Assessment of a Commercial Kit Collection for Diagnosis of the Fish Viruses: IHNV, IPNV, SVCV and VHSV.

Transcription

1 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 6 Assessment of a Commercial Kit Collection for Diagnosis of the Fish Viruses: IHNV,, and. Ellen Ariel and Niels Jørgen Olesen Community Reference Laboratory for Fish Diseases, Danish Verinary Laboratory, Department of Poultry, Fish and Fur Animals, Hangøvej 2, DK Århus N, Denmark. Abstract A commercial kit collection for the dection of the fish pathogenic viruses,, IHNV,, and, was assessed for its ability to dect isolates in selected panels of the respective viruses. The kit collection, which was based on fluorescence staining of infected cell cultures in tissue culture plates, fulfilled the promised requirements for the IHN kit only. The IPN, the SVC and especially the VHS kit were lacking in either specificity or sensitivity. The findings stress the need for commercial companies to carry out proper validation before mark release. Introduction Diagnostic kits for identification of fish pathogens are commercially available. Such kits are marked for the identification of Infectious Haematopoiic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (), Spring Viraemia of Carp Virus () and Viral Haemorrhagic Septicaemia Virus (VHS). The kits assessed here are based on the direct identification of virus in infected cell cultures in 24-well plates by use of unspecified monoclonal antibodies against the respective viruses as primary antibodies. Visualisation is based on a fluorescein-coupled secondary antibody where an inverse microscope equipped with fluorescence is needed to read the results in the plates. Several laboratories in Europe use these kits for the diagnosis of fish viruses. It is therefore important to dermine if the kits recognize all isolates of the respective viruses or if certain virus isolates remain undected. This paper reports on an assessment of the kits using a panel of isolates for each virus and reference reagents from the Community Reference Laboratory for Fish Diseases (CRL). The assessment revealed that the kits received at the CRL did not fulfil the requirements. Materials and Mhods Kits were supplied by the local distributor in Denmark. The kits are designed for the dection of four different fish viruses: IHNV,, and. Five to nine isolates were selected for each virus to produce a panel covering a range of different host species, serotypes and geographical locations (Table 1). Each panel of viruses was tested in their respective kits. Before testing, all isolates were identified by the standard diagnostic procedures as described in Commission Decision 96/240 (ELISA and/or IFAT). The antibodies used for this identification were monoclonal antibodies (MAb) against :

2 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 7 Virus Isolate Reference I HNV 4008 (First Italian isolate) Bovo 1987 I HNV 32/87 (First French isolate) Hattenberger-Baudouy I HNV RBH (US) I HNV HAG (US) I HNV TR (US) L apatra, 1990 L apatra, 1990 LaPatra 1993 I HNV 193/110 (US) N ichol, 1995 VR 299 reference strain Western Hemisphere Committee 1970 Ab reference strain and Kehl, 1971 I PNV DK-6110 Eel (Type Ab) Unpublished Sp reference strain and Grauballe, 1971 I PNV DK c16 Cod (Type Sp) Unpublished Birnavirus II DK Ising 9B Olesen al /70 reference strain Fijan al / / 3 DK-3587 PFR de Kinkelin 1973 V76 S64 DK-F1 reference strain, type I Jensen, 1965 DK-5131 Klapmølle Type II DK-1p8 Herring isolate Olesen Mortensen V HSV D17/95 (Germany) V HSV F (Ireland turbot isolate) Schau, Unpublished McArdle, Unpublished DK-61 Hededam 1972 V HSV 23/76 (French) de Kinkelin and Le Berre 1977 DK-3592B Voldbjerg Type I DK-5151 Rindsholm Type III Lorenzen Olesen 1990 al, 1993 Table 1: Virus isolates used for the assessment of the commercial kits.

3 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 8 A Virus isolate CPE Control CRL-MAb anti Kit Mab IHNV Hyb IHNV 4008 (Italian) 32/87 (French) R BH (US) H AG (US) T R (US) 193/110 (US) I PNV S VCV V HSV B Virus isolate CPE Control CRL- Rabbit anti Sp/Ab Kit MAb I PNV VR I PNV Ab DK Eel I PNV Sp DK c16 Cod Birnavirus II Dab isolate I HNV S VCV V HSV Table 2: Outcome of the testing panels of IHNV isolates (a), isolates (b), isolates (c) and isolates (d) in the commercial kits, with CRL reagents and by controls of cytopathic effect (CPE control) Herologous control isolates for each panel consisted of reference strains of the following viruses: IHNV: 32/87, : Sp and Ab, 56/70 and : DK-3592B. The cell monolayers in uninfected wells were used as negative controls. a. IHNV Kit; b. Kit; c. Kit; d. Kit IP5B11 (Lorenzen 1988); IHNV: Hyb (Olesen, not published); and polyclonal rabbit antisera against : F46-51 (Lorenzen, not published); birnavirus II: K101 17/12/85 (Olesen al, 1988); : K42; and Pike fry rhabbdovirus (PFR): K44 ( ), respectively. The virus panels were examined according to the guidelines enclosed with the kits. In brief, each isolate was cultured (Eagles MEM, 10% Foal calf serum, Tris buffer and antibiotics in standard concentrations, 15 C incubation temperature) and titrated in its preferred cell line:,, and Birnavirus II in BF2 cells (Wolf 1966), IHNV and in EPC cells (Fijan 1983). For each virus, the members of the selected panel were inoculated onto 24 hours old cell monolayers in 24-well plates in concentrations ranging from 10 5 to 10 8 TCID 50 /ml, and diluted 10-fold twice into adjacent wells. One or two representative members of the other three virus panels were also inoculated to provide a control for possible cross-reaction of MAbs bween the four fish viruses. All virus panels were inoculated into two 24-well plates: one for the test and one to control that all isolates induced cytopathic effect (CPE controls). In each plate three non-infected wells were used as negative control. After 48 hours incubation at 15 C all cells in one of the plates were fixed with isopropanol, and then rinsed. The virus specific kit MAb or the CRL antibodies described above were added to separate wells and rinsed off again after incubation. This step was followed by the addition of goat-antimouse immunoglobulin (Ig) conjugated to fluorescein (FITC). After incubation, rinsing and addition of mounting medium, the cell layer was observed under an inverted microscope equipped with fluorescence. Virus infected cells displayed fluorescence. CRL reagents (secondary antibody and chromogen/ substrate) were used as controls in one of the

4 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 9 C Virus (IFAT) isolate CPE Control CRL-MAb anti IP5B11 CPE Control CRL- Rabbit anti SVC K42 CRL-Rabbit anti PFR K44 Kit Mab S VC - reference strain + S VCV 12840/7 + S VCV 14286/3 + S VCV DK P FR - reference strain + P FR V76 - P FR S64 - I HNV - I PNV - V HSV Kit Mab D K - F1 Reference strain D K (Klapmølle) I p8 Herring F (Irish) D 17/95 (German) D K - 61 (Hededam) 23/75 (French) D K B (Voldbjerg) D K (Rindsholm) I HNV I PNV S VCV D Table 2 continued... 3 infected wells. The CPE control plates were kept for 1 week at 15 and examined daily for the development of CPE. Results The CRL reagents recognised all isolates in the respective panels, but not the negative control isolates included in each test. All MAbs from the four kits appeared to be specific to one virus type only, no cross-reaction bween the 4 different fish viruses were observed. No reactions could be observed in any of the noninfected wells. All CPE controls in all tests were positive.

5 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 10 The kit anti-ihnv MAb reacted with all the isolates in the IHNV panel (Table 2a). The kit anti- MAb reacted with all the isolates in the panel, but it also classes Birnavirus II as (Table 2b). The kit anti- MAb dected all isolates and the reference strain of PFRV, but not all PFRV isolates (Table 2c). Five out of nine isolates did not react with the kit anti- MAb (Table 2d). Discussion Compared to CRL reagents, the IHNV kits performed well for the selected IHNV panel. The other 3 kits were lacking in either specificity or sensitivity. The kit could not distinguish bween and Birnavirus II. These 2 serogroups can be discriminated by most rabbit antisera against (Personal observation). This is not possible if only the IPN kit is used. Thus, the kit cannot perform the necessary differential diagnosis. As expected, the SVC kit could not distinguish bween and PFR, which is true for many antibodies against these viruses when used in immunofluorescence ( al ). The SVC kit, however, was only able to identify the PFR reference strain, while 2 other PFR-like viruses remained undected. Of major concern, however, are the findings that several VHS isolates remained undected in the VHS kit. Should this kit be applied for surveying the disease status of a farm, the farm may unjustly be declared free from VHS. On a different note, a disadvantage in the format of these kits, is the need for an inverse fluorescence microscope to read the results. Such costly equipment may not be justifiable in a laboratory that only handles small sample numbers. The findings stress the need for commercial companies to carry out proper validation before mark release, including testing the monoclonal antibodies against a wide range of isolates and possibly instruct users to include positive and CPE controls in addition to negative controls. References Bovo, G., Giorgti, G.,, P.E.V. and Olesen, N.J. (1987) Infectious haematopoiic Necrosis: First dection in Italy. Bull. Eur.Ass.Fish.Pathol. 7(5):124 de Kinkelin, P. and Le Berre, M. (1977) Isolement d un rhabdovirus pathogene de la truite fario Salmo trutta L C. R. Acad. Sci. Paris 284: de Kinkelin P, Galimard B, Bootsma R (1973) Isolation and identification of the causative agent of red-disease of pike (Esox lucius L 1766). Nature, London, 241: Fijan, N., Prinec, Z., Sulimanovic, D. and Zwillenberg, L.O. (1971) Isolation of the causative agent from the acute form of the infectious dropsy of carp. V.Arhiv 41: Fijan, N., Sulimanovic, D., Bearzotti, M., Muzinic, D., Zwillenberg, L.O., Chilmonczyk, S., Vautherot, J.F. and de Kinkelin, P. (1983) Some properties of the epithelioma papulosum cyprinid (EPC) cell line from carp Cyprinus Carpio. Ann. Virol. (Inst Pasteur) 134E:

6 Bull. Eur. Ass. Fish Pathol., 21(1) 2001, 11 Hattenberger-Baudouy, A.M., Danton, M., Merle, G., Torchy, C., de Kinkelin, P. () Serological evidence of infectious haematopoiic necrosis in rainbow trout from a French outbreak of disease. J. Aquat. Ani. Health 1: Jensen, M.H. (1965) Research on the virus of Egtved Disease. Ann. N. Y. Acad. Sci. 126: , P.E.V. (1972) Egtved virus: Antigenic variation in 76 isolates examined in neutralization tests and by means of the fluorescent antibody technique. In: Mawdesley-Thomas, L.E: (ed.) Diseases of fish. Academic Press, London, p , P.E.V. and Grauballe, P.C. (1971) Problems in serological typing of IPN virus. Acta. V. Scand. 12: , P.E.V. and Kehl, N.P. (1971) Infectious Pancreatic Necrosis (IPN) viruses in Danish rainbow trout. Their serological and pathogenic properties. Nord.V. Med. 23: , P. E. V., Olesen, N. J., Ahne, W., Lorenzen, N. () and PFR viruses: Serological examination of 22 strains indicates close relationship bween the two rhabdoviruses. In Ahne, W., Kurstak, E. (eds.): Viruses of lower vertebrates. Springer Verlag, Berlin, Heidelberg LaPatra, S.E., Fryer, J.L. and Rohovec, J.S. (1993) Virulence comparison of different electropherotypes of infectious hematopoiic necrosis virus. Dis. Aquat. Org. 16: LaPatra, S.E., Groberg, W.J., Rohovec, J.S. and Fryer, J.L. (1990) Size-related susceptibility of salmonids to two strains of Infectious Hematopoiic Necrosis Virus. Transactions of the Am. Fish. Soc. 119: Lorenzen, N., Olesen, N. J.,, P. E. V. (1988) Production and Characterization of Monoclonal Antibodies to Four Egtved Virus Structural Proteins. Dis.Aquat.Org. 4: Lorenzen, N., Olesen, N.J.,, P.E.V. (1990) Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. J. Gen. Vir. 71: Mortensen, H.F., Heuer, O.E., Lorenzen, N., Otte, L. and Olesen, N.J. (1999) Isolation of viral haemorrhagic septicaemia virus () from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea. Vir. res. 63: Nichol, S.T., Rowe, J.E. and Winton, J.R. (1995) Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoiic necrosis virus, a fish rhabdovirus. Virus Res. 38(2-3): Olesen, N. J.,, P. E. V., Bloch, B., Mellergaard, S. (1988) Isolation of an IPN-like virus belonging to the serogroup II of the aquatic birnaviruses from dab (Limanda limanda). J. Fish Dis. 11: Olesen, N.J., Lorenzen, N. and, P.E.V. (1993) Serological differences among isolates of viral haemorrhagic septicaemia virus dected by neutralizing monoclonal and polyclonal antibodies. Dis. Aquat. Org. 16: Western Hemisphere Committee on Animal Virus Characterization: Animal reference virus recommendations (1970) Am. J.V. Res. 31: Wolf, K., Gravell, M. and Malsberger, R.G. (1966) Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151: