Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.15.

RED SEA BREAM IRIDOVIRAL DISEASE

---

SUMMARY

Red sea bream iridoviral disease (RSIVD) is a significant cause of mortality among cultured marine fish (1). The causative agent is the red sea bream iridovirus (RSIV) (1, 4, 12). Overt infections have been recognised not only in red sea bream (Pagrus major), but also among other cultured marine fish including yellowtail (Seriola quinqueradiata), sea bass (Lateolabrax sp.) and Japanese parrotfish (Oplegnathus fasciatus) (5). Currently, RSIVD has only been reported from cultured marine fish in Japan, though a serologically and genetically related iridovirus has been isolated from brown-spotted grouper (Epinephelus malabaricus) from Thailand. This latter isolate did not induce disease in experimentally exposed red sea bream (2, 6, 7).
 
The first outbreak of an RSIVD was recorded in cultured red sea bream in Shikoku Island, Japan in 1990 (1). Since 1991, the disease has produced mass mortalities in cultured fish populations in the western part of Japan, mainly among juvenile red sea bream (5). However, mortality of market-sized fish has also been reported.
 
Affected fish are lethargic, exhibit severe anaemia, petechiae of the gills, and enlargement of the spleen (1, 2, 7). The disease is characterised by the appearance of enlarged cells stained deeply with Giemsa solution on microscopic observation of tissue sections of the spleen, heart, kidney, liver and gills of infected fish (1).
 
A similar disease has also seriously damaged stocks of more than 20 species of cultured marine fish in 18 prefectures located in south-western part of Japan. The infected fish include fish belonging to the orders Perciformes, Pleuronectiformes and Tetradontiformes (5).
 
The principal mode of transmission of RSIVD is by horizontal means via the water.
 
The RSIV showed some weak cross-reactivity in indirect fluorescent antibody tests (IFAT) using polyclonal rabbit anti-RSIV serum with the systemic Ranaviruses resembling epizootic haematopoietic necrosis virus (EHNV) or FV-3 (11). However, monoclonal antibodies (MAbs) against RSIV (13) do not recognise the systemic ranaviruses by IFAT, and two members of the systemic Ranavirus group (EHNV and European catfish virus) did not infect red sea bream after experimental challenges (7).
 
Diagnostic methods, such as the observation of stained impression smears or tissue sections, an immunofluorescence test with an MAb, and a polymerase chain reaction, have been reported for RSIV (3, 8, 11, 14, 15).
 
Control methods currently rely on the implementation of hygiene practices at the farm. A commercial vaccine may soon be available for RSIVD in red sea bream (9-11). Vaccination for other marine fish species is at the experimental stage.
 

DIAGNOSTIC PROCEDURES

The diagnosis of red sea bream iridoviral disease (RSIVD) is based on direct methods, which are either the isolation of red sea bream iridovirus (RSIV) in cell culture followed by its identification with anti-RSIV monoclonal antibodies (MAbs) or direct demonstration of RSIV antigens in infected fish tissue using MAbs.
 
Infected fish material suitable for virological examination is:
 

.	Clinically affected fish: The kidney and spleen tissues from fish of any size suffering characteristic signs of the disease.
.	Asymptomatic fish (apparently healthy fish): Kidney and spleen tissues.
1.	Standard Screening Method for RSIVD
	1.1.	Isolation of RSIV in cell culture
		Cell line to be used: GF cells
		Cells should be grown at 25°C in a temperature-controlled incubator to ensure subsequent success in the isolation of RSIV.
		a)	Inoculation of cell monolayers
			i)	Make an additional tenfold dilution of the 1/10 spleen 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 drained cell monolayer with 100 µl of each dilution.
			ii)	Allow to adsorb for 0.5-1 hour at 25°C and, without withdrawing the 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 25°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 from x40 to x100 magnification for 10 days. The use of a phase-contrast microscope is recommended.
			ii)	If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of tested homogenate supernatants, identification procedures have to be undertaken immediately (see 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 RSIV.
			iii)	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)	Inoculate cell monolayers as described above in Section 1.1.a.
			iii)	Incubate and monitor as described in Section 1.1.b.
			iv)	If no CPE occurs, the test may be declared negative.
	1.2.	Identification of RSIV
		RSIV cannot be identified by neutralisation tests as the antisera generated by the immunisation of rabbits have few neutralising antibodies.
		a)	Indirect fluorescent antibody test
			The indirect fluorescent antibody tests (IFAT) is to be conducted directly after virus isolation in cell culture
			i)	Prepare monolayers of cells on cover-slips in order to reach around 80% confluency, which is usually achieved within 24 hours of incubation at 25°C. The FCS content of cell culture medium can be reduced to 2-4%.
			ii)	When the cell monolayers are ready for infection inoculate the virus suspension to be identified by making tenfold dilution steps directly in the cell culture wells or flasks.
			iii)	Incubate at 25°C for 24-72 hours.
			iv)	When CPE appears, remove the cell culture medium, rinse three times with phosphate buffered saline (PBS). Air-dry the infected cells, then fix with cold acetone (stored at -20°C) for 10 minutes.
			v)	Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or freeze at -20°C.
			vi)	Prepare a solution of MAb (M10) to RSIV.
			vii)	Treat the cell monolayers with the solution of antibody to RSIV for 30 minutes at 37°C in a humid chamber.
			viii)	Rinse the cells three times for 5 minutes with PBS.
			ix)	Incubate with a suitable FITC-conjugated specific antibody for 30 minutes at 37°C in darkness in a humid chamber (FITC = fluorescein isothiocyanate).
			x)	Rinse three times for 5 minutes with PBS.
			xi)	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.
			xii)	Examine under incident UV light using a microscope with x10 eye pieces and x20 to x40 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 diffuse fluorescence throughout the cytoplasm.
2.	Diagnostic Methods for RSIVD
	2.1.	Virus isolation with subsequent identification
		As in Section 1.1 and 1.2.
	2.2.	Indirect fluorescent antibody test
		i)	Bleed the fish thoroughly.
		ii)	Make spleen imprints on the cleaned glass.
		iii)	Store the spleen pieces together with the other organs required for virus isolation in case this becomes necessary later.
		iv)	Allow the imprints to air-dry for 20 minutes.
		v)	Fix with cold acetone.
		vi)	Treat the imprints with the solution of MAb (M10) to RSIV for 30 minutes at 37°C.
		vii)	Rinse three times with PBS.
		viii)	Treat the imprints for 30 minutes at 37°C with a solution of FITC-conjugated antibody to the immunoglobulin used in step vi and prepared according to the instructions of the supplier. These FITC antibodies are most often rabbit or goat antibodies.
		ix)	Rinse three times with PBS.
		x)	Mount the cover-slips using glycerol saline prior to microscopic observation.
		xi)	Examine under indirect UV light using a microscope with x10 eye pieces and x20 to x40 objective lens having high numeral aperture. Positive and negative controls must be found to give the expected results prior to any other observation.
	2.3.	Polymerase chain reaction amplification
		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.
			B.3.2. for virus extraction and obtaining of organ homogenates.
		b)	Polymerase chain reaction amplification and sequencing
			RSIV has a large double-stranded DNA genome. Two primers, a forward primer (5'-CGG-GGG-CAA-TGA-CGA-CTA-CA-3') and reverse primer (5'-CCG-CCT-GTG-CCT-TTT-CTG-GA-3') are used for the amplification of the gene (568 bases) sequence of RSIV DNA by polymerase chain reaction (PCR).
			Fish samples are prepared as described in Kurita et al. (3). Nucleic acid (1 µl) is added to Taq polymerase buffer containing 1 mM of each primer, 200 mM of deoxynucleotide triphosphate, 1.25 U of ExTaq DNA polymerase in 20 mM Mg2+ PCR buffer. The mixture is incubated in an automatic thermal cycler programmed for 30 cycles at 94°C for 30 seconds, 58°C for 60 seconds, and 72°C for 60 seconds, and finally held at 72°C for 5 minutes. Amplified DNA (568 bp) is analysed by agarose gel electrophoresis.

REFERENCES

1.	Inouye K., Yamano K., Maeno Y., Nakajima K., Matsuoka M., Wada Y. & Sorimachi M. (1992). Iridovirus infection of cultured red sea bream, Pagrus major. Fish Pathol., 27, 19-27.
2.	Jung S., Miyazaki T., Miyata M., Danayadol Y. & Tanaka S. (1997). Pathogenicity of iridovirus from Japan and Thailand for the red sea bream Pagrus major in Japan, and histopathology of experimentally infected fish. Fisheries Sci., 63, 735-740.
3.	Kurita J., Nakajima K., Hirono I. & Aoki T. (1998). Polymerase chain reaction (PCR) amplification of DNA of red sea bream iridovirus (RSIV) Fish Pathol., 33, 17-23.
4.	Kusuda R., Nagato K. & Kawai K. (1994). Characteristics of an iridovirus isolated from red sea bream, Pagrus major. Suisanzoshoku, 42, 151-156.
5.	Matsuoka S., Inouye K. & Nakajima K. (1996). Cultured fish species affected by red sea bream iridoviral disease from 1991 to 1995. Fish Pathol., 31, 233-234.
6.	Miyata M., Matsuno K., Jung S.J., Danayadol Y. & Miyazaki T. (1997). Genetic similarity of iridoviruses from Japan and Thailand. J. Fish Dis., 20, 127-134.
7.	Nakajima K. & Maeno Y. (1998). Pathogenicity of red sea bream iridovirus and other fish iridoviruses to red sea bream. Fish Pathol., 33, 143-144.
8.	Nakajima K., Maeno Y., Fukudome M., Fukuda Y., Tanaka S., Matsuoka S. & Sorimachi M. (1995). Immunofluorescence test for the rapid diagnosis of red sea bream iridovirus infection using monoclonal antibody. Fish Pathol., 30, 115-119.
9.	Nakajima K., Maeno Y., Honda A., Yokoyama K., Tooriyama T. & Manabe S. (1999). Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test. Dis. Aquat. Org., 36, 73-75.
10.	Nakajima K., Maeno Y., Kurita J. & Inui Y. (1997). Vaccination against red sea bream iridoviral disease in red sea bream. Fish Pathol., 32, 205-209.
11.	Nakajima K., Maeno Y., Yokoyama K., Kaji C. & Manabe S. (1998). Antigen analysis of red sea bream iridovirus and comparison with other fish iridoviruses. Fish Pathol., 33, 73-78.
12.	Nakajima K. & Sorimachi M. (1994). Biological and physicochemical properties of the iridovirus isolated from cultured red sea bream, Pagrus major. Fish Pathol., 29, 29-33.
13.	Nakajima K. & Sorimachi M. (1995). Production of monoclonal antibodies against red sea bream iridovirus. Fish Pathol., 30, 47-52.
14.	Oshima S., Hata J., Hirasawa N., Ohtaka T., Hirono T., Aoki T. & Yamashita S. (1998). Rapid diagnosis of red sea bream iridovirus infection using the polymerase chain reaction. Dis. Aquat. Org., 32, 87-90.
15.	Oshima S., Hata J., Segawa C., Hirasawa N. & Yamashita S. (1996). A method for direct DNA amplification of uncharacterized DNA viruses and for development of a viral polymerase chain reaction assay: Application to the red sea bream iridovirus. Anal. Biochem., 242, 15-19.

[retour au début]

Copyright Office international des épizooties (OIE) - scientific.dept@oie.int