Serum neutralization test for epidemiological studies of salmonid rhabdoviroses in France

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1 Serum neutralization test for epidemiological studies of salmonid rhabdoviroses in France Am Hattenberger-Baudouy, M Danton, G Merle, P De Kinkelin To cite this version: Am Hattenberger-Baudouy, M Danton, G Merle, P De Kinkelin. Serum neutralization test for epidemiological studies of salmonid rhabdoviroses in France. Veterinary Research, BioMed Central, 1995, 26 (5-6), pp <hal > HAL Id: hal Submitted on 1 Jan 1995 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Serum neutralization test for epidemiological studies of salmonid rhabdoviroses in France AM Hattenberger-Baudouy G Merle M Danton P de Kinkelin 1 CNEVA, laboratoire central de recherches vétérinaires, Maisons-Alfort cedex; 2 INRA, laboratoire de virologie et immunologie moléculaires, Jouy-en-Josas cedex, France Summary &horbar; Serological examination is not yet accepted as being a suitable diagnostic method for fish that are asymptomatic virus carriers. Nevertheless, encouraging preliminary results using an endpoint serum neutralization test (SNT) in several French trout farm populations have demonstrated an excellent correlation between the SNT and the previously established virus histories of the tested populations. Following the isolation of infectious haematopoietic necrosis virus (IHNV) in France, serological screening of fish for a neutralizing antibody (NAb) to IHN was conducted on a national scale. This survey confirmed the relationship between the serum-neutralizing immune response of the fish and the presence of IHNV in a given trout farm population. Insofar as many trout populations underwent dual rhabdovirus infections with both IHNV and viral haemorrhagic septicemia virus (VHSV), NAbs to both viruses were also detected in the fish from such populations, often in distinct individuals. NAb-responding fish became detectable 2-3 months post-infection (pi). The number of responding fish reached a mean prevalence of 20% between 3 and 6 months pi and disappeared after 8 months. The neutralizing serum titres (NST) were considered positive at > 32 and 64 for VHSV and IHNV, respectively. Both the NST results and the prevalence varied greatly according to individuals, populations and the number of repeated stimuli involved in a given serum sampling series. Conversely, the thousands of sera collected from trout in virus-free farms did not display any neutralizing activity against either VHSV or IHNV. The SNT thus seems to constitute a reliable tool for the assessment of the health status of trout farm populations for rhabdoviruses, and would be useful in the implementation of fish health surveillance programmes. rhabdovirose / salmonid / serology / serum neutralization / epidemiology Résumé &horbar; Épreuve de séroneutralisation pour l étude épidémiologique des rhabdoviroses chez les salmonidés. La sérologie n est pas encore une méthode diagnostique reconnue en ichtyopathologie. Cependant, nous avions enregistré, dans le passé, des résultats très encourageants dans le sérodiagnostic des rhabdoviroses par une microtechnique de séroneutralisation (SN). C est pourquoi, après que la nécrose hématopoïétique infectieuse (NHI) eut été identifiée en France, une enquête épidémiologique fondée sur la SN a été conduite au niveau national. Elle a confirmé une bonne corrélation entre la détection des anticorps neutralisants (AcN) et la présence des virus de la NHI dans les truites d une population donnée. Comme de nombreuses populations de truites connaissaient une * Correspondence and reprints

3 double infection rhabdovirale par les virus de la NHI et de la septicémie hémorragique virale, l épreuve de SN a révélé des AcN spécifiques de chacun des virus. Inversement, aucune réponse positive n a été obtenue à partir des milliers de sérums prélevés chez des truites originaires de piscicultures reconnues indemnes de rhabdoviroses pendant au moins les 4 années précédant l enquête. La SN apparaît donc comme une technique de choix dans la mise en service des programmes de surveillance sanitaire des piscicultures vis-à-vis des rhabdoviroses, maladies réputées contagieuses de la législation vétérinaire française. rhabdo viroses / salmonidés / sérologie l séroneutralisation l épidémiologie INTRODUCTION Serological examination is widely used as a diagnostic method for both human and animal infectious and parasitic diseases. It is not yet used for fish disease diagnosis. Antibodies (Abs) to various fish pathogens and especially to salmonid rhabdoviruses were discovered over 20 years ago, under both natural and experimental conditions (Vestergaard-Jorgensen, 1971, 1974; Amend and Smith, 1974; de Kinkelin et al, 1977a, b; Bernard etal, 1983; Hattenberger- Baudouy ef al, 1989; Olesen et al, 1991 ). The demonstration of trout complement dependence of serum neutralization (SN) of viral haemorrhagic septicaemia virus (VHSV) (Dorson and Torchy, 1979) enabled the routine use of an SN test (SNT). Nevertheless, the use of serological examination as a diagnostic tool for determining the virus status of trout populations was not included in the guidelines for either the national and international animal health authorities, despite its recommendations in the International Animal Health Code of the Office international des epizooties (1986). This reluctance towards using serological diagnosis for certain fish diseases may be due in part to the somewhat limited Ab repertoire reported for fish (Du Pasquier, 1982). In addition, the current dogma inherited from the early fish pathologists is a belief that the direct isolation of the virus is the best diagnostic method. There is also a real need for more information about the immune responses of fish to rhabdoviruses, based on more field data. The preliminary results of an end-point SNT used in field assays and experimental conditions, demonstrated the utility of serological studies at the population level for the detection of viral infections. This work aims to demonstrate, using thousands of SNT results from fish populations with previously known and unknown virus histories, the correlation between the results of virological and serological methods. The vast scope of the study also allowed us to assess the virus status of trout farm populations and thus provided a convenient tool for the descriptive epidemiology of trout rhabdoviroses. MATERIALS AND METHODS Cell lines The fish cell line, epithelioma papulosum cyprini, (EPC), derived from the common carp Cyprinus carpio (Fijan et al, 1983) was used for the isolation, propagation and identification of the virus isolates used in this study, as well as for the serum-neutralization tests. The cell line, rainbow trout gonad (ATCC CCL55) RTG-2 (Wolf and Quimby, 1962), was used for certain virus isolation procedures. Both lines were propagated in Stoker s medium (Gibco Life Technologies Ltd, Parsley, Scotland, UK), buffered to ph 7.45 with 0.16 M Tris-HCI (Sigma Chemical Company, Saint Louis, MO, USA) and supplemented with 10% fetal bovine serum (FBS) (Gibco) and other ingredients as previously described (Hattenberger-Baudouy etal, 1989). For the routine cell propagation, the EPC and RTG-2 cells were incubated at 29 and 20 C, respectively.

4 Virus strains, virus propagation and isolation The virus strains used, and the techniques for their propagation and isolation were identical to those previously reported (Hattenberger et al, 1989). Briefly, the French isolate of VHSV, INRA belonging to serotype 1 (Vestergaard-Jorgensen, 1972) and 2 IHNV isolates were used in the SN tests. The IHNV isolates were the North American virus isolate 1266 from rainbow trout donated by D Amend (Seattle, WA, USA) in 1970 and the French isolate (de Kinkelin et al, 1987). All 3 virus isolates were propagated in EPC cells incubated at 15 C in Stoker s medium with 2% FBS. The supernatants from the infected cells were centrifuged, filtered, aliquoted and frozen at -70 C to await use as SNT antigens. Virus isolation and identification from either diseased fish or asymptomatic virus carriers were performed according to the previously described methods (de Kinkelin et al. 1985). Serum neutralization tests The SNT were performed according to an endpoint technique conducted in cell culture microplates. The technique was derived from one previously described (Hattenberger-Baudouy et al, 1989) with modifications consisting of the use of flat-bottomed microplate wells, immunized trout sera as positive controls for the SN to IHNV instead of rabbit serum, and the adjustment of the virus titres of neutralization virus antigen in order to generate a clearcut cytopathic effect in the serum-negative control cells within h. These virus titres were usually lower than those previously observed, ie pfu and pfu per 25 pl. Fish, trout farms, sampling procedures and objectives Blood samples were collected from more than fish. The majority of the fish were rainbow trout (Oncorhynchus mykiss) but 5% of the samples were from brown trout (Salmo trutta) and less than 1 % were from brook trout (Salvelinus fontinalis). The number of serum samples varied from 18 to more than 180 per population. At 196 of the sites, 60 samples were taken. In most cases the blood samples were taken from fish more than one year old and were generally (60% of blood sample) from broodfish. When the dual NAb screening for VHSV and IHNV is taken into consideration, the final number of SNT performed reached slightly more than The materials sampled for virological examination were taken from the kidney, spleen and brain or were sexual fluids. The organs from 5 fish were pooled before processing, the brains being sometimes kept apart from the kidneys and spleens. The ovarian fluids and milt were always individually tested for the virus. This resulted in more than virological examinations. The sampling campaign included trout populations from 229 farming sites located in all the regions of France involved in salmonid fish culture. Prior to the beginning of the study, the virus history of about 2/3 of the farming sites visited had been previously established (within the last 4 years) on the basis of virological examinations made either at the farmer s request during periods of disease outbreaks or as part of an official fish health surveillance programme. This, therefore, allowed comparisons between the data resulting from the serological examination and the information provided by internationally agreed methods which utilized virus isolation as their central criterion. Once the sampling for the national survey was completed, particular aspects of the serological response of trout populations to rhabdoviruses were reinvestigated. This was conducted at several farming sites (table I) in order to gather data that supported the use of the SNT as a diagnostic method for the implementation of fish health surveillance programmes. At sites Ne and Lu, the course of the Nab response in trout fingerlings to VHS following an outbreak of overt disease, was studied and at the site Lu again, the SNT was used 3 years later for casual diagnosis of VHS. Finally, at site Ro in 1993, simultaneous screening for rhabdoviruses was conducted in brood fish using both virological examination and the SNT. This last study was aimed at demonstrating the efficacy of using SNT alone as a diagnostic method for evaluating the virus status of a trout population.

5 RESULTS Serological survey for IHN and the detection of dual NAb responses to rhabdoviruses The screening of rainbow trout populations from 229 farming sites for NAbs to the IHNV provided positive results with groups of serum batches from 35 of the sites (table II). Eighteen of these sites had between 5 and 20% of the responding fish providing titres ranging from 64 to 512. In 6 of the responding trout populations tested, IHNV was isolated prior to the serological samplings. Conversely, due to the constraints of French legislation, only 13 out of the 29 were checked for the virus during the months following the positive serological examination. Nevertheless, IHNV was recovered from fish at all the sites, either during overt IHN outbreaks or during the screening of sexual fluids from broodfish. Ten of the sites with IHN-responding fish were checked for NAbs to VHSV. Five of these sites were also found positive for this virus, sometimes with a higher prevalence than that found for IHNV. Indeed the example shown in table III indicates prevalences of Nab responses at 27.5 ± 10 and 8.5 ± 6% for VHS and IHN, respectively. In contrast, none of the sera collected in trout populations from the 103 trout farms operating in rhabdovirus-free zones displayed neutralizing activity against IHNV. Similarly, the virus screening of the ovarian fluids from rainbow and brown trout broodfish from this zone was negative. Sampling for VHSV performed with 10 sera from each of 60 trout farm populations was also negative. Response of naive rainbow trout fingerlings to VHSV In 1989, because of the restocking of VHSV carriers upstream, overt disease occurred

6 in a formerly virus-free trout farm (Ne) that had been supplying our laboratory with normal trout serum as the source of trout complement since VHSV was isolated from the diseased trout about 8 d after the onset of mortality. The periodic SNT tests of 60 fish were negative both 1 month prior to the virological diagnosis and 3 weeks afterwards. Seroconversion was demonstrated at month 4 in 29/60 fish with titres of , whereas the virological diagnosis was negative and dropped to 1 at month 7. The rainbow trout fingerlings response was also studied in 1989 at another trout farm that had a long history of VHS (Lu). The fish became infected after they were transferred from the virus-free indoor facilities to the outdoor ponds. Virological diagnosis performed under the same conditions as those indicated above was initially positive and became negative at month 4 after the onset of clinical signs, whereas NAbs were detectable in 12/60 fish within titres ranging from 32 to 512. At month 8, only 1 fish serum reacted to VHSV with a titre of 64 (not shown). Casual use of serological diagnosis of VHS at a trout farm During a visit to the above farm, Lu, in 1992, the fish farmer randomly netted 12 ninemonth-old rainbow trout that had survived a VHS attack 5 months earlier (lot 1) and similarly captured 30 six-month-old trout from another lot (2) in which some individuals had undergone overt VHS (table IV).

7 No virus was recovered from the fish in lot 1 but half of them responded serologically, whereas in lot 2, VHSV was easily isolated from the infected organ homogenates and low titres of NAbs were detected in only 2 sera. Both virological and serological examinations were IHN negative. Simultaneous use of virus isolation and SNT in the diagnosis of asymptomatic rhabdovirus carriers A virus assay of the ovarian fluids and a SNT were performed on 60 rainbow trout broodfish at trout farm Ro, a site with a wellestablished history of rhabdoviroses. The results revealed that 14 fish harboured VHSV. Ten fish displayed high NAb titres to VHSV. The fish 11 displayed a positive response to both the serological and the virological tests. Two fish, 47 and 48, reacted serologically with IHNV with titre of 64 and >_ 512 respectively (table V). All samples were negative for IHNV isolation. DISCUSSION The serological screening of trout using the end-point neutralization technique, which was the basis for the national survey for IHN conducted in France in , confirmed the correlation between the NAb response and the presence of IHNV in a given trout farm stock as previously reported (Hattenberger-Baudouy et al, 1989, 1995). Insofar as several trout populations underwent dual rhabdoviroses (VHS and IHN), NAbs to both viruses were also detected in the fish from such populations. In contrast, no neutralizing titres higher than 32 and 16 for IHNV and VHSV, respectively, were demonstrated in sera collected from the trout populations of virus-free origin. This allowed us to consider the responses of NAb titres 64 for IHNV and 32 for VHSV as positive. Thus, each time the SNT and virus assay in cell culture were used either simultaneously or separately with material from a given trout population, the virological and serological results correlated well, establishing the health status of the given population. The scale of our field investigations constitutes one of the largest epidemiological studies of rhabdoviroses ever undertaken. These studies were made necessary by the implementation of fish surveillance programmes at both the national and international levels. The data collected were both informative and predictive. Indeed, in several cases, the NAb response of one trout population, especially in the broodfish, preceded

8 the onset of overt rhabdovirus infection some months later. An example of this occurred at site Ro where the response of asymptomatic fish to IHNV was followed by several episodes of clinical infection among the young trout that were the progeny of the broodfish that had responded positively. The results of the SNT used during the course of field VHSV infections in naive rainbow trout fingerlings confirmed the acquired aspect of the NAb response to VHSV previously found during the national survey. At water temperatures of 9-11 C, the NAbs that were detected 4 months post-infection at sites Ne and Lu 89 likely appeared after 6-8 weeks as had been found at site Lu in These results are in agreement with those previously reported under experimental and natural conditions (Bernard et al, 1983; Olesen et al, 1991 The data in table IV show that the virus assay method encounters limitations when used for the

9 causal diagnosis of VHS in an asymptomatic fish population. Conversely, limitations to STN appear if it is used too early in the course of a natural infection. Nevertheless, at the population level on trout farms there was a good correlation between the virological and serological results which demonstrates the utility of the SNT as a screening method for VHSV in surveillance programs. The SNT method we used in this study was specific but less sensitive than the SN microplaque technique (data not shown) described by Olesen and Vestergaard-Jorgensen (1986). The 2 tests provide similar neutralization patterns although the titres and number of positive are lower with our end-point SNT. However, the ability of the end-point SNT to examine large numbers of samples makes it highly informative at the population level. In addition, we have shown that this test provides results that correlate well with those generated by the virus isolation technique (table V) particularly when the sample sizes are sufficiently large. According to existing sampling charts, 60 is a suitable sample size (Ossiander and Wedemeyer, 1973). We found that the neutralizing titres and the prevalence of positive responses varied greatly with individuals, the frequency of virus exposure and the timing of a given fish sample. The risk of obtaining false negative responses could be reduced by selecting a specific class of fish for the sampling from a population rather than by testing randomly. Older fish, particularly broodfish, are the most suitable targets. If this age group does not exist at a given farming site, an alternative is to sample those fish that have been transferred to the outdoor ponds 3-6 months prior to the serological sampling date. When comparing the serological versus virus isolation approaches it is apparent that the SNT is more effective with asymptomatic virus carriers unless the virus can be recovered from the sexual products at the time of spawning. The virological examinations of (nonspawning) carriers are laborious, costly and less reliable than the SNT to assess the virus status of such populations. Furthermore, the somewhat lower sensitivity of the end-point SNT diminishes the risk of finding false positive responses compared with the plaque neutralization approach. Attempts at substituting a quicker and perhaps more up-to-date serological diagnostic method for rhabdoviruses, such as the ELISA test, have been made (Vestergaard-Jorgensen et al, 1991 These tests, however, lack antivirus specificity. This supports the continued use of the SNT as a serological diagnostic method. SNT is gaining acceptance as an indicator of exposure to IHNV in the USA (La- Patra et al, 1993, 1994), but so far no offi- cial agreement has been made for its use as a fish health assessment method for rhabdoviruses, despite the utility of this technique for examining the fish during periods when virus isolation would not be productive. On the basis of our present work, SNT, as a serological diagnostic method, should be considered as an approach that is of equal value and also complementary to the virus isolation method for use in virus surveillance programmes. ACKNOWLEDGMENTS The collaboration and help of the following persons, for the collection and processing of the samples is warmly acknowledged: J Castric and staff CNEVA/Brest, J Catel, M Morand, P Nougayrbde, A Vigouroux and the respective staffs of the veterinary laboratories of Pas-de- Calais, Jura, Landes and Finistere. I Clement is also acknowledged for her excellent typing work.

10 REFERENCES Amend DF, Smith L (1974) Pathophysiology of infectious hematopoietic necrosis virus disease in rainbow trout (Salmo gairdnen): early changes in blood and aspects of the immune response after injection of IHN virus. J Fish Res Board Can 31, Bernard J, de Kinkelin P, Bearzotti M (1983) Viral hemorrhagic septicemia of trout: relation between the G polypeptide, antibodies production and protection of the fish following infection with the F25 attenuated variant strain. Infect and Immun 39, 7-14 Dorson M, Torchy C (1979) Complement dependent neutralization of Egtved virus by trout antibodies. J Fish Dis 2, Du Pasquier L (1982) Antibody diversity in lower vertebrates - Why is it so restricted? Nature (Lond) 296, 31 1 Fijan N, Sulimanovic D, Bearzotti M et al (1983) Some properties of the epithelioma papulosum cyprini (EPC) cell line from carp (Cyprinus carpio). Ann Virol (Institut Pasteur) 134 E, Hattenberger-Baudouy AM, Danton M, Merle G, Torchy C, de Kinkelin P (1989) Serological evidence of infectious hematopoietic necrosis in rainbow trout from a French outbreak of disease. J Aquat Anim Health 1, Hattenberger-Baudouy AM, Danton M, Merle G, de Kinkelin P (1995) Epidemiologie de la necrose hbmatopoibtique infectieuse (NHI) des salmonides en France : suivi de l infection naturelle par des techniques virologiques et s6rologiques et tentatives d eradication. Vet Res 26, de Kinkelin P, Baudouy AM, Le Berre M (1977a) Reaction de la truite Fario (salmo trutta) et arc-en-ciel (Salmo gairdnen) a I infection par un nouveau rhabdovirus. CR Acad Sc Paris S6rie D, 284, de Kinkelin P, Gerard JP, Dorson M, Le Berre M (1977b) Viral haemorrhagic septicaemia: demonstration of a protective immune response following natural infection. Fish Health News 6, 3-4 de Kinkelin P, Hattenberger AM, Torchy C, Lieffrig F (1987) Infectious haematopoietic necrosis (IHN): first report in Europe. European Association of Fish Pathologists, Third international conference (Abstract 57) de Kinkelin P, Michel C, Ghittino P (eds) (1985) Precis de pathologie des poissons. INRA, Office international des epizooties. Paris, 348 p LaPatra SE, Turner T, Lauda KA, Walker SC, Jones GR (1993) Characterization of the humoral response of rainbow trout to infectious hematopoietic necrosis virus. J Aquat Anim Health 5, LaPatra SE, Lauda KA, Jones GR, Shewmaker WD, Walker SC (1994) Development of passive immunotherapy for control of infectious hematopoietic necrosis. Dis Aquat Org 20, 1-6 Office international des 6pizooties (1986) International Animal Health Code, Recommended norms for the pathogens of fish. Vth edition, 364, 1-23 Olesen NJ, Vestergaard-Jorgensen PE (1986) Detection of neutralizing antibodies to Egtved virus in rainbow trout (Salmo gairdnen) by plaque neutralization test with complement additions. J/tpp/ /enrhyo<. 2, Olesen NJ, Lorenzen N, Vestergaard-Jorgensen PE (1991) Detection of rainbow trout antibody to Egtved virus by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and plaque neutralization tests (50% PNT). Dis Aquat Org 10, Ossiander JF, Wedemeyer G (1973) Computer program for sampling sizes required to determine disease incidence in fish populations. J Fish Res Board Can 30, Vestergaard-Jorgensen PE (1971) Egtved virus: demonstration of neutralizing antibodies in serum from artificially infected rainbow trout. J Fish Res Board Can 28, Vestergaard-Jorgensen PE (1972) Egtved virus: antigenic variations in 76 virus isolates examined on neutralization tests and by the means of the fluorescent antibody technique. In: Symposia of the Zoological Society of London (LE Mawdeslex-Thomas, ed), Academic Press, UK, 30, Vestergaard-Jorgensen PE (1974) Indirect fluorescent antibody techniques for demonstration of trout viruses and corresponding antibodies. Acta Vet Scand 15, Vestergaard-Jorgensen PE, Olesen NJ, Lorenzen N, Winton JR, Ristow SS (1991) Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): detection of trout antibodies to the causative viruses by the means of plaque neutralization, immunofluorescence and enzyme-linked immunosorbent assay. J Aquat Anim Health 3, Wolf K, Quimby MC (1962) Established eurythermic line of fish cell in vitro. Science 135,