Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.1.

EPIZOOTIC HAEMATOPOIETIC NECROSIS

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SUMMARY

Epizootic haematopoietic necrosis (EHN) is a systemic iridovirus (Ranavirus) infection of redfin perch (Perca fluviatilis), rainbow trout (Oncorhynchus mykiss), sheatfish (Silurus glanis) and catfish (Ictalurus melas) (3, 4, 12, 14). The disease is caused by three similar viruses: epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV) and European catfish virus (ECV) (3, 7, 11, 14, 15). The geographical range of EHNV is currently restricted to Australia (10, 24). The ECV and ESV agents have only been detected among fish in Europe (1, 3, 14). Although inducing similar diseases in their respective hosts, EHNV, ECV and ESV are distinct agents distinguished by molecular techniques (10, 13, 17).
 
EHN infection in redfin perch is generally lethal (19). Rainbow trout are relatively resistant and only a small proportion of individuals become infected (20-22). Infections with ESV and ECV can induce high morbidity and mortality in their catfish hosts (3, 14). The disease caused by all three iridoviruses is characterised by mortalities due to necrosis in the liver, spleen, haematopoietic tissue of the kidney and other tissues. Experimental exposure studies have shown that rainbow trout can be infected with ECV and ESV without showing morbidity and mortality (1).
 
The viruses do not induce differentiating neutralising antibodies in mammals or fish (10). The viruses can be identified by immunofluorescence, enzyme-linked immunosorbent assays (ELISAs) or immunoelectron microscopy (7-10, 16). EHNV has several antigens in common with ESV and ECV from Europe and at least one antigen in common with the amphibian iridoviruses from North America (frog virus 3) and Australia (Bohle iridovirus) (2, 7, 8). Antigen-detection tests with polyclonal antibodies against EHNV detect each of these viruses. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and polymerase chain reaction (PCR) can differentiate to varying degrees between the different viruses (10, 13).
 
The epidemiology of EHNV in rainbow trout is incompletely understood (20-22). Due to their extreme susceptibility, it is unlikely that redfin perch are a natural host (18, 19). Infection may recur annually at a rainbow trout production site and this may be due to reinfection from wild redfin perch in the catchment area. A carrier state in naturally infected rainbow trout appears to be very uncommon as neither EHNV antigen nor anti-EHNV antibody are routinely detected in rainbow trout surviving an outbreak (20, 22). However, the disease can occur at a very low frequency in an infected population, and mortalities may not exceed the usual background rate. Thus infected fish could easily be included in batches of healthy translocated fish and there is good evidence that this has occurred. No epidemiological data are available for ESV or ECV.
 
The factors modulating the susceptibility of fish to EHNV, ESV and ECV infection are poorly understood. Clinical outbreaks due to EHNV are associated with poor water quality. In rainbow trout, infection occurs naturally at water temperatures from 11 to 17°C and experimentally from 8 to 21°C (20). Disease in redfin perch does not occur at temperatures below 12°C. The following fish species were found to be susceptible to EHNV following bath exposure: redfin perch, rainbow trout, Macquarie perch (Macquaria australasica), mosquito fish (Gambusia affinis), silver perch (Bidyanus bidyanus) and mountain galaxias (Galaxias olidus) (11). Both juvenile and adult redfin perch may be affected in outbreaks, but juveniles may be more susceptible to the disease. EHNV has now been detected in diseased rainbow trout ranging from hatchery fry to table-sized fish, although mortalities are most often seen in 0+ fish up to 125 mm fork-length.
 
The screening and diagnostic procedures for EHNV, ESV and ECV are based on isolation of virus in cell culture, ELISA, indirect fluorescent antibody tests (IFAT), and electron microscopy (3, 5, 7, 9, 12, 14, 16). Antigen-capture ELISA has a sensitivity of 60-80% depending on the stage of infection when used to test tissues from both rainbow trout and redfin perch (9, 16, 20, 23). Antigen-capture ELISA is the method of choice for confirming the cause of cytopathic effect in cell culture; IFAT and electron microscopy are also useful. IFAT or immunoperoxidase staining may also be used for diagnosis on formalin-fixed tissues. PCR may be used to detect Ranavirus DNA in infected tissues (6). Western blots, SDS-PAGE and PCR sequencing can be used to specifically identify EHNV, ESV and ECV (10, 13).
 

DIAGNOSTIC PROCEDURES

The diagnosis of epizootic haematopoietic necrosis (EHN) is based on direct methods that are either the isolation of virus (epizootic haematopoietic necrosis virus [EHNV], European sheatfish virus [ESV] and European catfish virus [ECV]) in cell culture followed by its immunological identification (conventional approach), or the immunological demonstration of virus antigen in infected fish tissues.
 
Due to insufficient knowledge of the serological responses of fish to virus infections, the detection of fish antibodies to viruses has not thus far been recognised as a valuable diagnostic method for assessing the virus status of fish populations. In the case of EHNV, preliminary investigations using enzyme-linked immunosorbent assay (ELISA) in known infected rainbow trout populations have revealed up to 1% prevalence of seropositive fish in adult age classes. Further investigation of the epidemiology of EHNV in rainbow trout is needed before serological results can be interpreted.
 
Infected fish material suitable for virological examination is:
 

.	Clinically affected fish: whole alevin (body length < or = 4 cm), viscera including kidney (4 cm < or = body length < or = 6 cm) or, for larger size fish, kidney, spleen and liver.
.	Asymptomatic fish (apparently healthy fish): kidney, liver, spleen, heart, milt and ovarian fluid at spawning time.

Sampling procedures: see Chapter I.1. Section B.
 

1.	Standard Screening Method for EHNV, ESV and ECV
	1.1.	Isolation of EHNV, ESV and ECV in cell culture
		Cell line to be used: BF-2, CHSE-214 or EPC
		BF-2 cells are preferred for EHNV and should be grown at 22°C in a temperature-controlled refrigerated incubator to ensure subsequent success in isolation of EHNV. Incubation at higher or fluctuating temperatures may reduce the susceptibility of the cells to infection, regardless of the incubation temperature after inoculation with virus. In the isolation of iridovirus of catfish (ECV), the best results have been obtained using EPC cells. Each laboratory should confirm the susceptibility of cell line(s) in current use and the suitability of cell lines other than those specified here. The most sensitive cell line should always be used. Although EHNV growth is optimum at 22°C, laboratories may need to determine optimum incubation temperatures to suit local conditions, and for temperate regions and viruses other than EHNV (such as ESV and ECV) this may be 15°C. In all cases, appropriate positive and negative controls should be included with the virus isolation tests.
		a)	Inoculation of cell monolayers
			i)	Make two additional tenfold dilutions of the 1/10 organ homogenate supernatants and transfer an appropriate volume of each of the three dilutions on to 24-hour-old cell monolayers. Inoculate at least 2 cm2 of drained cell monolayer with 100 µl of each dilution.
			ii)	Inoculate directly or allow to adsorb for 0.5-1 hour at 22-25°C and, without withdrawing inoculate, add cell culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum (FCS) (1 ml/well for 24-well cell culture plates) and incubate at 22°C using a temperature-controlled refrigerated incubator to ensure successful isolation.
		b)	Monitoring incubation
			i)	Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination at magnification x40-100, for 14 days. The use of a phase-contrast microscope is recommended.
			ii)	Maintain the pH of the cell culture medium at between 7.3 and 7.6 over the whole incubation phase. This can be achieved by addition to the inoculated cell culture medium of sterile bicarbonate buffer (for tightly closed cell culture flasks) or 2 M Tris buffer solution (for cell culture plates) or, preferably, by using HEPES-buffered media (HEPES = N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid).
			iii)	If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of the tested homogenate supernatants, identification procedures have to be undertaken immediately (see Section 1.2. below).
				If a fish health surveillance/control programme is being implemented, provisions have to be taken to suspend the approved health status of the production unit and/or the zone (if it was approved previously) from which the virus positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not EHNV, ESV or ECV.
			iv)	If no CPE develops in the inoculated cultures (despite normal progression of CPE in the virus controls), the inoculated cultures should be subcultured for a further 7 days and, if no CPE has developed by then, tests for ESV and ECV can be declared negative. However, for EHNV testing, the cultures should be subjected to two further blind passages (giving a total of three passes, a primary culture and two subcultures). Should the virus control fail to develop CPE, the process should be repeated with fresh susceptible cells and new batches of organ samples.
		c)	Subcultivation procedures
			i)	Collect aliquots of cell culture medium from all monolayers inoculated with dilutions of each supernatant of organ homogenates.
			ii)	Inoculate cell monolayers as described above (1.1.a.).
			iii)	Incubate and monitor as in 1.1.b.
	1.2.	Identification of EHNV, ESV and ECV
		Note: All antibodies used in the tests described below have been generated against a defined strain of EHNV at the OIE Reference Laboratories in Australia. The antibodies cross-react with ESV, ECV and all other known Ranaviruses. Techniques including but not limited to indirect fluorescent antibody staining, immunoperoxidase staining and antigen-capture ELISA can be used to identify EHNV, ESV, ECV and other Ranaviruses in cell culture.
		a)	Neutralisation test
			EHNV, ESV and ECV cannot be identified by neutralisation as the antisera generated by the immunisation of rabbits have few neutralising antibodies.
		b)	Indirect fluorescent antibody test
			This indirect fluorescent antibody test (IFAT) is to be conducted directly after virus isolation in cell culture.
			i)	Prepare monolayers of susceptible cells in 2 cm2 wells of cell culture plastic plates or on cover-slips in order to reach around 80% confluency, which is usually achieved within 24 hours of incubation at 22°C (seed six cell monolayers per virus isolate to be identified, plus two for positive and two for negative controls). The FCS content of the cell culture medium can be reduced to 2-4%. If numerous virus isolates have to be identified, the use of Terasaki plates is strongly recommended.
			ii)	When the cell monolayers are ready for infection, i.e. on the same day or on the day after seeding, inoculate the virus suspensions to be identified by making tenfold dilution steps directly in the cell culture wells or flasks.
			iii)	Dilute the control virus suspension of EHNV, ESV or ECV in a similar way, in order to obtain a virus titre of about 5000-10,000 plaque-forming units per ml in the cell culture medium.
			iv)	Incubate at 22°C for 24 hours.
			v)	Remove the cell culture medium, rinse once with 0.01 M phosphate buffered saline (PBS), pH 7.2, then three times briefly with cold fixative. This fixative will be acetone (stored at -20°C) for cover-slips or a mixture of acetone 30%/ethanol 70% (v/v) for plastic, also stored at -20°C.
			vi)	Let the fixative act for 15 minutes. A volume of 0.5 ml is adequate for 2 cm2 of cell monolayer.
			vii)	Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or freeze at -20°C.
			viii)	Prepare a solution of purified polyclonal antibody or serum to EHNV (also provided by OIE Reference Laboratories) in 0.01 M PBS, pH 7.2, containing 2% skim milk, at the appropriate dilution (which has been established previously or is given by the reagent supplier).
			ix)	Rehydrate the dried cell monolayers by rinsing four times with PBS and remove this buffer completely after the last rinsing.
			x)	Treat the cell monolayers with the antibody solution for 1 hour at 37°C in a humid chamber. The volume of solution to be used is 0.25 ml/2 cm2 well.
			xi)	Rinse four times with PBS as above.
			xii)	Incubate the cell monolayers in appropriately diluted biotinylated anti-rabbit antibody (in 2% skim milk in PBS) at 37°C for 1 hour.
			xiii)	Rinse four times with PBS as above.
			xiv)	Treat the cell monolayers for 1 hour at 37°C with a solution of FITC-streptavidin conjugate (FITC = fluorescein isothiocyanate).
			xv)	Rinse four times with PBS.
			xvi)	Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using glycerol saline at pH 8.5 prior to microscopic observation.
			xvii)	Examine under incident UV light using a microscope with x10 eye pieces and x20-40 objective lens having numerical aperture >0.65 and >1.3, respectively. Positive and negative controls must be found to give the expected results prior to any other observation. Positive results are indicated by a granular fluorescence within the cytoplasm, fluorescent inclusion bodies are also evident. An overall fluorescence indicates nonspecific reactivity.
		c)	Enzyme-linked immunosorbent assay
			i)	Purify rabbit anti-EHNV immunoglobulins by affinity chromatography with Protein A. Coat the wells of microplates designed for ELISAs with 100 µl of an appropriate dilution of purified rabbit immunoglobulins in an alkaline coating buffer, pH 9.6, designed for ELISA, such as borate or carbonate buffer.
			ii)	Incubate overnight at 4°C.
			iii)	Rinse four times with 0.01 M PBS containing 0.05% Tween 20 (PBST).
			iv)	Block with skim milk (5% in PBST) or other blocking solution for 30 minutes at 22°C.
			v)	Rinse four times with PBST.
			vi)	Dispense 100 µl/well of the cell culture fluid containing the virus to be identified, and of EHNV control virus, and allow to react with the coated antibody for 60-90 minutes at 22°C.
			vii)	Rinse four times with PBST. Collect waste fluids and autoclave. (Note: all waste materials should be treated this way.)
			viii)	Add to the wells 100 µl of an appropriate dilution of a second antibody, usually sheep antibodies to EHNV (provided by OIE Reference Laboratories). These may or may not be conjugated to biotin. Allow to react for 60-90 minutes at 22°C.
			ix)	Rinse four times with PBST.
			x)	Add to the wells 100 µl of appropriate dilutions of either streptavidin-conjugated horseradish peroxidase, or a commercial anti-sheep immunoglobulin conjugated to horseradish peroxidase, as appropriate. Allow to react for 60-90 minutes at 22°C.
			xi)	Rinse four times with PBST.
			xii)	Add the substrate (H2O2) and chromogen (O-phenylenediamide or other approved chromogen). Stop the course of the test when positive controls react, and read the results.
			Note: Dilutions of second antibody and enzyme conjugate should be prepared in PBST to which 0.1% (w/v) gelatin, ovalbumin or other suitable blocking agent is added.
2.	Diagnostic Methods for EHNV, ESV and ECV
	2.1.	Virus isolation with subsequent identification
		As in Section 1.1. and 1.2.
	2.2.	Indirect fluorescent antibody test for fish tissues
		i)	Bleed the fish thoroughly.
		ii)	Make kidney imprints on cleaned glass slides or at the bottom of the wells of a plastic cell culture plate.
		iii)	Store the kidney pieces (as indicated in Chapter I.1. Section B.3.1.), together with the other organs required for virus isolation in case this becomes necessary later.
		iv)	Allow the imprint to air-dry for 20 minutes.
		v)	Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.b. steps v-vii.
		vi)	Rehydrate the above preparations (see Section 1.2.b. step ix) and block with 5% skim milk or 1% bovine serum albumin, in PBS for 30 minutes at 37°C.
		vii)	Rinse four times with PBS.
		viii)	Treat the imprints with the solution of antibody to EHNV and rinse as indicated in Section 1.2.b.
		ix)	Block and rinse as previously in steps vi and vii.
		x)	Treat the imprints for 1 hour at 37°C with a solution of FITC-streptavidin conjugate as in Section 1.2.b.
		xi)	Rinse four times with PBS.
		xii)	Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using glycerol saline at pH 8.5 prior to microscopic observation.
		xiii)	Examine under incident UV light. Positive and negative controls must be found to give the expected results prior to any other observation. Positive results are indicated by a granular fluorescence within the cytoplasm, fluorescent inclusion bodies are also evident. An overall fluorescence indicates nonspecific reactivity. If the immunofluorescence test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as in Section 1.1.
	2.3.	Enzyme-linked immunosorbent assay for fish tissues
		a)	Microplate processing
			As described in Section 1.2.c. of this chapter up to step iv (inclusive).
		b)	Sampling procedures
			EHNV, and presumably ESV and ECV are highly resistant viruses and withstand freezing in fish carcasses for prolonged periods. Fish may be frozen at the farm and dissected later in the laboratory.
			In order to detect EHNV in rainbow trout only dead or moribund fish should be examined as it is almost impossible to find EHNV among healthy in-contact fish, even during an outbreak of EHN.
			See the following sections in Chapter I.1:
			B.1. for the selection of fish specimens
			B.2. for the selection of materials sampled.
		c)	Processing of organ samples
			See the following sections in Chapter I.1.:
			B.3.1. for transportation, note that organ samples can be frozen prior to transport to the laboratory as Ranaviruses (e.g. EHNV) are resistant viruses.
			B.3.2. for virus extraction and obtaining of organ homogenates.
		d)	The enzyme-linked immunosorbent assay procedure
			i)	Set aside an aliquot of 1/4 of each homogenate and store at -20°C or -80°C in case virus isolation in cell culture is required.
			ii)	Treat the remaining part of the homogenate with 2% Triton X-100 (v/v) and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
			iii)	Complete the other steps of the procedure described in Section 1.2.c.
			iv)	The test has been validated only for EHNV in tissues from redfin perch and rainbow trout. Positive results in ELISA from tissues of species other than redfin perch and rainbow trout should be confirmed by virus culture, electron microscopy of fish tissues and polymerase chain reaction (PCR).
	2.4.	Polymerase chain reaction amplification for fish tissues
		a)	Processing of organ samples
			See the following sections in Chapter I.1.:
			B.3.1. for transportation, note that organ samples can be frozen prior to transport to the laboratory as Ranaviruses (e.g. EHNV) are resistant viruses.
			B.3.2. for virus extraction and obtaining organ homogenates.
		b)	Polymerase chain reaction amplification and sequencing
			Ranaviruses such as EHNV, ESV and ECV have a large double-stranded DNA genome of approximately 125 kb. Two primers, a reverse primer (5'-AAA-GAC-CCG-TTT-TGC-AGC-AGC-AAA-C-3') and a forward primer (5'-CGC-AGT-CAA-GGC-CTT-GAT-GT-3'), are used for amplification of the target capsid sequence (580 base pairs [bp]) of EHNV DNA by PCR. This PCR procedure can be used for detection of iridoviruses from redfin perch, rainbow trout, sheatfish, catfish, guppy fish (Poecilia reticulata), doctor fish (Labroides dimidatus) and a range of amphibian Ranaviruses. Fish samples are prepared as per Gould et al. (6). Nucleic acid (1 µl) is added to Taq polymerase buffer containing 0.1 µM of each primer, 2.5 U Taq polymerase (Promega) and 2.5 mM MgCl2. The mixture is incubated in an automatic thermal cycler programmed for 35 cycles at 95°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds, and finally held at 72°C for 15 minutes. Amplified DNA (580 bp) is analysed by agarose gel electrophoresis, excised and sequenced using the Cyclone Sequencing Kit (Bresatec, Australia). Each viral species is identified by its unique DNA sequence.
			Samples should be submitted to the OIE reference laboratory for specific identification.
		c)	Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
			EHNV, ESV and ECV can be differentiated from other iridoviruses by analyses of viral proteins in reducing polyacrylamide gels (10%). The major coat protein of EHNV is always slightly larger (approximately 51 kDa) compared with other iridoviruses such as ESV and ECV (approximately 49 kDa).

REFERENCES

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