Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.9.

INFECTIOUS SALMON ANAEMIA

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SUMMARY

Infectious salmon anaemia (ISA) is an infectious disease of Atlantic salmon (Salmo salar) (28) caused by an orthomyxo-like virus (7, 15). Initially reported in Norway in the mid-1980s, ISA has to date been reported to occur in Canada (New Brunswick and Nova Scotia), the United Kingdom (Scotland and the Shetland Islands), the Faroe Islands and USA (Maine) (1, 12, 21, 24) and the causal virus has been isolated from samples from Coho salmon from Chile (9) and from rainbow trout in Ireland. For more detailed information on the condition see refs 3, 5, 8, 26 and 27. Atlantic salmon is the only susceptible fish species known to develop the disease, but, following experimental infection, the ISA virus (ISAV) may survive and replicate in sea trout and brown trout, (Salmo trutta) and in rainbow trout (Oncorhynchus mykiss) (16, 17, 19), which thus may act as carriers of the virus for an unknown period of time.
 
Clinically, the infection appears as a systemic and lethal condition that is characterised by anaemia, ascites, congestion and enlargement of the liver (dark in colour) and spleen, as well as peritoneal petechiae. Haemorrhages in the eyes may also be seen. Hepatocellular degeneration and necrosis, tubular necrosis and haemorrhages in the kidneys are consistent histopathological findings in typical outbreaks. The infection is mainly observed in fish held in sea water or in fish exposed to sea water, but indications of disease outbreaks in fish held in fresh water has also been reported (18). The infection spreads slowly and the virus is considered to be of relatively low virulence. However, mortality may ensue in some outbreaks of the disease.
 
The infective agent, ISAV, is an enveloped virus, 100-130 nm in diameter, with morphological, biochemical and genomic properties consistent with those of the Orthomyxoviridae (7, 11, 15). The genome of ISAVconsists of eight single-stranded RNA segments with negative polarity (15).
 
The reservoirs of ISAV are not known, but spread of the disease has occurred as a result of the purchase of subclinically infected Atlantic salmon smolts, from farm to farm, and from fish slaughterhouses or industries where organic material (especially blood and processing water) from ISA-infected fish has been discharged directly into sea water without further treatment. Transfer of ISA by salmon lice (Lepeophtheirus salmonis) has been demonstrated under experimental conditions (20). Evidence for the presence of ISAV in wild salmonids is increasing (22).
 
Few environmental factors have been identified that can be directly linked to outbreaks of the disease. In a latent carrier population, various stress factors, such as treatment against salmon lice, cestodes or other infectious diseases may be followed by disease outbreaks some 2-3 weeks later.
 
Screening of potential carriers for ISAV have hitherto been made by polymerase chain reaction (PCR)-based technology, or by virus isolation in cell culture followed by confirmatory testing by immunological methods or by PCR (4, 22, 23). The current diagnostic procedures for ISA are based on clinical, pathological, histopathological and haematological changes, detection of ISAV by means of virus isolation or detection of the virus in tissues by means of an indirect immunofluorescent antibody test. PCR may be a useful diagnostic tool in cases with inconclusive disease signs. The incidence of ISA may be greatly reduced by implementation of general legislatory measures regarding the movement of fish, mandatory health control, and slaughterhouse and transport regulations, as well as specific measures including restrictions on affected, suspected and neighbouring farms, epizootiological studies, enforced sanitary slaughtering, generation segregation ('all in/all out'), and disinfection of offal and wastewater, etc., from fish slaughterhouses and fish processing plants.
 

DIAGNOSTIC PROCEDURES

The diagnosis of infectious salmon anaemia (ISA) was initally based on clinical and patholgoical findings, but direct methods for detection of virus have recently been established. These include isolation of virus in cell culture followed by immunological identification (2, 6), immunological demonstration of ISA virus (ISAV) antigen in tissues (6) and polymerase chain reaction (PCR) techniques (4, 13, 15).
 
The first isolation of ISAV was performed in the salmonid cell line SHK-1 (3), but other salmonid cell lines such as CHSE-214, AS and ASK may also support virus propagation (4, 10, 25, 29). A monoclonal antibody (MAb) (3H6F8) against ISAV that reacts with the viral haemagglutinin has been produced for confirming a diagnosis of ISA (6, 7).
 
Sampling procedures: See Chapter I.1. Section B.
 

1.	Standard Screening Method for ISAV
	Detection of latent carriers of ISAV has been demonstrated by PCR, but detection of carriers by virus isolation has also been reported. It is generally assumed that PCR is a more sensitive technique than isolation in cell culture for detection of carriers or subclinical cases (14), but relatively few validation studies of the different methods have been performed. A recently reported reverse-transcription PCR (RT-PCR) procedure has proved to be at least ten times more sensitive than isolation in cell culture (13). The standard procedures for virus isolation and PCR are described in Sections 2.2.1. and 2.3.2., respectively.
2.	Diagnostic Methods for ISA
	Fish affected with ISA may show a range of signs from none to severe, depending on factors such as infective dose, temperature, age, immune status, virus strain and pathogenicity, seasonal variation, etc. A pathological diagnosis can be made from identification of typical pathological changes. None of the described lesions is pathognomonic to ISA. The following changes have been described to be consistent with ISA: exophthalmus, distended abdomen (ascites), anaemic gills, sometimes skin haemorrhages and scale oedema, pale heart, dark liver, congested spleen and kidneys, and petechia in perivisceral fat and internal organs. Liver pathology and anaemia were the central and classical diagnostic criteria.
	2.1.	Diagnosis based on clinical signs and post-mortem findings
		The following requirements should be fulfilled when making a pathological diagnosis: macroscopic findings, histological findings and haematological findings consistent with the descriptions given for ISA as detailed below.
		.	2.1.1. Macroscopic findings

Dark livers	Not necessarily present in all individuals, but there must be some fish with dark livers. Alternatively, livers may become yellow with haemorrhagic spots or may be pale
Pale gills and heart	Result of anaemia
Ascites	Always present, early sign
Enlarged spleen	Always present, early sign
Visceral fat petechiae	Usually present
Dark foregut	Sometimes present

 

		All of these findings are typical of ISA, although some variation will occur in their severity. Confidence in the diagnosis increases with the increased observation of the occurrence of these signs. Dark livers are an essential sign because this finding is most specific to ISA. (Dark livers are also seen in cardiomyopathic syndrome [CMS], which is distinguished from ISA by typical gross and histological heart lesions.) Furthermore, pale livers with nonhaemorrhagic necroses have been observed both in ISA and 'winter ulcers'.
		.	2.1.2. Histological findings
			The following are typical histological findings:
			.	Multifocal haemorrhagic hepatic necroses that may become confluent to give the changes a 'zonal' appearance, leaving areas around large veins intact (late stage of disease development);
			.	Focal congestion and dilatation of hepatic sinusoids, sometimes with distribution as described for the necroses (early stage). Rupture of sinusoidal endothelium with the presence of erythrocytes within the space of Disse (early sign);
			.	Kidney lesions are characterised by either or both acute tubular necrosis with eosinophilic casting and moderate sinusoidal congestion and interstitial haemorrhages;
			.	In the spleen, moderate to severe sinusoidal congestion and erythrophagia may be seen.
			Findings described as supportive are present in the early stages of disease development at haematocrit values of 15-25. The ISA-typical liver changes are observed at haematocrit values below 10. Comparable, although not identical, liver changes (usually without rupture of endothelium) may be seen in advanced stages of CMS.
			ISA is diagnosed most often in spring. Mortality may be low for months following the introduction of ISA, until an 'outbreak' occurs. Necropsy and examination of a large number of diseased or dead fish increase the probability of detecting ISA.
			Always suspect ISA when extremely dark livers are observed.
		.	2.1.3. Haematological findings
			The following are typical haematological findings:
			.	Haematocrit <10;
			.	Blood smears with degenerate and vacuolised erythrocytes and the presence of erythroblasts with irregular nuclear shape. A reduction in the proportion of leukocytes relative to erythrocytes, with the largest reduction being among lymphocytes and thrombocytes. A reduction in several plasma parameters, except for some enzymes indicating liver damage, and for electrolytes (fish in sea water).
			.	Other evidence of anaemia e.g. pale gills and heart.
			A haematocrit value below 10 is not a unique finding for ISA. Fish with ulcerations and fish with erythrocytic inclusion body syndrome, may regularly demonstrate haematocrit values this low.
	2.2.	Isolation of virus in cell culture
		Infected material suitable for virological examination is: spleen, heart, liver, and preferably kidney from clinically infected fish.
		.	2.2.1. Isolation of ISA virus in cell culture
			Cell line to be used: SHK-1 or other susceptible cell line
			SHK-1 or other susceptible cell lines, such as TO, ASK and CHSE-214, may be used, but strain variability and the ability to replicate in different cell lines should be taken into consideration. The SHK-1 cells seem to support growth of the hitherto known virus isolates (14).
			The SHK-1 cells are grown at 20°C in Leibovitz's L-15 cell culture medium supplemented with fetal bovine serum (5%), L-glutamine (4 mM), gentamicin (50 µg/ml) and 2-mercaptoethanol (40 µM) (may be omitted).
			For virus isolation, cells grown in 25 cm2 tissue culture flasks or multi-well cell culture plates, which may be sealed with parafilm to stabilise the pH of the medium, may be used. Cells grown in 24-well plates may not grow very well into monolayers, but this trait may vary between laboratories and according to the type of cell culture plates used. Serially diluted ISAV-positive controls should be inoculated in parallel with the tissue samples as a test for cell susceptibility to ISAV.
		a)	Inoculation of cell monolayers
			Inoculate actively growing monolayers (1-3-day-old cultures) with a small volume of tissue homogenate supernatant (e.g. 1 ml per well in a six-well plate) after removal of the growth medium. Dilute tissue supernatant with L-15 medium without serum to final dilutions of tissue material of 1/50 and/or higher. Allow 3-4 hours' incubation at 15ºC followed by removal of the inoculate and addition of fresh, fully supplemented growth medium. Using this procedure, cytotoxicity is seldom observed even at the lowest dilution of supernatant. Alternatively, a 1/1000 dilution and direct inoculation without medium replacement can be used.
			When samples come from production sites where infectious pancreatic necrosis virus (IPNV) is regarded as ubiquitous, the tissue homogenate supernatant should be incubated (for a minimum of 1 hour at 15ºC) with a pool of antisera to the indigenous serotypes of IPNV prior to inoculation.
		b)	Monitoring incubation
			Inoculated cell cultures (kept at 15ºC) are examined at regular intervals (at least every 7 days) for the occurrence of a cytopathic effect (CPE). Typical CPE due to ISAV appears as vacuolated cells that subsequently round up and loosen from the growth surface. If a CPE appears that is consistent with that described for ISAV or IPNV, remove an aliquot of the medium for virus identification as described below (Section 2.2.2.). In the case of an IPNV infection, re-inoculate cells with tissue homogenate supernatant that has been incubated with a lower dilution of IPNV antisera. If no CPE has developed after 14 days, subcultivate to fresh cell cultures.
		c)	Subcultivation procedure
			i)	Aliquots of medium (supernatant) from the primary cultures are collected 14 days (or earlier when obvious CPE appears) after inoculation. Supernatants from wells inoculated with different dilutions of identical samples may be pooled.
			ii)	The supernatants are inoculated into fresh cell cultures as described for the primary inoculation: remove growth medium, inoculate monolayers with a small volume of diluted supernatants (1/5 and higher dilutions) for 3-4 hours before addition of fresh medium. Alternatively, add supernatants (final dilutions 1/10 and higher) directly to cell cultures with growth medium.
			iii)	The inoculated cell cultures are incubated for at least 14 days and examined at regular intervals as described for the primary inoculation. At the end of the incubation period, or earlier if obvious CPE appears, medium is collected for virus identification as described below (Section 2.2.2.). Cell cultures with no CPE should always be examined for the presence of ISAV by indirect fluorescent antibody test (IFAT), haemadsorption (7) or by PCR because virus replication may occur without the development of apparent CPE.
		.	2.2.2 Virus identification
		a)	Indirect fluorescent antibody test
			i)	Prepare monlayers of cells in appropriate tissue culture plates (e.g. 96-well or 24-well plates), in slide flasks or on plastic cover-slips dependent on the type of microscope available (inverted microscope equipped with UV light is necessary for monolayers grown on tissue culture plates). The SHK-1 cells grow rather poorly on glass cover-slips. Include the necessary monolayers for negative and positive controls.
			ii)	Inoculate the monolayers with the virus suspensions to be identified in tenfold dilutions, two monolayers for each dilution. Add positive virus control in dilutions known to give good staining reaction.
			iii)	Incubate inoculated cell cultures at 15ºC for 7 days or, if CPE appears, for shorter times.
			iv)	Remove cell culture medium and rinse once with 80% acetone. Add 80% acetone and let the fixative act for 20 minutes at room temperature. Remove the fixative and air dry for 1 hour. The fixed cell cultures may be stored dry for less than a week at 4ºC or at -20ºC for longer storage.
			v)	Prepare a solution of antibody against ISAV (MAb 3H6F8) at an appropriate dilution in phosphate buffered saline (PBS) containing 0.5% skimmed dry milk. Treat the cell monolayers with the antibody solution for 1 hour at room temperature or 30 minutes at 37°C.
			vi)	Rinse twice with PBS/0.05% Tween 20. Keep the monolayers in the dark for 1-2 minutes before removal of the last washing solution.
			vii)	Treat the cell monolayers with fluoescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (or if antibody raised in rabbits is used as the primary antibody, use FITC-conjugated antibody against rabbit immunoglobulin) according to the instructions of the supplier. Dilute the conjugate with PBS to appropriate working dilution and incubate for 1 hour at room temperature or 30 minutes at 37°C in the dark.
			viii)	Rinse once with PBS/0.05% Tween 20 as described above.
			ix)	To stain the nuclei (red colour) add propidiumiodid (100 µg/ml in aqua dest.) and incubate for 1-2 minutes at room temperature in the dark followed by rinsing once with PBS/0.05% Tween 20.
			x)	If the plates cannot be examined immediately, add a solution of 1,4-diazabicyclooctane (DABCO 2.5% in PBS, pH 8.2) or similar reagent as an anti-fade solution.
			xi)	Examine under UV light immediately or store the stained monolayers in the dark at 4ºC for 2-3 days before exmination (some reduction in the fluorescent signal may be observed by storage).
		b)	Polymerase chain reaction
			As an alternative to identification of isolated virus by IFAT, infected cell monolayers may be examined by RT-PCR according to the procedure described in Section 2.3.2.
	2.3.	Direct detection of virus in clinical material
		.	2.3.1. Indirect fluorescent antibody test
			An IFAT using anti-ISAV MAb (3H6F8) on frozen tissue sections of kidney, heart and liver or on kidney imprints has given positive reactions in both experimentally and naturally infected Atlantic salmon. Cases suspected from pathological signs are verified with a positive IFAT.
		a)	Preparations of tissue imprints
			A small piece of the mid-kidney is briefly blotted against absorbant paper to remove excess fluid, and several imprints are made on poly-L-lysine-coated microscope slides. The imprints are air-dried, fixed in chilled 100% acetone for 10 minutes and stored either at 4°C for a few days or at -80°C until use.
		b)	Preparations of cryosections
			Tissue samples from kidney, liver and heart are collected from moribund fish, frozen in isopentane, chilled in liquid nitrogen, and stored at -80°C. Sections are cut on a cryostat, placed on poly-L-lysine-coated slides, fixed in chilled 100% acetone for 10 minutes and stored at -80°C until use.
		c)	Staining procedure
			After blocking with 5% nonfat dry milk in PBS for 30 minutes, the preparations are incubated for 1 hour with diluted (e.g. 1/100) anti-ISAV MAb supernatant, followed by three washes. For the detection of bound antibodies, the preparations are incubated with FITC-conjugated anti-mouse Ig for 1 hour. PBS with 0.1% Tween 20 is used for washing. All incubations are performed at room temperature.
		.	2.3.2. Polymerase chain reaction
			Amplification of a 155 bp fragment of ISAV cDNA is performed using primers derived from sequences of segment 8: 5'-GGC-TAT-CTA-CCA-TGA-ACG-AAT-C-3' (forward primer) and 5'-GCC-AAG-TGT-AAG-TAG-CAC-TCC-3' (reverse primer) (13, 14). The RT reaction and the PCR are performed in two separate steps. This method may be regarded as a basic procedure for detection of ISAV. Improved methods and methods using other sets of primers have recently been reported (14).
		a)	Isolation of RNA
			Total RNA from tissue homogenate or cell culture is extracted using, for example, the phenol-chloroform method. RNA is dissolved in distilled water treated with 0.1% diethyl pyrocarbonate (DEPC) and preheated at 55°C for 10 minutes (2 µg RNA in a total volume of 11 µl).
		b)	RT reaction
			For cDNA synthesis (RT reaction), the following reagents are added giving a final volume of 20 µl (all concentrations are final): 50 mM Tris/HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 1 mM (each) of four deoxynucleoside triphosphates (dNTP), 5 mM dithiothrietol (DTT), 10 mM of the RNase inhibitor ribonucleoside vanadyl complex (RVC), 4.5 µM random hexamers primers and 200 U of murine leukaemia virus (M-MLV) reverse transcriptase. The mixture is incubated at 37°C for 1 hour.
		c)	PCR
			Following centrifugation, 5 µl of the mixture from the RT-reaction is transferred to 45 µl PCR mix consisting of (final concentrations in a total volume of 50 µl): 20 mM Tris/HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.02% polyoxyethylene ether, 0.2 mM of each dNTP, 25 pmol of each forward and reverse ISAV-specific primers and 1.25 U Taq DNA polymerase. This mixture is incubated in an automatic thermal cycler programmed for one cycle at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and is held finally at 72°C for 7 minutes. Amplified DNA (155 bp) is analysed by agarose gel electrophoresis or by spectrophotometer if real-time PCR is used. The optimal incubation times at the different temperatures must be adjusted to the thermal cycler in use.
			An optimised version of this method has recently been described where the RT reaction and the PCR reaction are carried out in one tube (13).
			Alternatively, the RT-PCR method described by Devold et al. (4) may be used. In this method the primers are also derived from genomic segment 8 and the size of the amplified product is estimated to be 211 bp when the primer set FA-3/RA-3 is used.
3.	Criteria For Suspicion and Verification of ISA
	3.1.	General principles for the diagnosis of ISA
		Reasonable grounds for fish to be suspected of being infected with ISAV are outlined in Section 3.2. below. The Competent Authorities shall ensure that, following the suspicion of fish on a farm being infected with ISAV, an official investigation to confirm or rule out the presence of the disease be carried out as quickly as possible applying inspection and clinical examination, as well as the collection and selection of samples and the methods for laboratory examination as described in Section 2.
	3.2.	Suspicion of infection with ISA
		The presence of ISA should be suspected if the criteria listed in points a) or b) or c) or d) or e) below are met.
		a)	The presence of post-mortem findings consistent with ISA, as detailed in Section 2.1., with or without clinical signs.
		b)	Isolation and identification of ISAV in cell culture, as described in Section 2.2., from a single sample from any fish on the farm.
		c)	Reasonable evidence of the presence of ISAV from two independent laboratory methods, such as IFAT (see Section 2.3.1.) and RT-PCR (see Section 2.3.2.).
		d)	Transfer of live fish into a farm where there are reasonable grounds to suspect that ISA was present at the time of the fish transfer.
		e)	Where an investigation reveals other substantial epidemiological links to farms suspected or confirmed to be infected with ISA.
	3.3.	Excluding suspicion of infection with ISA
		Suspicion of ISA can be officially excluded if continual investigations involving at least 1 clinical inspection per month for a period of 6 months reveals no further significant evidence of the presence of ISA.
	3.4.	Confirmation of infection with ISA
		The presence of ISA is officially confirmed if the criteria in Sections 3.4.1. or 3.4.2. or 3.4.3. are met:
		.	3.4.1. Clinical signs and post-mortem findings of ISA in accordance with Sections 2.1.1., 2.1.2. and 2.1.3. are detected, and ISAV is detected by one or more of the following methods:
		a)	Isolation and identification of ISAV in cell culture as described in Section 2.2.
		b)	Detection of ISAV in tissues or tissue preparations by means of specific antibodies against ISAV (e.g. IFAT on kidney imprints) as described in Section 2.3.1.
		c)	Detection of ISAV by RT-PCR using the methods described in Section 2.3.2.
		.	3.4.2. Isolation and identification of ISAV in two samples from one or more fish at the farm tested on separate occasions using the methods described in Section 2.2.
		.	3.4.3. Isolation and identification of ISAV from at least one sample from any fish on the farm using the methods described in Section 2.2. with corroborating evidence of ISAV in tissue preparations from any fish on the farm using either IFAT (Section 2.3.1.) or RT-PCR (Section 2.3.2.).

REFERENCES

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