Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.12.

ENTERIC SEPTICAEMIA OF CATFISH
(Edwardsiella ictaluri)

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SUMMARY

Enteric septicaemia of catfish (ESC) is caused by the bacterium Edwardsiella ictaluri, which belongs to the Enterobacteriaceae family (11). ESC is one of the most important infectious disease problems in the commercial catfish industry in the United States of America (USA). Most reported cases of disease caused by E. ictaluri are in channel catfish (Ictalurus punctatus), but the bacterium has been isolated from related North American catfish including blue catfish (I. furcatus), white catfish (Ameiurus catus), and brown bullhead (A. nebulosus) (11). ESC has also been reported from Clarias batrachus in Thailand (12) and from several ornamental species (13, 37). The susceptibility of other species including salmonids has been shown experimentally (4). Edwardsiella ictaluri should not be confused with E. tarda, another member of the same genus that is frequently found in aquatic animals and is responsible for opportunistic infections in fish and mammals, including humans.
 
Several studies have shown that E. ictaluri is a very biochemically and antigenically homogeneous species (5, 21, 33, 38).
 
Acute outbreaks of ESC occur within a limited temperature range, from 18 to 28°C. This critical temperature window makes spring and autumn the most common periods for outbreaks in regions where channel catfish are normally cultured. However, low-level mortality due to ESC can occur in carrier populations outside of this temperature range. Other environmental factors (poor water quality, high stocking density and other stressors) predispose the host to ESC. Edwardsiella ictaluri is considered to be a true obligate pathogen.
 
Two clinical forms of ESC occur in channel catfish, a chronic encephalitis and an acute septicaemia (20, 22, 30). In the chronic form the bacterium infects the olfactory sacs, and migrates along the olfactory nerves to the brain, generating granulomatous inflammation. This meningo-encephalitis causes abnormal behaviour, with alternating listlessness and chaotic swimming. In late stages of this disease, swelling develops on the dorsum of the head as the inflammatory process erodes the connective tissue in this region. This swelling ulcerates exposing the brain. This has lead to the term 'hole in the head disease', used in the industry. In the acute form of ESC the bacterium is thought to infect through the intestinal mucosa (3), and then to establish a bacteraemia. The affected fish display petechial haemorrhages around the mouth, on the throat, the abdomen and at the base of the fins. Multifocal distinct 2 mm diameter raised haemorrhagic cutaneous lesions that progress to depigmented ulcers also occur. Anaemia, moderate gill inflammation and exophthalmia are common signs. Internally, haemorrhages and necrotic foci are scattered in the liver and other internal organs. Haemorrhagic enteritis, systemic oedema, accumulation of ascitic fluid in the body cavity and enlargement of the spleen are nonspecific signs. Histological examination reveals a systemic infection of all organs and skeletal muscles, with the most severe changes being diffuse interstitial necrosis of the anterior and posterior kidney. Focal necrosis in the liver and spleen are also generally seen.
 
Fish from a population that has recovered from the disease are considered to be carriers. These fish will have protective immunity and may have high levels of E.-ictaluri-specific antibodies. Occasional losses due to recurrent ESC will occur in these populations, especially after a stress is induced. Edwardsiella ictaluri has been detected in the kidney of such fish well over 4 months after exposure (2, 14), suggesting that carrier fish act as the natural reservoir for the organism. It is believed that shedding with faeces is the main means of dissemination into the environment. The pathogen persistence and the common practice of continual partial harvest and stocking within a production pond have contributed to the success of this pathogen and the prevalence of ESC in the industry. Moreover, the agent can survive in pond sediments for an extended period of time (27), and this may be another important factor in disease recurrence in given areas. Researchers have found the bacterium in the gut of fish-eating birds by performing fluorescent antibody tests on ingesta, but generally no E. Ictaluri could be cultured indicating that the bacteria were not viable (34, 40). This suggests that birds are not an important means of disseminating this pathogen.
 
ESC may be controlled through chemotherapy and/or prophylactic measures. The most common antimicrobial treatments are oral application of potentiated sulfonamide sulfadimethoxine ormethoprim or oxytetracycline, but plasmid-mediated resistance to these antibiotics does occur (6). Many producers are now focusing on alternative methods to reduce losses. This relies on management to reduce stress in fish, the cessation of feeding when ESC-induced losses are detected (41) and on vaccination.
 

DIAGNOSTIC PROCEDURES

1.	Identification of the Agent
	The identification of Edwardsiella ictaluri is based on the isolation of the causative agent and characterisation by biochemical tests. Edwardsiella ictaluri can easily be differentiated from E. tarda by its inability to produce indole and hydrogen sulfide (E. tarda produces both). Additionally, the two species do not cross-react serologically.
	1.1.	Isolation and bacteriological identification
		a)	Sampling and isolation of the agent
			Bacteriological samples from freshly dead or moribund fish should be taken aseptically from the brain and kidney tissue. The samples should be streaked for isolation on to blood agar plates, brain-heart infusion (BHI) agar or nutrient 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. A selective medium (EIM) has been developed (31) that may prove useful when samples are taken from heavily contaminated environments, but it is not essential under normal conditions. For detecting a carrier state in a healthy population, kidney tissue has been homogenised in 0.5% triton-X 100, filtered on to 0.45 µm nitrocellulose and grown on EIM agar medium (8) or homogenised kidney tissue was cultured overnight in liquid EIM and 100 µl of this sample was plated on to BHI agar plates (14). 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). Optimal temperature for incubation is 28-30°C.
			For routine sampling of fish populations, see Chapter I.1. Section B.1.
		b)	Characteristics
			Following incubation for 36-48 hours, E. ictaluri appears as smooth, circular (1-2 mm diameter), slightly convex nonpigmented colonies with entire edges. It is a Gram-negative rod, measuring 0.75-2.5 µm, and is weakly motile by means of a peritrichous flagellation and is cytochrome oxidase negative. This bacterium grows slowly or not at all at 37°C.
			After isolation, the bacterium should be identified by biochemical and serological characteristics (10, 11). Table 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 Bergey's Manual of Determinative Bacteriology (9). The optimal growth temperature 28-30°C should be used for evaluating biochemical characteristics. Edwardsiella ictaluri is biochemically less active than the other Edwardsiella species, but it appears to be homogeneous (38). 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 sulfide (E. tarda produces both, while E. ictaluri does not). Also E. tarda, Yersinia ruckeri, Hafnia alvei and E. hoshinae grow well at 37°C whereas E. ictaluri does not. Edwardsiella ictaluri degrades chondroitin sulfate and this may be an important virulence factor (7, 32, 38).

Table 1. Differentiation of the species and biogroups of the genus Edwardsiella
and other Enterobacteriaceae found in fish*

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 sulfide production in triple sugar iron (agar)	-	-	+	-	-	-
Motility	-	+	+	+	+	-**
Citrate (Christensen's)	+	-	+	+	(+)	-

* Bergey's Manual of Determinative Bacteriology.

** Weakly motile at 28°C Hawke, 1979.

*** Citrate may be negative on rapid test strips (API 20E). Assay should be run at 25°C.

	1.2.	Detection of the bacterial antigen by serological methods
		In addition to well defined biochemical characteristics, there is antigenic homogeneity of the species and serology can easily differentiate E. ictaluri from other Enterobacteriacae (5, 26, 29). Slide agglutination with specific antisera against E. ictaluri, fluorescent antibody techniques (FATs), enzyme-linked immunostaining and enzyme-linked immunosorbent assays (ELISAs) have been used to provide confirmatory diagnosis. Characterised specific monoclonal antibodies (MAbs) are generally used in these assays, but polyclonal antisera obtained using formalin-killed bacterins to immunise rabbits according to classical standards may also be used (1, 16, 28).
		a)	Agglutination test
			Isolated colonies are gently mixed into a drop of sterile saline on a clean glass slide, and a drop of antiserum is added. The agglutination of the bacteria can be evaluated by comparison with a similar suspension in normal rabbit serum (control). The appropriate dilution of reacting serum will have been previously determined by testing a control E. ictaluri strain in twofold dilutions of this serum.
		b)	Specific immunofluorescence
			The indirect fluorescent antibody test (IFAT) may be employed on bacterial smears, or smears from infected organs, for rapid confirmation of a clinical diagnosis (28). Smears are air-dried and heated for 2 minutes at 60°C before being flooded and incubated for 5 minutes with specific rabbit antibody. They are washed in phosphate buffered saline (PBS), pH 7.2, flooded for 5 minutes with the rabbit-Ig-specific secondary antibody conjugated with fluorescein isothiocyanate (FITC). After rinsing, the slides are mounted with cover-slips using phosphate-buffered mounting medium and observed microscopically for bright green fluorescence under blue epi-illumination. Use undiluted cell culture supernate when using MAbs that are produced from cell culture in the first step, and a commercially available FITC-conjugated mouse-Ig-specific secondary antibody at the suggested working concentration (1). Smears of bacterial suspensions must be very thin. Positive and negative controls (such as E. tarda) should be stained on separate slides.
		c)	Enzyme-linked immunostaining
			An enzyme-linked immunostaining technique to directly identify E. ictaluri in tissue smears from infected fish has been described (28). Smears are prepared as for IFAT - the first steps are similar, but the second incubation step uses heterospecific immunoglobulin against rabbit antiserum, 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 under normal trans-illumination. 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.
	1.3.	Nucleic-acid-based diagnosis
		Assays based on PCR amplification of structural RNA sequences from bacterial colonies and direct sequencing the products are being adapted by several diagnostic bacteriology laboratories and some of these assays are commercially available (MicroSeq, Applied Biosystems). Species confirmation can be done by amplifying and sequencing the 16S portion of the ribosomal RNA operon and comparing the sequence with GenBank accession AF310622.
2.	Standard Screening Methods for ESC
	2.1.	Detection of the agent
		The techniques are the same as described in Section 1. Unless performed on bacteria isolated and purified first, any positive result obtained using a detection method will have to be confirmed, preferably by plating and isolation of the bacteria.
	2.2.	Serological tests
		Although antibody detection tests are rarely used for routine diagnostic purposes and are not yet approved as official procedures, they could be of value for the mass screening of large numbers of fish that is required with the development of health control policies. The specificity of the bacterium and the demonstration of circulating antibodies against E. ictaluri in the serum of fish recovering from the disease support this hypothesis.
		a)	Microagglutination test
			Direct microagglutination, performed in 96-well round-bottom microplates as described for other bacterial pathogens (19) can provide quick quantitative data at minimal cost when high sensitivity is not required. All that is needed is a formalin-killed bacterial suspension prepared according to the usual techniques (i.e. formalin [0.35% (v/v)] overnight) and adjusted to an optical density of 0.8 at 525 nm (about 5 x 108 colony-forming units/ml). Twofold dilutions of the sera are made in isotonic saline, so that the final volume is 25 µl/well. Antigen is added (75 µl/well) and the plates are incubated for 2 hours at 37°C and overnight at 4°C before being read. Controls include a rabbit standard serum of previously established titre and antigen incubated in saline.
		b)	Passive haemagglutination
			This technique has been described using E. ictaluri lipopolysaccharide (1 mg/ml in PBS, pH 7.2) passively coated on human Group O red blood cells at 4% (29). Tested sera must be heated for 30 minutes at 45°C to inactivate complement, and absorbed with Group O human red blood cells to remove nonspecific agglutinins before dilutions are done in buffered saline. Coated blood cells adjusted to 1% concentration are used. Incubation is for 6 hours at room temperature and overnight at 4°C. Controls include coated and uncoated red cells in buffer and coated cells in serum.
		c)	Indirect enzyme-linked immunosorbent assay
			ELISA methods have been developed to detect catfish antibodies to E. ictaluri and are the most widely used assays in research (15, 39). The first method uses whole heat-killed bacteria at 4 x 108 cells/ml PBS, 50 µl/well to coat poly-L-lysine-treated ELISA plates (39). The plates are washed with PBS and then blocked by the addition of 100 µl 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 can be used), 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 is similar but uses a soluble major antigen (16) obtained by sonicating the bacteria, or merely by dialysing the supernatants of 24-hour broth cultures then concentrating the sample to 25 µg/ml of protein content. Approximately 100 µl 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.

REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

The possibility of protecting channel catfish populations by vaccination has been reviewed by Plumb in 1988 (25) and Thune et al. in 1997 (36). Edwardsiella ictaluri is known to induce an antibody response after natural disease or immunisation. A commercial vaccine consisting of an inactivated bacterin was provisionally licensed in the USA against ESC and marketed in 1991. The effectiveness of this vaccine was low and it is no longer marketed. Age-related factors and the induction of a cellular immune response could be of critical importance in inducing strong anti-E.-ictaluri defences. Recent studies by Petrie-Hanson and Ainsworth show that catfish fry under 3 weeks of age are immunologically unresponsive to E. ictaluri (23), and this is thought to be due to poorly developed lymphoid organs in the young fish (24). New attenuated agents show promise in inducing more effective immune responses (7, 17, 18, 35). One such mutant is commercially available (17) and when administered at a high dose to 12-day-old fry, persists long enough to induce protective immunity (42). Additional research in developing genetically resistant strains of catfish show promise (43) and may help to reduce losses caused by this important disease.
 

REFERENCES

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