Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.4.

SPRING VIRAEMIA OF CARP

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SUMMARY

Spring viraemia of carp (SVC) is a rhabdovirus infection (6) of several carp species and of some other cyprinid and ictalurid fish species (5). Natural infections have been recognised in common carp and koi carp (Cyprinus carpio), grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), crucian carp (Carassius carassius), goldfish (C. auratus), roach (Rutilus rutilus), ide (Leuciscus idus), tench (Tinca tinca) and sheatfish (Silurus glanis). However, common carp is the most susceptible of these species and is the principal host. The geographical range of SVC is currently limited to countries of the European continent that experience low water temperatures during winter. For a detailed review of the condition, see Fijan (4) and Wolf (9).
 
As with other rhabdoviruses of fish, infection by SVC virus (SVCV) can be lethal due to the impairment of the salt-water balance, which occurs in a clinical context of oedema and haemorrhages. Virus multiplication, especially in endothelial cells of blood capillaries, haematopoietic tissue and nephron cells, underlies the clinical signs.
 
Overcoming SVCV infection results in a strong protective immunity associated with the presence of circulating antibodies detectable by methods such as virus neutralisation (VN), immunofluorescence or enzyme-linked immunosorbent assay (ELISA). In certain individuals, this health status also results in a covert virus carrier state.
 
On the basis of antigenic studies conducted with rabbit polyclonal neutralising antibodies, SVCV was found, using the VN test, to present only one serotype, but both the indirect fluorescent antibody test and the ELISA have revealed that SVCV shares common antigenic domain(s) with the pike-fry rhabdovirus (7). Differences in virulence of virus strains have been recorded during both natural cases of disease and experimental infections.
 
The reservoirs of SVCV are clinically infected fish and covert virus carriers from either cultured, feral or wild fish. Virulent virus is shed via faeces, urine, gill and skin mucus and exudate of skin blisters or oedematous scale pockets. However, liver, kidney, spleen, gill and encephalon are the organs in which SVCV is most abundant during the course of overt infection (3).
 
The mode of transmission for SVCV is horizontal, but an 'egg-associated' transmission (usually called 'vertical') cannot be ruled out. Horizontal transmission may be direct or vectorial, water being the major abiotic vector. Animate vectors and fomites are also involved in transmission of SVCV. Among animate vectors, the parasitic invertebrates Argulus foliaceus (Crustacea, Branchiura) and Piscicola piscicola (Annelida, Hirudinea) are able to transfer SVCV from diseased to healthy fish (1). Once SVCV is established in pond stock or pond farm stock, it may be very difficult to eradicate without destroying all kinds of life on the fish production site.
 
There is a high variability in the degree of susceptibility to SVC among individuals of the same fish species. Apart from the physiological state of the fish, the role of which is poorly understood, age or age-related status of innate immunity appears to be extremely important: the younger the fish, the higher the susceptibility to overt disease, but even adult broodfish can be susceptible to infection. It is clear that the environmental factor that is critical for virulence of an SVC infection is water temperature (5): in yearling or older fish overt infection is not often observed above 17°C whereas fry can be affected at temperatures as high as 22-23°C. Apart from the formerly cited cyprinid and ictalurid species, it seems that very young fish of various pond fish species are susceptible to SVC under experimental conditions over a wide temperature range. The most striking example is that of the pike (Esox lucius), which can be easily infected via the water route (2).
 
The implementation of hygiene measures and control policy rules are the only control methods currently feasible. Vaccination is mostly still at the experimental stage.
 
The screening procedure for SVCV in asymptomatic (clinically healthy) fish is based on isolation of the virus in cell culture as described in Section 1.1. below. Confirmation of SVCV is by immunological identification using VN (Section 1.2.). However, rapid, presumptive identification of SVCV isolated in tissue culture may be achieved using immunofluoresence or ELISA as described in Section 2. Here, a positive test is sufficient to initiate fish health control measures while awaiting confirmation by VN test.
 

DIAGNOSTIC PROCEDURES

Diagnosis of spring viraemia of carp (SVC) in clinically affected fish may be achieved by virus isolation or more rapidly by direct immunofluorescence (IF) tests (3) or enzyme-linked immunosorbent assays (ELISAs) (8) on infected tissues (Section 2). Ideally, direct diagnosis by IF or ELISA should be confirmed by virus isolation followed by a VN test. However, virus isolation may not be possible from decomposed clinical samples (8), so the presence of signs of SVC disease and a positive direct IF test or ELISA may be considered sufficient to initiate control measures.
 
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 accepted as a routine screening method for assessing the viral status of fish populations. However, the validation of some serological techniques for certain fish virus infections could arise in the near future, rendering the use of fish serology more widely acceptable for health screening purposes.
 
Fish material suitable for virological examination is:
 

.	Asymptomatic fish (apparently healthy fish): Kidney, spleen, gill and encephalon (any size fish).
.	Clinically affected fish: Whole alevin (body length < or = 4 cm), entire viscera including kidney and encephalon (4 cm < or = body length < or = 6 cm) or, for larger size fish, liver, kidney, spleen and encephalon.

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

1.	Standard Screening Method for SVC
	1.1.	Isolation of SVCV in cell culture
		Cell line to be used: EPC or FHM
		a)	Inoculation of cell monolayers
			i)	Make two serial tenfold dilutions of the 1/10 organ homogenate supernatants and transfer an appropriate volume of each of these two 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 10-15°C and, without withdrawing inoculate, add the 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 20°C.
		b)	Monitoring incubation
			i)	Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination at x40-100 magnification for 7 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 during incubation. This can be achieved by the addition to the inoculated cell culture medium of sterile bicarbonate buffer (for tightly closed cell culture flasks) or HEPES-buffered medium (HEPES = N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid) or 2 M Tris/HCl buffer solution (for cell culture plates).
			iii)	If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of the tested homogenate supernatants, identification procedures must be undertaken immediately (see Section 1.2. below).
				If a fish health surveillance/control programme is being implemented, steps may 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 suspected virus-positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not SVCV.
			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. Should the virus control fail to develop CPE, the process should be repeated with fresh susceptible cells and new batches of samples.
		c)	Subcultivation procedures
			i)	Collect aliquots of cell culture medium from all monolayers inoculated with dilutions of each supernatant of organ homogenates.
			ii)	If required, repeat the neutralisation treatment for infectious pancreatic necrosis virus (IPNV) as previously described (see Chapter I.1. Section B.3.3.), with dilution of the above supernatant (1/1 to 1/100).
			iii)	Inoculate cell monolayers as described above in Section 1.1.a.
			iv)	Incubate and monitor as described above in Section 1.1.b.
			v)	If no CPE occurs, the test may be declared negative.
		1.2.	Identification of virus isolated in cell culture
		.	1.2.1. Rapid presumptive methods
		a)	Indirect fluorescent antibody test
			i)	Prepare monolayers of cells in 2 cm2 wells of cell culture plastic plates or on cover-slips in order to reach around 80% confluency within 24 hours of incubation at 30°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 SVCV in a similar way, in order to obtain a virus titre of about 5000-10,000 plaque-forming units (PFU)/ml in the cell culture medium.
			iv)	Incubate at 20°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 acetone (stored at -20°C) for cover-slips or a mixture of acetone 30%/ethanol 70%, also at -20°C, for plastic wells.
			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 antibody or serum to SVCV in 0.01 M PBS, pH 7.2, containing 0.05% Tween 80 (PBST), at the appropriate dilution (which has been established previously or is given by the reagent supplier).
			ix)	Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, 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 and do not allow evaporation to occur. The volume of solution to be used is 0.25 ml/2 cm2 well.
			xi)	Rinse four times with PBST as above.
			xii)	Treat the cell monolayers for 1 hour at 37°C with a solution of fluorescein isothiocyanate (FITC)-conjugated antibody to the immunoglobulin used in the first layer and prepared according to the instructions of the supplier. These FITC antibodies are most often rabbit or goat antibodies.
			xiii)	Rinse four times with PBST.
			xiv)	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.
			xv)	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.
		b)	Enzyme-linked immunosorbent assay
			i)	Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified immunoglobulins (Ig) specific for SVCV, in 0.02 M carbonate buffer, pH 9.5 (200 µl/well). Ig may be polyclonal or monoclonal Ig originating most often from rabbit or mouse, respectively. For the identification of SVCV, monoclonal antibodies (MAbs) specific for certain domains of the nucleocapsid (N) protein are suitable.
			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 carbonate buffer) or other blocking solution for 1 hour at 37°C (300 µl/well).
			v)	Rinse four times with PBST.
			vi)	Add 2% non-ionic detergent (Triton X-100 or Nonidet P-40) to the virus suspension to be identified.
			vii)	Dispense 100 µl/well of two- or four-step dilutions of the virus to be identified, and of the non-infected cell culture harvest (negative control), and the SVCV control virus (positive control); allow to react with the coated antibody to SVCV for 1 hour at 20°C.
			viii)	Rinse four times with PBST.
			ix)	Add to the wells, 200 µl of horseradish peroxidase (HRPO)-conjugated MAb or polyclonal antibody to SVCV; or polyclonal IgG to SVCV or MAb to N protein specific for a domain different from the one of the coating MAb and previously conjugated with biotin.
			x)	Incubate for 1 hour at 37°C.
			xi)	Rinse four times with PBST.
			xii)	Add 200 µl of HRPO-conjugated streptavidin or ExtrAvidin (Sigma) to those wells that have received the biotin-conjugated antibody and incubate for 1 hour at 37°C.
			xiii)	Rinse four times with PBST.
			xiv)	Add 200 µl of the substrate and chromogen. Stop the course of the test when positive controls react, and read the results.
			xv)	Alternatively, add substrate and chromogen to those wells containing the peroxidase- conjugated antibody and proceed as above.
		.	1.2.2. Confirmatory identification methods
		a)	Neutralisation test
			i)	Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at 2000 g for 15 minutes at 4°C, or filter through a 0.45 µm pore membrane to remove cell debris.
			ii)	Dilute the virus-containing medium from 10-2 to 10-4.
			iii)	Mix aliquots of each dilution with equal volumes of an antibody solution against SVCV, and similarly treat aliquots of each virus dilution with cell culture medium.
				(The neutralising antibody [NAb] solution must have a 50% plaque reduction titre of at least 2000.)
			iv)	In parallel, other neutralisation tests must be performed against:
				.	a homologous virus strain (positive neutralisation test)
				.	a heterologous virus strain (negative neutralisation test).
			v)	If required, a similar neutralisation test may be performed using antibodies to IPNV, to ensure that no IPNV contaminant has escaped the first anti-IPNV test.
			vi)	Incubate all the mixtures at 20°C for 1 hour.
			vii)	Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell cultures per dilution) and allow adsorption to occur for 0.5-1 hour at 15-20°C; 24- or 12-well cell culture plates are suitable for this purpose, using a 50 µl inoculum.
			viii)	When adsorption is completed, add cell culture medium, supplemented with 2% FCS and buffered at pH 7.4-7.6, to each well and incubate at 20°C.
			ix)	Check the cell cultures for the onset of CPE and read the results as soon as it occurs in non-neutralised controls (cell monolayers being protected in positive neutralisation controls). Results are recorded either after a simple microscopic examination (phase-contrast preferable) or after discarding the cell culture medium and staining the cell monolayers with a solution of 1% crystal violet in 20% ethanol.
			x)	The tested virus is identified as SVCV when CPE is prevented or noticeably delayed in the cell cultures that received the virus suspension treated with the SVCV-specific antibody, whereas CPE is evident in all other cell cultures.
			NOTE: In the absence of any neutralisation by NAb to SVCV, it is mandatory to conduct an indirect fluorescent antibody test (IFAT) with the suspect isolate.
2.	Diagnostic Methods for Clinically Diseased Fish
		2.1. Direct detection in fish tissues
		a)	Indirect fluorescent antibody test
			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 1.2.1.a. steps v-vii.
			vi)	Rehydrate the above preparations (see Section 1.2.1.a. step ix) and block with 5% skim milk or 1% bovine serum albumin, in PBST for 30 minutes at 37°C.
			vii)	Rinse four times with PBST.
			viii)	Treat the imprints with the solution of antibody to SVCV and rinse as indicated in Section 1.2.1.a. steps viii-xi.
			ix)	Block and rinse as previously in steps vi and vii.
			x)	Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as indicated in Section 1.2.1.a. steps xii-xv.
			If the test is negative, process the organ samples stored at 4°C, for virus isolation in cell culture as described in Section 1.1.
		b)	Enzyme-linked immunosorbent assay
			i)	Microplate processing
				As described in Section 1.2.1.b. of this chapter up to step iv (inclusive).
			ii)	Sampling procedures
				See the following sections in Chapter I.1.:
				B.1. for the selection of fish specimens
				B.2. for the selection of materials sampled.
			iii)	Processing of organ samples
				See the following sections in Chapter I.1.:
				B.3.1. for transportation
				B.3.2. for virus extraction and obtaining organ homogenates.
			iv)	The enzyme-linked immunosorbent assay procedure
				.	Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
				.	Treat the remaining part of the homogenate with 2% Triton X-100 or Nonidet P-40 and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
				.	Complete the other steps of the procedure described in Section 1.2.1.b.
				.	If the test is negative, process the organ samples stored at 4°C, for virus isolation in cell culture as described in Section 1.1.
	2.2.	Virus isolation in cell culture
		As described in Section 1.1. of this chapter.

REFERENCES

1.	Ahne W. (1985). Argulus foliaceus l. and Philometra geometra l. as mechanical vectors of spring viraemia of carp virus (SVCV). J. Fish Dis., 8, 241-242.
2.	Ahne W. (1985). Viral infection cycles in pike (Esox lucius l). Z. Angew. Ichthyol., 2, 90-95.
3.	Faisal M. & Ahne W. (1984). Spring viraemia of carp virus (SVCV): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen. J. Fish Dis., 7, 57-64.
4.	Fijan N. (1999). Spring viraemia of carp and other viral disease agents of warm water fish. In: Fish Diseases and Disorders, Volume 3; Viral, Bacterial and Fungal infections, Woo P.T.K. & Bruno D.W., eds. CABI Publishing, UK, 177-244.
5.	Fijan N. (1972). Infectious dropsy in carp - a disease complex. In: Diseases of Fish, Mawdesley T., ed. Symposia of the Zoological Society of London. Academic Press, London, UK, 39-51.
6.	Fijan N., Petrinec Z., Sulimanovic D. & Zwillenberg L.O. (1971). Isolation of the causative agent from the acute form of infectious dropsy of carp. Vet. Arch. Zagreb, 41, 125-138.
7.	Vestergaard-Jorgensen P.E., Olesen N.J., Ahne W. & Lorentzen N. (1989). SVC and PFR viruses. Serological examination of 22 isolates indicates close relationship between the two rhabdoviruses. In: Viruses of Lower Vertebrates, Ahne W. & Kurstak E., eds. Springer-Verlag, Berlin, Germany, 349-366.
8.	Way K. (1991). Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA). J. Appl. Ichthyol., 7, 95-107.
9.	Wolf K. (1988). Fish Viruses and Viral Diseases. Cornell University Press, Ithaca, New York, USA, 476 pp.

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