Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.6.

CHANNEL CATFISH VIRUS DISEASE

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SUMMARY

Channel catfish virus disease (CCVD) is caused by a herpesvirus designated Ictalurid herpesvirus 1 by the International Committee on Taxonomy of Viruses, but the commonly used name is channel catfish virus (CCV). CCV affects channel catfish (Ictalurus punctatus) in the United States of America. For more detailed reviews of the condition, see Wolf (22) or Plumb (15).
 
CCVD is of importance because of its clinical and economic consequences in channel catfish farming. CCVD results in high mortality rates in populations of fry and juvenile catfish. Diseased fish demonstrate ascites, exophthalmia and haemorrhage in fins and musculature. Histologically the most remarkable damage occurs in the kidney with extensive necrosis of renal tubules and interstitial tissue.
 
In survivors, CCVD results in a strong protective immunity, the synthesis of circulating antibodies to the virus and, a covert latent carrier state. During this latent carrier state the virus is undetectable by traditional culture or antigen-detection means, even when adults are immunosuppressed during spawning.
 
On the basis of antigenic studies conducted with polyclonal rabbit antibodies, CCV isolates form a homogeneous group. However, the use of monoclonal antibodies shows some variation in antigenic determinants between isolates (1). Some variation in the virulence of CCV strains has been recorded during natural outbreaks of disease and has been demonstrated experimentally. Additionally, molecular data indicate genetic variation within this species (7, 18).
 
Reservoirs of CCV are clinically infected fish and covert carriers. Infectious CCV can be detected in the water from tanks of experimentally infected fish, but the route of shedding has not been determined. The sites where the virus is most abundant during the course of overt infection are posterior kidney, skin, gills, spleen and intestine, respectively, in decreasing magnitude (12, 13). The transmission of CCV is horizontal and vertical. Horizontal transmission may be direct or vectorial with water being the main abiotic vector. The virus has been shown to readily adsorb to pond sediments (6) and interaction with suspended clay particles in pond water may influence horizontal transmission. Animate vectors and inanimate objects could also act in CCV transmission. Vertical transmission is thought to be common, but the mechanism of vertical transmission is not known, as infectious virus has not been detected on the skin or in the sexual products of spawning adults. Once CCVD occurs in a fish population, survivors of the disease become covert carrier fish.
 
Channel catfish and the closely related blue catfish (Ictalurus furcatus) have been the only fish found to be infected with CCV, and variations in susceptibility to CCV have been recorded depending on fish strain. The age of the fish is extremely important for overt infection. Although experimental data suggest that older fish are susceptible to natural outbreaks of acute CCVD (11), the disease occurs almost exclusively in fish that are less than 1 year of age, and generally less than 4 months of age. Water temperature is the critical environmental factor. The mortality rate is high above 27°C, but readily decreases and ceases below 18°C.
 
Diagnosis of CCVD is based on virus isolation in cell culture. Confirmatory testing is by immunological identification by neutralisation, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR). Rapid techniques by immunofluorescence tests or ELISA are suitable mainly for diagnosis in clinically infected fish. Because virus proteins or infectious virus is not produced, culture methods or antigen-based testing is of little use for carrier screening. Instead, detection of neutralising antibodies in a population of fish and, more recently, the use of PCR to detect latent CCV genomic DNA is of more use.
 
Control methods currently rely on maintaining relatively low stocking densities and avoiding stressful handling of young fish during the summer months. Also, control policies and hygiene practices have been used, where practical, in catfish husbandry. The incubation of eggs and rearing of fry and juveniles in facilities separated from carrier populations are critical to preventing the occurrence of CCVD in a CCV-free fish production site. Because virus is only detected during active outbreaks, defining CCV-free status has been done largely from historical data or identifying populations that are seronegative to the virus. Recent use of PCR and hybridisation probes to detect latent CCV genomic DNA suggests that CCV is present in many populations that have no history of the disease (2, 5, 9, 21). Vaccination, although experimentally promising (19, 20, 23, 24), is not in use at this time.
 

DIAGNOSTIC PROCEDURES

The diagnosis of channel catfish virus disease (CCVD) is generally based on the isolation of channel catfish virus (CCV) in cell culture followed by its immunological or nucleic-acid-based identification (conventional approach). Alternatively, the immunological demonstration of CCV antigen in infected fish tissues can be used. The conventional approach is most common because the virus produces rapid cytopathic effect (CPE) in cell culture and there are no commercial sources of CCV-specific antiserum, and custom-produced antisera to CCV is often of low titre or has cross-reaction with fish tissue.
 
Due to insufficient knowledge of the serology of fish virus infections, the detection of fish antibodies to viruses has not yet been recognised as a valuable diagnostic method for assessing the viral status of fish populations. However, the use of direct culture or detection of viral antigen is of little use in detecting carrier fish. Therefore, the identification of antibodies to CCV has more merit in screening carrier populations (8, 14). The antibody titres in carrier populations vary seasonally, with the lowest titres occurring in the late winter and early spring (3). The validation of some serological techniques for diagnosis of certain fish virus infections could arise in the near future, rendering the use of fish serology more widely acceptable for diagnostic purposes.
 
Infected fish material suitable for virological examination is:
 

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Clinically affected fish: Whole fry or small juveniles (body length < or = 3 cm), viscera including kidney (3 cm < or = body length < or = 6 cm) or, for larger size fish, kidney, and spleen.

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There is no definitive test for asymptomatic fish (apparently healthy fish): Cell culture or antigen detection of latent virus is not possible. Occasional recrudescence of latent infection has been detected in adult catfish after spawning (16) or during winter months or after dexamethazone treatment (4), but these methods are not reliable enough for inspection purposes. Detection of latent virus DNA can be done using polymerase chain reaction (PCR) on kidney, fin or gill tissue (2, 5, 10). However, these tests were developed on experimentally infected fish and have not been applied to production systems. Therefore, their use or reliability in detecting CCV in populations or predicting the potential for vertical transmission has not been documented.

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

1.	Standard Screening Method for CCVD
	1.1.	Isolation of CCV in cell culture
		Cell line to be used: CCO
		a)	Inoculation of cell monolayers
			i)	Make an additional tenfold dilution of the 1/10 organ homogenate supernatants and transfer an appropriate volume of each of the two dilutions on to 24-hour-old cell monolayers. Inoculate at least 2 cm2 of cell monolayer with 100 µl of each dilution.
			ii)	Allow virus to adsorb for 0.5-1 hour at 25-30°C. Then (without withdrawing the inoculum), add the cell culture medium buffered at pH 7.6 and supplemented with 10% fetal calf serum (FCS) (1 ml/well for 24-well drained cell culture plates), and incubate at 25-30°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 10 days. The use of a phase-contrast microscope is recommended. CPE is extensive and rapidly progressing in cultures from overtly diseased individuals. CPE consists of cell fusion (syncytium) formation and contraction leaving cytoplasmic spindles irradiating from the syncytium to points on the flask surface where the cells were originally attached.
			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 bicarbonate buffer (for tightly closed cell culture flasks or in a CO2 incubator), 2 M Tris buffer solution or by using 25 mM HEPES-buffered medium (HEPES = N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid).
			iii)	If 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, provisions 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 virus-positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not CCV.
			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 sample inoculated with 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)	Inoculate cell monolayers as described above in Section 1.1.a.
			iii)	Incubate and monitor as described in Section 1.1.b.
			iv)	Perform a second (and last) subcultivation step if the first one remains virus-negative.
	1.2.	Identification of CCV
		a)	Neutralisation test (Note: when developing antisera for CCV most researchers have found a weak neutralising antibody response in rabbits; cross-reaction with cellular components also often occurs)
			i)	Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at 2000 g for 15 minutes at 4°C to remove cell debris.
			ii)	Dilute virus-containing medium from 10-2 to 10-4.
			iii)	Mix aliquots (for example 200 µl) of each virus dilution with equal volumes of an antibody solution specific for CCV, 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 (positive neutralisation test)
				.	a heterologous virus (negative neutralisation test).
			v)	Incubate all the mixtures at 25°C for 1 hour.
			vi)	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 25°C; 24- or 12-well cell culture plates are suitable for this purpose, using a 50 µl inoculum.
			vii)	When adsorption is complete, add the cell culture medium, supplemented with 2% FCS and buffered at pH 7.3-7.6, into each well and incubate at 25-30°C.
			viii)	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 ethanol 20%.
			ix)	The tested virus is identified as CCV when CPE is abolished or noticeably delayed in the cell cultures that had received the virus suspension treated with the CCV-specific antibody, whereas CPE is evident in all other cell cultures.
			x)	In the absence of any neutralisation by NAb to CCV, conduct an indirect fluorescent antibody test (IFAT) with the suspect sample, perform an enzyme-linked immunosorbent assay (ELISA) or use a CCV-specific nucleic-acid-based assay.
		b)	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, which is usually achieved within 4 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).
			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 CCV 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 25°C for 18 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 CCV in 0.01 M PBS, pH 7.2, containing 0.05% Tween 80 (PBST), at the appropriate dilution (which has been established previously, there is no commercial source for CCV-specific antiserum).
			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. 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 a high numerical aperture. Positive and negative controls must be found to give the expected results prior to any other observation.
		c)	Enzyme-linked immunosorbent assay
			i)	Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified immunoglobulins (Ig) or serum specific for CCV, in 0.01 M PBS, pH 7.2 (200 µl/well). Ig may be polyclonal or monoclonal Ig originating most often from rabbit or mouse, respectively.
			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 1 hour at 37°C (200 µl/well).
			v)	Rinse four times with PBST.
			vi)	Add 2% Triton X-100 to the virus suspension to be identified.
			vii)	Dispense 100 µl/well of a two- or four-step dilution of the virus to be identified and of CCV control virus, and allow to react with the coated antibody to CCV for 1 hour at 20°C.
			viii)	Rinse four times with PBST.
			ix)	Add biotinylated polyclonal antibody to CCV to the wells.
			x)	Incubate for 1 hour at 37°C.
			xi)	Rinse four times with PBST.
			xii)	Add streptavidin-conjugated horseradish peroxidase to those wells that have received the biotin-conjugated antibody, and incubate for 1 hour at 20°C.
			xiii)	Rinse four times with PBST.
			xiv)	Add the substrate and chromogen. Stop the course of the test when positive controls react, and monitor the results.
		d)	Polymerase chain reaction
			There are several published PCR assays for CCV (2, 5, 10). As stated in Chapter I.1. Section C.3.5., PCR is very susceptible to false-positive and false-negative results. Therefore each assay and tissue extraction should include a negative control to rule out contamination. PCR reaction set-up should be done in a separate physical location from where the PCR products are evaluated. PCR is a useful method for identifying the virus after isolation in culture. The following is a modification of the method of Boyle & Blackwell (5) and uses a modified target as an internal control (12). The 5'-3' sequence of the upper primer is TCA-TCC-GAA-TCC-GAC-AAC-TGA and that of the lower primer is CCA-AGA-TCG-CGG-AGA-AAC. To minimise the potential for contamination, all sample preparation and reaction set-up should be done with aerosol-preventing pipette tips.
		.	Sample preparation
			i)	Collect 1-0.5 ml of cell culture supernatant from affected wells into a 1.5 ml microcentrifuge tube.
			ii)	Centrifuge at maximum speed in microfuge (18,000-20,000 g) for 30 minutes.
			iii)	Discard supernatant and resuspend the pellet in 10 µl proteinase K buffer (50 mM KCl, 15 mM Tris-HCl pH 8.3 and 0.5% Nonidet P-40) containing 0.5 mg/ml proteinase K and incubate at 55°C for 1 hour.
			iv)	Heat inactivate the proteinase K at 95°C for 10 minutes. Centrifuge at maximum speed for 10 seconds. Use 3 µl in the PCR reaction.
			Other standard DNA extraction methods may be used.
		.	PCR set-up
			The mastermix should be made-up in a separate location from areas where diagnostic samples are prepared and PCR product are analysed.
			i)	Make-up the mastermix. Prepare at least one extra aliquot for each 10 reactions planned. Include a positive and a negative (water blank) control for every 10 samples.
				Per sample: distilled water 33.6 µl
				10 x buffer	5.0 µl
				Upper primer (50 pmole/µl)	0.4 µl
				Lower primer (50 pmole/µl)	0.4 µl
				dNTPs (2.5 mM each)	2.0 µl
				25 mM MgSO4	5.0 µl
				Internal control (0.025 pg/µl)	0.3 µl
				Taq polymerase (5 U/µl)	0.3 µl
			ii)	Add 47 µl per reaction tube.
			iii)	Add 3 µl of sample or water (negative control).
			iv)	If using a thermocycler without a hot lid, add 50 µl of mineral oil.
			v)	Run the reaction. Use 30 cycles of 93°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.
			vi)	Electrophorese 10 µl of the product on a 10% polyacrylamide gel with a 100 bp size ladder. Stain the gel with ethidium bromide. Observe and photograph under UV transillumination (17).
		.	Evaluate results
			The internal control produces a 149 bp product. CCV produces a 136 bp product. Expect a 149 bp band within the negative control and on negative samples. Expect a 136 bp product and a 149 bp product on CCV-positive samples. If CCV is present at a high level, the 149 bp band may be missing. If no bands are present, the PCR reaction did not work indicating a failure of one of the PCR components or an inhibitor in the sample. If the negative control shows a 136 bp product, then there is a contaminant in the PCR set-up and the assay must be redone. If an aberrant size band is produced or to confirm that the PCR product is from CCV, the PCR can be redone using no internal standard and the product can be directly sequenced. The sequence can then be evaluated using BLAST on the National Center for Biological Information internet site (http://www3.ncbi.nlm.nih.gov/BLAST/) to identify sequences with high homology. The PCR amplifies the region from 107827 to 107962 of the CCV genome (GenBank accession M75136) representing a portion of open reading frame 73 within ORF (9). Expect at least 95% identity to this sequence.

TCA-TCC-GAA-TCC-GAC-AAC-TGA-CGC-GTC-GGT-AGC-CCG-ACC-GAT-CCG-
TAT-GTT-ACG-GGT-GCG-GGG-GTC-GAC-ACC-GTG-CTC-GCC-GCG-ATG-AGG-CTG-
ACC-GCG-GAC-ACG-GGG-GGT-CCC-CCT-CGT-TTC-TCC-GCG-ATC-TTG-G

Figure 1. Sequence of CCV PCR product. Italic underlined portions indicate primer sequences.

2.	Diagnostic Methods for CCV
	2.1.	Virus isolation with subsequent identification
		As in Section 1.1. and 1.2.
	2.2.	Indirect fluorescent antibody test
		.	Test procedure
			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 Section B.3.1. in Chapter I.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 PBST for 30 minutes at 37°C.
			vii)	Rinse four times with PBST.
			viii)	Treat the imprints with the solution of antibody to CCV and rinse as indicated in Section 1.2.b.
			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.b. 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.
	2.3.	Enzyme-linked immunosorbent assay
		a)	Microplate processing
			As described in Section 1.2.c. of this chapter up to step iv (inclusive).
		b)	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.
		c)	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.
		d)	The enzyme-linked immunosorbent assay procedure
			i)	Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
			ii)	Treat the remaining part of the homogenate with 2% Triton X-100 as described in Section 1.2.c. step v, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
			Complete the other steps of the procedure described in Section 1.2.c.
			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.
	2.4.	Polymerase chain reaction
		As stated in Chapter I.1. Section C.3.5., PCR is very susceptible to false-positive and false-negative results. Therefore each assay and tissue extraction should include a negative control to rule out contamination. PCR reaction set-up should be done in a separate physical location from that where the PCR products are evaluated. Thus the use of nested PCR protocols for inspection purposes is not recommended. The PCR assay given in Section 1.2.d. can be modified for direct detection of CCV in overtly infected fish. This is done by centrifuging 1.5 ml of a 1/10 dilution of the homogenate at 8000 g for 5 minutes. The supernatant is then used the same way as the cell culture supernatant in Section 1.2.d. If this assay is used to detect carrier fish, the DNA must be purified from the tissue (using a commercially available kit such as the Puregene DNA isolation kit, Gentra Systems, Minneapolis Minnesota, USA). To increase the detection limit to that needed to detect a carrier state, use 0.5 µg of DNA, leave out the internal control DNA, and use 35-40 cycles. In performing PCR on tissue extracts, it is recommended that a parallel sample be run that has been spiked with dilute purified CCV DNA or cloned target fragment (such as the internal standard used above) as a positive control to rule out the presence of inhibitors giving false-negative results.

REFERENCES

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