1. Sequencing 259



Assays based on polymerase chain reaction (PCR) amplification of structural RNA sequences from bacterial colonies and direct 260 sequencing of the products are being adapted by several diagnostic bacteriology laboratories and some of these assays are 261 already commercially available (MicroSeq, Applied Biosystems). Species confirmation can be carried out by amplifying and 262 sequencing the 16S portion of the ribosomal RNA operon and comparing the sequence with GenBank accession AF310622. 263 However, the E. tarda 16s sequence is very similar and the use of the 23S portion of the operon has been advocated (Genbank 264 Accession DQ211093). In particular, E. ictaluri has a 98 bp insert in the 23S fragment in at least six of the ribosomal RNA operons 265 that is not present in E. tarda (43). 266

4.3.1.2.3.2. Real-time PCR 267

An E. ictaluri-specific real-time PCR has been developed (5). This assay can be applied to isolated bacteria or for ESC diagnosis 268 on tissues from a suspect case. 269

DNA from trunk kidney or brain can be isolated using a commercial kit such as DNeasy Tissue Kit (Qiagen, Maryland, Delaware 270 USA). Bacteria (<1 µl) from a colony can be suspended in 200 µl of lysis buffer (20 mM Tris/HCl, pH 8.0, 2 mM EDTA (ethylene 271 diamine tetra-acetic acid), pH 8.0 and 1.2% Triton X-100) and lysed in a boiling water bath for 10 minutes. 272

Real-time PCR can be performed in 25 µl reactions containing 2 µl DNA (10–50 ng), 12.5 µl of Platimum Quantitative PCR Super 273 Mix-UDG (Invitrogen, Carlsbad, California USA), 200 nM of each primer and 200 nM of probe (5’ FAM flourophore and 3’ Black Hole 274 Quencher from Biosearch Technologies, Novato California, USA). Reactions are run for 2 minutes at 50°C, 2 minutes at 95°C and 275 40 cycles of 15 seconds at 95°C and 1 minute at 60°C on a real-time PCR machine. Positive results are indicated by a threshold 276 cycle of less than that of 0.1 pg purified E. ictaluri DNA. All reactions should include negative controls consisting of water for 277 bacteria samples or negative tissue that was extracted at the same time for tissue samples. 278

Table 4.2. Primers and probe for E. ictaluri specific real-time PCR (5) Forward primer ACT-TAT-CGC-CCT-CGC-AAC-TC

Reverse CCT-CTG-ATA-AGT-GGT-TCT-CG Probe CCT-CAC-ATA-TTG-CTT-CAG-CGT-CGA-C

4.3.1.2.3.3. Loop-mediated isothermal amplification method (LAMP) 279

Loop-mediated isothermal amplification method (LAMP) has been developed and evaluated for E. ictaluri from cultures and 280 infected tissue (42). The method can use the same DNA preparation procedures described above. The LAMP assay is performed in 281 a 25-µl reaction mixture: 1× ThermoPol buffer with 8 U Bst DNA polymerase (New England Biolabs, Beverly, MA), 6 mM MgSO4, 0.8 282 M betaine, 1.0 mM each deoxynucleotide triphosphate, 0.2 µM each of F3 and B3 primers, 1.6 µM each of FI and BI primers and 1 µl 283 bacterial lysate or 0.5 µg tissue DNA template. The amplification is carried out at 65°C for 1 hour. The product is electrophoresed 284 on 2% agarose gel and stained with ethidium bromide. 285

Table 4.3. Primers for E. ictaluri specific LAMP (42) F3 TAA-GAC-TCC-AGC-CCT-CGG B3 TTC-CCT-CGC-TGG-AAG-TGG FI GCC-CGC-AGG-AAA-CCA-TTG-ATT-TTT-TTC-CGC-CTT-ACC-GCT-CTG-AT BI GAG-GCC-CCG-GAG-CAG-TCA-TAT-TTT-GCG-ATA-AGT-TCG-CCT-TCT-GT

4.3.1.2.4. Agent purification 286

NA 287

4.3.2. Serological methods 288

Enzyme-linked immunosorbent assay (ELISA) methods have been developed to detect catfish antibodies to E. ictaluri and are widely 289 used in research (17, 36). The first method uses whole heat-killed bacteria at 4 108 cells/ml PBS, 50 µl/well to coat poly-L-lysine-290 treated ELISA plates (36). The plates are washed with PBS and then blocked by the addition of 100 µl of 100 mM glycine and 1% bovine 291 serum albumin in PBS for 30 minutes. The plates are then incubated for 30 minutes with dilutions of the sera to be tested, washed, 292 and incubated with anti-fish-species immunoglobulin serum (MAb 9E1 can be used for channel catfish), washed, and incubated with an 293 antibody conjugate (either horseradish peroxidase or alkaline phosphatase) specific to the secondary antibody. Then, the plate is 294 washed and the chromogenic enzyme substrate is added, the colour is allowed to develop and the plate is read. The second method (17) 295 is similar but uses a soluble major antigen (20) obtained by sonicating the bacteria, or merely by dialysing the supernatants of 24-296



hour broth cultures, then concentrating the sample to 25 µg/ml protein content. Approximately 100 µl is used to coat the wells of the 297 microplates. Optimal working concentrations must first be determined for each reagent used in the test. These techniques have 298 proven useful for investigating the immune response of channel catfish to E. ictaluri, and may be applicable for screening populations 299 of fish for previous exposure. However, the delay in the immune response after exposure, as well as any seasonal and genetic 300 variability in the immune responses makes these methods unreliable for inspection purposes. 301

5. Rating of tests against purpose of use 302

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 303 = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity; b = the method is a standard 304 method with good diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or other factors 305 severely limits its application; and d = the method is presently not recommended for this purpose. These are somewhat subjective as suitability 306 involves issues of reliability, sensitivity, specificity and utility. Not all of the tests listed as category a or b have undergone formal standardisation 307 and validation. 308

Table 5.1. Methods for targeted surveillance and diagnosis 309

Method Targeted surveillance Presumptive diagnosis

Confirmatory diagnosis

Clinical ESC Subclinical ESC

Gross signs c d c d

Histopathology c d c d

IFAT or immunostaining b c a a

Culture – biochemical a b a b

Selective culture – biochemical b a a b

PCR sequencing d d d a

Real-time PCR* b c a a

LAMP* b c a a

Antibody-specific ELISA d b d a

IFAT= indirect fluorescent antibody test; PCR = polymerase chain reaction; *= relatively new test with limited testing for routine diagnostic work; 310 LAMP= loop-mediated isothermal amplification; ELISA = enzyme-linked immunosorbent assay. 311

6. Test(s) recommended for targeted surveillance to declare freedom from enteric septicaemia of catfish (Edwardsiella 312 ictaluri) 313

Regular health inspections in spring and fall when the temperature is 20–26°C using sampling methods described and a selective enrichment 314 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 315 bacteriological culture of posterior kidneys and brain on nutrient rich agar at 28–30°C for 72 hours any time moribund fish are identified. 316

7. Corroborative diagnostic criteria 317

7.1. Definition of suspect case 318

ESC is suspected if any of the following occurs: 319

i) Channel catfish are found displaying clinical signs of ESC at any temperature. 320



ii) Any population that has been diagnosed with ESC in the past even if no clinical signs are present. 321

iii) Any population with detectable antibodies to E. ictaluri. 322

iv) Any population that produces bacterial colonies that are Gram negative, cytochrome oxidase negative, hydrogen sulphide negative, 323 indole negative and grows well at 30°C but poorly at 37°C. 324

7.2. Definition of confirmed case 325

ESC is confirmed if any other following are found: 326

i) Fish show clinical signs of disease and tissue smears contain IFAT or positive immunostaining bacterial rods or tissues test positive 327 by E. ictaluri-specific real-time PCR or LAMP. 328

ii) Bacteria are isolated by culture and test positive by specific antibody based detection or molecular methods. 329

8. References 330

1. AINSWORTH J., CAPLEY G., WATERSTREET P. & MUNSON D. (1986). Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for 331 the diagnosis of Edwardsiella ictaluri. J. Fish Dis., 9, 439–444. 332

2. ANTONIO-BAXTA D.B. & HEDRICK R.P. (1994). Effects of the corticosteroid kenalog on the carrier state of juvenile channel catfish exposed to 333 Edwardsiella ictaluri. J. Aquat. Anim. Health, 6, 44–52. 334

3. BALDWIN T.J. & NEWTON J.C. (1993). Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and 335 light electron microscopy findings. J. Aquat. Anim. Health, 5, 189–198. 336

4. BAXA D.V., GROFF J.M., WISHKOVSKY A. & HEDRICK R.P. (1990). Susceptibility of nonictalurid fishes to experimental infection with Edwardsiella 337 ictaluri. Dis. Aquat. Org., 8, 113–117. 338

5. 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 339 Edwardsiella ictaluri in channel catfish. J. Aquat. Anim. Health, 15, 80–86. 340

6. 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. 341 Anim. Health, 6, 234–241. 342

7. CRUMLISH M., DUNG T.T., TURNBULL J.F., NGOC N.T.N. & FERGUSON H.W. (2002). Identification of Edwardsiella ictaluri from diseased freshwater catfish, 343 Pangasius hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam. J. Fish Dis., 25, 733–736. 344

8. EARLIX D., PLUMB J.A. & ROGERS W.A. (1996). Isolation of Edwardsiella ictaluri from channel catfish by tissue homogenisation, filtration and 345 enzyme linked immuosorbant assay. Dis. Aquat. Org., 27, 19–24. 346

9. FARMER J.J. & MCWORTHER A.C. (1984). Genus Edwardsiella, Ewing & McWorther (1965). In: Bergey’s Manual of Determinative Bacteriology, Krieg 347 N.R. & Holt J.G., eds. William & Wilkins: Baltimore, Maryland, USA. pp 486–491. 348

10. HANSON L.A. & ROGERS W.A. (1989). Enzyme Immunoassay Identification of Edwardsiella ictaluri in Decomposing Channel Catfish. J. World 349 Aquacult. Soc., 20, 279–280. 350

11. HAWKE J.P. (1979). A bacterium associated with disease of pond cultured channel catfish, Ictalurus punctatus. J Fish Res Board Can, 36, 351 1508–1512. 352

12. HAWKE J.P., DURBOROW R.M., THUNE R.L. & CAMUS A.C., (1998). ESC-Enteric Septicemia of Catfish. USDA-CSREES, Southern Regional Aquaculture 353 Center. publication 477. 354

13. HAWKE J.P., MCWHORTER A.C., STEIGERWALT A.G. & BRENNER D.J. (1981). Edwardsiella ictaluri sp. nov., the causative agent of enteric septicemia of 355 catfish. Int. J. Syst. Bacteriol., 31, 396–400. 356



14. HUMPHREY J.D., LANCASTER C., GUDKOVS N. & MCDONALD W. (1986). Exotic bacterial pathogens Edwardsiella tarda and Edwardsiella ictaluri from 357 imported ornamental fish Betta splendens and Puntius conchonius, respectively: isolation and quarantine significance. Aust Vet J, 63, 369–358 71. 359

15. IWANOWICZ L.R., GRIFFIN A.R., CARTWRIGHT D.D. & BLAZER V.S. (2006). Mortality and pathology in brown bullheads Amieurus nebulosus associated 360 with a spontaneous Edwardsiella ictaluri outbreak under tank culture conditions. Dis Aquat Org., 70, 219–25. 361

16. KENT M.L. & LYONS J.M. (1982). Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News, 2, ii. 362

17. KLESIUS P. (1993). Rapid enzyme-linked immunosorbent tests for detecting antibodies to Edwardsiella ictaluri in channel catfish, Ictalurus 363 punctatus, using exoantigen. Vet. Immunol. Immunopathol., 36, 359–368. 364

18. KLESIUS P. (1994). Transmission of Edwardsiella ictaluri from from infected, dead to uninfected channel catfish. J. Aquat. Anim. Health, 6, 180–365 182. 366

19. KLESIUS P.H. (1992). Carrier state of channel catfish infected with Edwardsiella ictaluri. J. Aquat. Anim. Health, 4, 227–230. 367

20. KLESIUS P.H. & HORST M.N. (1991). Characterization of a major outer-membrane antigen of Edwardsiella ictaluri. J. Aquat. Anim. Health, 3, 181–368 187. 369

21. KLESIUS P., LOVY J., EVANS J., WASHUTA E. & ARIAS C. (2003). Isolation of Edwardsiella ictaluri from tadpole madtom in a southwestern New Jersey 370 river. J. Aquat. Anim. Health, 15, 295–301. 371

22. KLESIUS P.H. & SHOEMAKER C.A., Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In: 372 Veterinary Vaccines and Diagnostics, Schultz R.D., Editor. 1999, Academic Press: San Diego, California USA, pp. 523–537. 373

23. MITCHELL A.J. & GOODWIN A.E. (1999). Evidence that Enteric Septicemia of Catfish (ESC) was Present in Arkansas by the Late 1960s: New Insights 374 into the Epidemiology of ESC. J. Aquat. Anim. Health, 11, 175–178. 375

24. NAHMS, (2003). Highlights of NAHMS Catfish 2003: Part II. 2003, National Animal Health Monitoring System, Centers of Epidemiology and 376 Animal Health, USDA:APHIS.: Ft. Collins, CO. 377

25. 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 378 Septicemia of Catfish. J. Aquat. Anim. Health, 19, 151–158. 379

26. PLUMB J.A. & QUINLAN E.E. (1986). Survival of Edwardsiella ictaluri in pond water and bottom mud. Progress. Fish Cult., 48, 212–214. 380

27. PLUMB J.A. & SANCHEZ D.J. (1983). Susceptibility of five species of fish to Edwardsiella ictaluri. J. Fish Dis., 6, 261–266. 381

28. PLUMB J.A. & VINITNANTHARAT S. (1989). Biochemical, Biophysical, and Serological Homogeneity of Edwardsiella ictaluri. J. Aquat. Anim. Health, 1, 382 51–56. 383

29. ROGERS W.A. (1981). Serological detection of two species of Edwardsiella infecting catfish. In International symposium on fish biologics: 384 serodiagnostics and vaccines. Dev. Biol. Stand., 49, 169–172. 385

30. 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 386 days post hatch. Aquaculture, 176. 387

31. SHOTTS E.B. & WALTMAN W.D. (1990). A medium for the selective isolation of Edwardsiella ictaluri. J. Wildl. Dis., 26, 214–218. 388

32. TAYLOR P.W. (1992). Fish-eating birds as potential vectors of Edwardsiella ictaluri. J. Aquat. Anim. Health, 4, 240–243. 389

33. 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. 390 Health, 14, 263–272. 391

34. WALTMAN W., SHOTTS E.B. & HSU T.C. (1986). Biochemical characteristics of Edwardsiella ictaluri. Appl. Environ. Microbiol., 51, 101–104. 392

35. WALTMAN W.D., SHOTTS E.B. & BLAZER V.S. (1985). Recovery of Edwardsiella ictaluri from Danio (Danio devario). Aquaculture, 46, 63–66. 393



36. WATERSTRAT P., AINSWORTH J. & CHAPLEY G. (1989). Use of an indirect enzyme-linked immunosorbent assay (ELISA) in the detection of channel 394 catfish, Ictalurus punctatus (Rafinesque), antibodies to Edwardsiella ictaluri. J. Fish Dis., 12, 87–94. 395

37. WATERSTRAT P.R., DORR B., GLAHN J.F. & TOBIN M.E. (1999). Recovery and viability of Edwardsiella ictaluri from great blue herons Ardea herodias 396 fed E. ictaluri-infected channel catfish Ictalurus punctatus fingerlings. J. World Aquacult. Soc., 30, 115–122. 397

38. WISE D., KLESIUS P., SHOEMAKER C. & WOLTERS W. (2000). Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against 398 Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33). J. World Aquacult. Soc., 31, 206–212. 399

39. WISE D.J. & JOHNSON M.R. (1998). Effect of feeding frequency and Romet-medicated feed on survival, antibody response and weight gain of 400 fingerling channel catfish (Ictalurus punctatus) after natural exposure to Edwardsiella icaluri. J. World Aquacult. Soc., 29, 170–176. 401

40. WISE D.J. & JOHNSON M.R. (1998). Effect of feeding frequency and Romet-medicated feed on survival, antibody response, and weight gain of 402 fingerling channel catfish Ictalurus punctatus after natural exposure to Edwardsiella ictaluri. J. World Aquacult. Soc., 29, 169–175. 403

41. WOLTERS W.R., WISE D.J. & KLESIUS P.H. (1996). Survival and antibody response of channel catfish, blue catfish and channel catfish female × blue 404 catfish male hybrids after exposure to Edwardsiella ictaluri. J. Aquat. Anim. Health, 8, 249–254. 405

42. YEH H.Y., SHOEMAKER C.A. & KLESIUS P.H. (2005). Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel 406 catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. J Microbiol Methods, 63, 36–44. 407

43. ZHANG Y. & ARIAS C.R. (2007). Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of 408 Edwardsiella ictaluri. Syst. Appl. Microbiol., 30, 93–101. 409

* 410 * * 411

NB: There is an OIE Reference Laboratory for Enteric septicaemia of catfish (Edwardsiella ictaluri) (see Table at the end of this Aquatic Manual or 412 consult the OIE Web site for the most up-to-date list: www.oie.int). 413

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