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Susceptibility of genotyped marble trout Salmo marmoratus (Cuvier, 1829) strains to experimental challenge with European viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV)
Aquaculture 435 (2015) 152–156
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Susceptibility of genotyped marble trout Salmo marmoratus (Cuvier, 1829) strains to experimental challenge with European viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) F. Pascoli a,⁎, F. Bilò b, F. Nonnis Marzano c, F. Borghesan a, M. Mancin d, A. Manfrin a, A. Toffan a a
Istituto Zooprofilattico Sperimentale delle Venezie, Centro di Referenza Nazionale (NRL) per le patologie dei pesci, molluschi e crostacei, Legnaro (PD), Italy Veneto Agricoltura, Legnaro (PD), Italy c Dipartimento di Bioscienze, Università degli Studi di Parma, Viale delle Scienze 11, Parma, Italy d Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio di Analisi del rischio e sistemi di sorveglianza in sanità pubblica, Legnaro (PD), Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 21 September 2014 Accepted 22 September 2014 Available online 28 September 2014 Keywords: Virus VHS IHN Salmo marmoratus Susceptibility
a b s t r a c t The marble trout Salmo marmoratus (Cuvier, 1829) is an endemic species of great conservation concern, given its geographical restricted distribution and the high risk of hybridization with brown trout Salmo trutta L. Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are predominantly diseases of freshwater salmonids, which may trigger off severe disease outbreaks causing high mortality. Marble trout has not been inserted in the vector species list for those pathogens, but at present, little information on its susceptibility and on the possible carrier status is available. The aim of the present study was to assess the ability of European strains of VHSV and IHNV to cause disease and associated mortality in experimentally infected marble trout, and to determine whether a carrier status can result after experimental infection. Three genetically different strains of marble trout were challenged with VHSV and IHNV by immersion. Rainbow trouts were used as positive controls. Fish were checked twice a day, and dead fish were removed and sampled for subsequent virological analyses. With respect to rainbow trout, results demonstrated a very low susceptibility of marble trout to both VHSV and IHNV under simulated natural conditions. The presence of a certain number of chronically infected marble trout confirms that some individuals can survive the infection and eventually act as carrier of the disease, suggesting that marble trout should be definitely added to the list of susceptible species according to the European legislation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The marble trout Salmo marmoratus (Cuvier, 1829) is an endemic species of great conservation concern, given the high risk of hybridization with brown trout Salmo trutta L. (Meraner et al., 2010) and its geographical distribution restricted to the Po basin in northern Italy (Forneris et al., 1990; Kottelat and Freyhof, 2007; Sommani, 1961), the Adriatic basin of Slovenia, Croatia, Bosnia-Herzegovina (Povz et al., 1996) and to Albania (Schoffmann, 1994). Since the beginning of last century, brown trout have been introduced for commercial and recreational purposes in the same geographic regions, quickly leading to the creation of hybrid populations (Giuffra et al., 1996; Povz et al., 1996). Due to the severe hybridization with the introduced brown trout (Berrebi et al., 2000; Meraner et al., 2010), marble trout is therefore ⁎ Corresponding author at: Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università, 10, 35020, Legnaro (PD), Italy. Tel.: +39 0498084388. E-mail address: [email protected] (F. Pascoli).
http://dx.doi.org/10.1016/j.aquaculture.2014.09.038 0044-8486/© 2014 Elsevier B.V. All rights reserved.
considered highly endangered. This species is now farmed in northern Italy with the main purpose of restocking freshwater basins with selected individuals by means of appropriate investigations of population genetics. At the same time, intensive aquaculture practices drive major attention to sanitary issues regulated by European directives. In particular, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are among the most important pathogens for freshwater aquaculture. They are RNA viruses belonging to the family Rhabdoviridae, which is the largest group of pathogenic fish viruses. Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are predominantly diseases of freshwater salmonids, which may cause severe disease outbreaks with high mortality (Hoffmann et al., 2005; Wolf, 1988). In particular, VHS is the most serious viral disease affecting the production of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) in continental Europe (Olesen et al., 1999), causing high mortality rates in all sizes of fish. IHN was originally endemic in North America but has gradually transferred to Europe and Asia by the movement of infected fish or eggs. It typically
F. Pascoli et al. / Aquaculture 435 (2015) 152–156
occurs in rainbow trout fry and fingerlings, but the disease has also been reported in various life stages of sockeye, chinook and Atlantic salmon (Lapatra, 1998). The virus was first detected in Italy in 1987 (Bovo et al., 1987). Both diseases are listed as being notifiable, according to the European legislation (2006/88/EC) and to the World Organization for Animal Health (Office International des Epizooties, OIE). A list of susceptible and vector species for both diseases is available (Dir. 2006/88/EC and Reg. 2008/1251/EC). Marble trout has not been inserted in the vector species list, but at present, little information is available on the susceptibility of marble trout to those pathogens, nor on the possible carrier status of this species. The aim of the present study was to assess the ability of European strains of VHSV and IHNV to produce disease and associated mortality in experimentally infected marble trout and to determine whether a carrier status can result in this species after experimental infection. The investigation was carried out on three different marble trout strains previously genotyped with mitochondrial and nuclear markers. The susceptibility of marble trout to each pathogen was compared to that of an already known sensitive species such as the rainbow trout O. mykiss. 2. Materials and methods 2.1. Fish Juvenile marble trouts were hatched in a category I regional facility, according to the 2006/88/EC: the Centro Ittico Valdastico (Veneto Agricoltura, Valdastico-VI, Italy). Eight hundred and ten marble trouts were used in this study, all belonging to three different local strains named after the parents river basin of provenience and genetic characterization: Adige, Brenta and Piave. The fish were transferred to the experimental aquarium at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Legnaro-PD, Italy) at the mean size of 4.3 ± 1 g. Two hundred and seventy rainbow trouts (mean weight 3.8 ± 1 g) were provided from a commercial category I fish farm of northern Italy, according to the 2006/88/EC. Upon arrival, all fish (rainbow and marble trouts) were screened for VHS and IHN infection by virus isolation and Real time RT-PCR (rRT-PCR). Fish were gradually acclimatized to the aquarium tanks and to the trial temperatures (12 ± 1 °C) for 2 weeks before challenge. 2.2. Mitochondrial and nuclear DNA analyses Ninety specimens of wild marble trout collected from 3 different Italian rivers (30 from the river Adige, 30 from the Brenta, and 30 from the Piave) were moved to the Centro Ittico Valdastico and adapted to captivity. Samples were collected by means of non-invasive procedures. In particular, a small fin fragment (about 1 mm2) was sampled from each individual submitted to narcosis and stored in 70% ethanol under refrigerated conditions. After DNA extraction and purification, samples underwent genetic characterization by means of mitochondrial D-loop SNPs analyses, besides nuclear LDH-C1. Subsequent AFLP and microsatellite genotyping was performed to assess population differentiation. 2.3. Virus Viruses were selected according to their high pathogenicity for fish: both strains, VHS 80/V10 and IHN 409/V06, were isolated from severely affected rainbow trout during an outbreak in commercial farms in northern Italy in 2010 and 2006, respectively. Both viruses had been previously used in the IZSVe experimental aquarium, showing a mortality rate of 50% for VHS and 30% for IHN by bath exposure in rainbow trout. VHSV 80/V10 genetically belongs to the Ia genotype, the most common in European fresh water isolates according to the current
153
classification (Einer-Jensen et al., 2004); IHNV 409/V06 clustered in the M genotype, European lineage (Kurath, 2012), again the most widespread genotype in Europe. Isolates were propagated on EPC cells (Fijan et al., 1983) for IHNV and on BF-2 cells (Wolf and Quimby, 1962) for VHSV, in 150 cm2 tissue culture flasks. The collected viruses were subjected to titration by end point dilutions assays. Titres were calculated according to the SpearmanKarber formula (Finney, 1978) and expressed as TCID50/ml. 2.4. Challenge procedures Experimental fish were challenged with VHSV (80/V10) and IHNV (409/V06) by immersion. Rainbow trouts were used as positive controls. Each strain of marble trout (Adige, Brenta and Piave) was treated separately. Ninety fish groups of marble trout and rainbow trout were infected by adding 50 mL of virus solution (approximately 107.05 TCID50/ml VHSV and 107.55 TCID50/ml IHNV) to 10 L of water and adding the fish for 4 h in closed systems with aeration. Each group was then placed in a separate 200-L aquarium and maintained at a temperature of 12 ± 1 °C. An additional group of each fish species and strain was mock infected by immersion containing MEM-10 without virus to act as negative control. Fish were checked twice a day and dead fish were removed. Samples of spleen, head kidney and heart were collected individually from each dead fish and stored at −80 °C. The experiment was ended on day 30. All remaining fish were euthanized by an overdose of MS-222, sampled and stored at −80 °C until analyses. The experimental protocol was evaluated and approved by the Internal Ethic Commission and the Italian Ministry of Health. 2.5. Virological analysis Samples were examined by standard virological techniques, as described in Commission Decision 2001/183/EC. Briefly, spleen, heart and head kidney were homogenized in a mortar with a pestle and sterile sand. The samples were diluted 1:10 in transport medium (Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 2% of antibiotic/antimycotic solution–10.000 IU/ml penicillin G, 10 mg/ml streptomycin sulfate, 25 μg/ml amphotericin B–0.4% of 50 mg/ml kanamycin solution) and centrifuged at 4000 g at 4 °C for 15 min. Ten percent of antibiotic/antimycotic solution was added to the supernatant and the samples incubated at 4 °C overnight. The 24-wells plates with 24 h old EPC and BF-2 cell lines were inoculated with samples in final dilutions 1:100 and 1:1000. The inoculated cell cultures were incubated at 15 °C for 1 week, with regular examination for cytopathic effect (CPE). After 1 week, samples with no CPE were re-inoculated into a new culture of EPC cells. If no CPE was observed at the end of the 2nd week, the sample was considered as being negative for the virus. Cell cultures with positive CPE were stored at −80 °C. The rRT-PCR analyses were performed according to the published protocol (Jonstrup et al., 2013; Overturt et al., 2001) for VHS and IHN. 2.6. Statistical analysis Mortality data were reported as mean ± SD in term of percentage. Each fish in the study was followed over time and the event “death” was recorded and verified by PCR to make a distinction between “death because of infection” and “death for other causes.” Two different survival analyses were performed to evaluate the event “dead” and “ dead for infection.” In this second case and in accordance to the Cox model, the dead fish with positive PCR were considered as the “event,” while those with negative PCR were considered as “censured data.” To analyze the survival function from lifetime data, the Kaplan– Meier estimator was used, which allows drawing the survival curve as
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a step curve for each group, measuring the length of time the fish survives the infection. In the graph, the y-axis plots the cumulative probability of the surviving fish at each time (van Belle et al., 2004). The semi-parametric Cox model was used to compare the survival function in the case of verified proportional hazards, taking into account the possible effect of species, treatment and interaction between these two variables. In the case of overlapped curves, the nonparametric log-rank test was applied to verify the equality of survivor functions across the groups (van Belle et al., 2004). Statistical analyses were performed using STATA 12 software. 3. Results 3.1. Mitochondrial and nuclear DNA analyses Fig. 2. Kaplan–Meier survival function from lifetime data for VHS infected fish.
Of 90 samples of marble trout, 4 (3 hybrid trouts from the Adige strain and 1 from the Piave population) were discharged after molecular characterization, which resulted in an introgression with brown trout alleles either at the D-Loop mitochondrial region or in nuclear LDHC1. The remaining 86 samples underwent additional genotyping at 12 microsatellite loci and AFLPs to define population differentiation. The three groups were completely separated, as assessed by bioinformatics parameters. These fish were used as broodstock for the production of genetically known fingerlings to restock rivers of origin and to perform the experimental infection described in this paper (Fig. 1).
3.2.2. IHN Also in the group challenged with IHNV a low mortality was recorded for all the strains of marble trout (3.2 ± 2.1%), with no differences among strains (p N 0.05; Fig. 3). Challenge of rainbow trout resulted in 28.4% of mortality (Fig. 3). Mortality started 4 days post-challenge and continued until the termination of the trial, with similar but milder clinical signs than those noted for VHS. In all challenged fish, IHNV was confirmed by rRT-PCR, as reported in Table 1. IHNV was re-isolated in all the rainbow trout tested, whereas a mean of 30% occurred in marble trout.
3.2. Challenge with VHSV and IHNV
4. Discussion
Among all the marble trouts, a little higher mortality was recorded in the Piave strain of all groups, including the negative control. The Cox regression analysis showed that the Piave strain runs a significantly higher risk of mortality if compared to the other strains (p b 0.05), independently from the treatment. Therefore, this event may be considered as a physiological mortality, probably due to a higher susceptibility of that strain to husbandry, as recorded at the Centro Ittico Valdastico in the same period (personal communication). For this reason, we reported the mortality rate considering “death for infection” only the rRT-PCR positive dead fish.
Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are the two most economically important viral fish diseases, particularly in salmonid farming. Viruses with a broad hostrange may pose a risk for new species in fish farming; however, strains which are virulent in one species may have low pathogenicity in another. In aquaculture, the identification of disease resistant species of fish is an important step toward the development of healthier stocks. Marble trout are of great economic importance in northern Italy, considering the widespread release into the wild for conservation purposes. This species is also valued by anglers for its shiny and wary mood, which makes it difficult to fish. Due to the ecological and economic significance of this species, a preliminary study on the resistance of marble trout to viral diseases was carried out some years ago in Italy (Borghesan et al., 2004). This species appeared more resistant than rainbow trout to VHS infection, but as susceptible as this latter for IHN infection. Unfortunately, results were biased by testing dead fish in pool and only with viral isolation methods; therefore, the obtained data cannot be considered exhaustive and additional investigations are necessary. As a matter of fact, the present study demonstrated a lower susceptibility of marble trout to both VHSV and IHNV under simulated natural conditions if compared to the one registered in rainbow trout. The low
3.2.1. VHS In the group challenged with VHSV, a low mortality was recorded for all the tested strains of marble trout (7.1 ± 0.5%), with no differences among strains (p N 0.05; Fig. 2). Challenge of rainbow trout resulted in 82.5% of mortality (Fig. 2), which started 4 days post-challenge in those fish manifesting the typical clinical signs (i.e., darkening, pop eyes, abnormal swimming) and stopped at the termination of the trial. In all challenged fish, VHSV was confirmed by rRT-PCR as reported in Table 1. VHSV was re-isolated in all rainbow trout tested, whereas a mean of 40% occurred in marble trout.
Fig. 1. Specimen of a broodstock marble trout (Salmo marmoratus) farmed in Centro Ittico Valdastico (Veneto Agricoltura, Valdastico, Vicenza) showing the typical color pattern. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Table 1 Summary of mortality and positivity to rRT-PCR for VHSV and IHNV in rainbow trout and marble trout experimentally challenged. Data are reported as positive to rRT PCR/total fish. Species
Strain
Virus
Positive fish/dead fish
% positive fish/dead fish
Positive fish/survivors fish
% positive fish/survivors
Rainbow trout Marble trout Marble trout Marble trout Rainbow trout Marble trout Marble trout Marble trout Rainbow trout Marble trout Marble trout Marble trout
– Adige Brenta Piave Adige Brenta Piave – Adige Brenta Piave
IHN IHN IHN IHN VHS VHS VHS VHS Negative control Negative control Negative control Negative control
26/26 2/4 5/7 1/12 74/74 6/7 7/7 6/14 0/1 0/0 0/3 0/14
100.0 50.0 71.4 8.3 100.0 85.7 100.0 42.9 0 0 0 0
2/64 1/86 0/83 0/74 12/16 0/83 1/83 2/76 0/89 0/90 0/87 0/76
3.8 1.0 0.0 0.0 75.0 0.0 1.2 2.6 0 0 0 0
susceptibility was assessed by means of two independent laboratory tests (virus isolation and rRT-PCR) and supported by statistical analysis. Similar results were obtained in other species and other viruses (Follett et al., 1997; McAllister et al., 2000), where very low mortality rates were found in experimentally infected fish, which highlighted the importance of subclinical reservoir species in transferring viral diseases. Moreover, precautions should be taken to prevent possible adaptation of the viruses to this species, as occurred elsewhere (Follett et al., 1997). Marble trout is a species of great conservation concern due to the high risk of hybridization with the introduced brown trout, as highlighted by the mitochondrial and nuclear DNA analyses performed. As a matter of fact, 4.5% of the tested fish proved to be hybrids although a strict phenotype selection had been carried out. Despite the clear genetic differences observed among the three marble trout populations under study, no differences were detected for disease susceptibility, at least not for the two viruses tested in the present study. Unfortunately, the presence of a certain number, although small, of chronically infected marble trout confirms that some individuals can survive the infection and eventually act as carrier of the disease. Additional work could be required to confirm whether IHNV and VHSV can persist in marble trout for a period of time longer than the one considered in this work (30 day post infection) and to assess the potential risk of IHN and VHS survivors to act as a reservoir. It could be important, for example, to determine the ability of chronically infected fish to transmit the viruses to susceptible species through cohabitation trial, as suggested by St-Hilaire et al. (2001) for Atlantic salmon. Anyway, according to the definition given by EFSA (2007) and by the Aquatic Animal Health code (OIE 2014; Chapter 1.5), in which a susceptible species means any species in which infection by a disease agent has been demonstrated by natural cases or by experimental infection that mimics the natural pathways, marble trout should definitely be added to the list of species susceptible to VHS and IHN.
Fig. 3. Kaplan–Meier survival function from lifetime data for IHN infected fish.
5. Conclusions In conclusion, this study showed the very low susceptibility of marble trout to European strains of VHSV and IHNV under experimental conditions. These data are of great interest if we consider the importance of this species in the restocking programs. Releasing into the wild a species less susceptible to viral diseases than the rainbow trout could reduce the prevalence of these pathogens and maintain the local biodiversity. Acknowledgments Authors want to thank all the laboratory technicians for their precise support, especially Dr. Rosita Quartesan. Special thanks also to Francesca Ellero for the English review of the manuscript. This study was financially supported by Italian Ministry of Health RC IZSVE 21/11. References 2001/183/EC, 2001. Commission Decision of 22 February 2001 laying down the sampling plans and diagnostic methods for the detection and confirmation of certain fish diseases and repealing Decision 92/532/EEC. 2006/88/EC, 2006. Council Directive of 24 October 2006 on animal health requirements for aquaculture animals and products thereof, and on the prevention and control of certain diseases in aquatic animals. 2008/1251/EC, 2008. Commission Regulation of 12 December 2008 implementing Council Directive 2006/88/EC as regards conditions and certification requirements for the placing on the market and the import into the Community of aquaculture animals and products thereof and laying down a list of vector species.. Berrebi, P., Povz, M., Jesensek, D., Crivelli, A.J., 2000. The genetic diversity of native, stocked and hybrid populations of Marble trout in the Soca River, Slovenia. Heredity 85, 277–287. Borghesan, F., Schiavon, E., Selli, L., Manfrin, A., Cappellozza, E., Quartesan, R., Bovo, G., 2004. Susceptibility of marble trout (Salmo [trutta] marmoratus) to viral haemorrhagic septicemia and infectious haematopoietic necrosis: preliminary observations. Ittiopatologia 1, 5–14. Bovo, G., Giorgetti, G., Jørgensen, P.E.V., Olesen, N.J., 1987. Infectious haematopoietic necrosis: first detection in Italy. Bull. Eur. Assoc. Fish Pathol. 7 (5), 124. EFSA, 2007. Scientific Opinion of the Panel on Animal Health and Welfare on a request from the European Commission on possible vector species and live stages of susceptible species not transmitting disease as regards certain fish diseases. EFSA J. 584, 1–163. Einer-Jensen, K., Ahrens, P., Forsberg, R., Lorenzen, N., 2004. Evolution of the fish rhabdovirus viral haemorragic septicaemia virus. J. Gen. Virol. 85, 1167–1179. Fijan, N., Sulimanovic, D., Bearzotti, M., Muzinic, D., Zwillenberg, L.O., Chilmonczyk, S., Vautherot, J.F., de Kinkelin, P., 1983. Some properties of the epithelioma papulosum cyprini (EPC) cell line from carp (Cyprinus carpio). Ann. Inst. Pasteur Virol. 134 E, 207–220. Finney, D.J., 1978. Statistical method in biological assay, third ed. Macmillan Publishing Co. Inc., New York. Follett, J.E., Meyers, T.R., Burton, T.O., Geesin, J.L., 1997. Comparative susceptibilities of salmonid species in Alaska to Infectious Hematopoietic Necrosis Virus (IHNV) and North American Viral Hemorrhagic Septicemia Virus (VHSV). J. Aquat. Anim. Health 9, 34–40. Forneris, G., Paradisi, S., Specchi, M., 1990. Pesci d'acqua dolce. Carlo Lorenzini Ed, Udine, Italy. Giuffra, E., Guyomard, R., Forneris, G., 1996. Phylogenetic relationships and introgression patterns between incipient parapatric species of Italian brown trout (Salmo trutta L. complex). Mol. Ecol. 5, 207–220. Hoffmann, B., Beer, M., Schütze, H., Mettenleiter, T.C., 2005. Fish rhabdoviruses: molecular epidemiology and evolution. Curr. Top. Microbiol. Immunol. 292, 81–117.
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Jonstrup, S.P., Kahns, S., Skall, H.F., Boutrup, T.S., Olesen, N.J., 2013. Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral hemorragic septicaemia virus. J. Fish Dis. 36, 9–23. Kottelat, M., Freyhof, J., 2007. Handbook of European Freshwater Fishes. Kottelat, Cornol, Switzerland and Freyhof, Berlin. ,pp. 416–417. Kurath, G., 2012. Fish novirhabdoviruses. In: Dietzgen, R.G., Kuzmin, I.V. (Eds.), Rhabdoviruses: Molecular Taxonomy, Evolutuion, Genomics, Ecology, Host-Vector Interactions, Cytopathology and Control. Caister Academic Press, pp. 89–116. Lapatra, S.E., 1998. Factors affecting pathogenicity of infectious hematopoietic necrosis virus (IHNV) for Salmonid fish. J. Aquat. Anim. Health 10, 121–131. McAllister, P.E., Bebak, J., Wagner, B.A., 2000. Susceptibility of Arctic char to experimental challenge with Infectious Hematopoietic Necrosis Virus (IHNV) and Infectious Pancreatic Necrosis Virus (IPNV). J. Aquat. Anim. Health 12, 35–43. Meraner, A., Baric, S., Pelster, B., Dalla Via, J., 2010. Microsatellite data point to extensive but incomplete admixture in a marble and brown trout hybridisation zone. Conserv. Genet. 11, 985–998. OIE, 2014. Aquatic Animal Health Code, 17th edition. (http://www.oie.int). Olesen, N.J., Lorenzen, N., LaPatra, S.E., 1999. Production of neutralizing antisera against viral hemorragic septicemia (VHS) virus by intravenous injections of rabbits. J. Aquat. Anim. Health 11, 10–16.
Overturt, K., LaPatra, S., Powell, M., 2001. Real-time PCR for the detection and quantitative analysis of IHNV in salmonids. J. Fish Dis. 24, 325–333. Povz, M., Jesensek, D., Berrebi, P., Crivelli, A.J., 1996. The marble trout, Salmo trutta marmoratus, Cuvier 1817, in the Soca River Basin, Slovenia. Tour du Valat Publication, Arles. Schoffmann, J., 1994. Zur gegenwatigen Situation des marmorierten Forelle (Salmo marmoratus Cuvier, 1817) in Albanien, ihrem su¨ dlichsten Verbreitungsraum. Osterr. Fisch. 47, 132–136. Sommani, E., 1961. Il Salmo marmoratus Cuv: sua origine e distribuzione nell'Italia settentrionale. Loro caratteristiche ecologiche e considerazioni relative ai ripopolamenti. Boll. Pesca Pisc. Idrobiol. 15, 40–47. St-Hilaire, S., Ribble, C.S., LaPatra, S.E., Chartrand, S., Kent, M.L., 2001. Infectious hematopoietic necrosis virus antibody profiles in naturally and experimentally infected Atlantic salmon Salmo salar. Dis. Aquat. Org. 46, 7–14. van Belle, G., Fisher, L.D., Heagerty, P.J., Lumley, T.S., 2004. Biostatistics: A Methodology for the Health Sciences, second ed. John Wiley & Sons, Inc. Wolf, K., 1988. Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca. Wolf, K., Quimby, M.C., 1962. Established eurythermic line of fish cells in vitro. Science 135, 3508.
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Susceptibility of genotyped marble trout Salmo marmoratus (Cuvier, 1829) strains to experimental challenge with European viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) F. Pascoli a,⁎, F. Bilò b, F. Nonnis Marzano c, F. Borghesan a, M. Mancin d, A. Manfrin a, A. Toffan a a
Istituto Zooprofilattico Sperimentale delle Venezie, Centro di Referenza Nazionale (NRL) per le patologie dei pesci, molluschi e crostacei, Legnaro (PD), Italy Veneto Agricoltura, Legnaro (PD), Italy c Dipartimento di Bioscienze, Università degli Studi di Parma, Viale delle Scienze 11, Parma, Italy d Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio di Analisi del rischio e sistemi di sorveglianza in sanità pubblica, Legnaro (PD), Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 21 September 2014 Accepted 22 September 2014 Available online 28 September 2014 Keywords: Virus VHS IHN Salmo marmoratus Susceptibility
a b s t r a c t The marble trout Salmo marmoratus (Cuvier, 1829) is an endemic species of great conservation concern, given its geographical restricted distribution and the high risk of hybridization with brown trout Salmo trutta L. Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are predominantly diseases of freshwater salmonids, which may trigger off severe disease outbreaks causing high mortality. Marble trout has not been inserted in the vector species list for those pathogens, but at present, little information on its susceptibility and on the possible carrier status is available. The aim of the present study was to assess the ability of European strains of VHSV and IHNV to cause disease and associated mortality in experimentally infected marble trout, and to determine whether a carrier status can result after experimental infection. Three genetically different strains of marble trout were challenged with VHSV and IHNV by immersion. Rainbow trouts were used as positive controls. Fish were checked twice a day, and dead fish were removed and sampled for subsequent virological analyses. With respect to rainbow trout, results demonstrated a very low susceptibility of marble trout to both VHSV and IHNV under simulated natural conditions. The presence of a certain number of chronically infected marble trout confirms that some individuals can survive the infection and eventually act as carrier of the disease, suggesting that marble trout should be definitely added to the list of susceptible species according to the European legislation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The marble trout Salmo marmoratus (Cuvier, 1829) is an endemic species of great conservation concern, given the high risk of hybridization with brown trout Salmo trutta L. (Meraner et al., 2010) and its geographical distribution restricted to the Po basin in northern Italy (Forneris et al., 1990; Kottelat and Freyhof, 2007; Sommani, 1961), the Adriatic basin of Slovenia, Croatia, Bosnia-Herzegovina (Povz et al., 1996) and to Albania (Schoffmann, 1994). Since the beginning of last century, brown trout have been introduced for commercial and recreational purposes in the same geographic regions, quickly leading to the creation of hybrid populations (Giuffra et al., 1996; Povz et al., 1996). Due to the severe hybridization with the introduced brown trout (Berrebi et al., 2000; Meraner et al., 2010), marble trout is therefore ⁎ Corresponding author at: Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università, 10, 35020, Legnaro (PD), Italy. Tel.: +39 0498084388. E-mail address: [email protected] (F. Pascoli).
http://dx.doi.org/10.1016/j.aquaculture.2014.09.038 0044-8486/© 2014 Elsevier B.V. All rights reserved.
considered highly endangered. This species is now farmed in northern Italy with the main purpose of restocking freshwater basins with selected individuals by means of appropriate investigations of population genetics. At the same time, intensive aquaculture practices drive major attention to sanitary issues regulated by European directives. In particular, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are among the most important pathogens for freshwater aquaculture. They are RNA viruses belonging to the family Rhabdoviridae, which is the largest group of pathogenic fish viruses. Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are predominantly diseases of freshwater salmonids, which may cause severe disease outbreaks with high mortality (Hoffmann et al., 2005; Wolf, 1988). In particular, VHS is the most serious viral disease affecting the production of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) in continental Europe (Olesen et al., 1999), causing high mortality rates in all sizes of fish. IHN was originally endemic in North America but has gradually transferred to Europe and Asia by the movement of infected fish or eggs. It typically
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occurs in rainbow trout fry and fingerlings, but the disease has also been reported in various life stages of sockeye, chinook and Atlantic salmon (Lapatra, 1998). The virus was first detected in Italy in 1987 (Bovo et al., 1987). Both diseases are listed as being notifiable, according to the European legislation (2006/88/EC) and to the World Organization for Animal Health (Office International des Epizooties, OIE). A list of susceptible and vector species for both diseases is available (Dir. 2006/88/EC and Reg. 2008/1251/EC). Marble trout has not been inserted in the vector species list, but at present, little information is available on the susceptibility of marble trout to those pathogens, nor on the possible carrier status of this species. The aim of the present study was to assess the ability of European strains of VHSV and IHNV to produce disease and associated mortality in experimentally infected marble trout and to determine whether a carrier status can result in this species after experimental infection. The investigation was carried out on three different marble trout strains previously genotyped with mitochondrial and nuclear markers. The susceptibility of marble trout to each pathogen was compared to that of an already known sensitive species such as the rainbow trout O. mykiss. 2. Materials and methods 2.1. Fish Juvenile marble trouts were hatched in a category I regional facility, according to the 2006/88/EC: the Centro Ittico Valdastico (Veneto Agricoltura, Valdastico-VI, Italy). Eight hundred and ten marble trouts were used in this study, all belonging to three different local strains named after the parents river basin of provenience and genetic characterization: Adige, Brenta and Piave. The fish were transferred to the experimental aquarium at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Legnaro-PD, Italy) at the mean size of 4.3 ± 1 g. Two hundred and seventy rainbow trouts (mean weight 3.8 ± 1 g) were provided from a commercial category I fish farm of northern Italy, according to the 2006/88/EC. Upon arrival, all fish (rainbow and marble trouts) were screened for VHS and IHN infection by virus isolation and Real time RT-PCR (rRT-PCR). Fish were gradually acclimatized to the aquarium tanks and to the trial temperatures (12 ± 1 °C) for 2 weeks before challenge. 2.2. Mitochondrial and nuclear DNA analyses Ninety specimens of wild marble trout collected from 3 different Italian rivers (30 from the river Adige, 30 from the Brenta, and 30 from the Piave) were moved to the Centro Ittico Valdastico and adapted to captivity. Samples were collected by means of non-invasive procedures. In particular, a small fin fragment (about 1 mm2) was sampled from each individual submitted to narcosis and stored in 70% ethanol under refrigerated conditions. After DNA extraction and purification, samples underwent genetic characterization by means of mitochondrial D-loop SNPs analyses, besides nuclear LDH-C1. Subsequent AFLP and microsatellite genotyping was performed to assess population differentiation. 2.3. Virus Viruses were selected according to their high pathogenicity for fish: both strains, VHS 80/V10 and IHN 409/V06, were isolated from severely affected rainbow trout during an outbreak in commercial farms in northern Italy in 2010 and 2006, respectively. Both viruses had been previously used in the IZSVe experimental aquarium, showing a mortality rate of 50% for VHS and 30% for IHN by bath exposure in rainbow trout. VHSV 80/V10 genetically belongs to the Ia genotype, the most common in European fresh water isolates according to the current
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classification (Einer-Jensen et al., 2004); IHNV 409/V06 clustered in the M genotype, European lineage (Kurath, 2012), again the most widespread genotype in Europe. Isolates were propagated on EPC cells (Fijan et al., 1983) for IHNV and on BF-2 cells (Wolf and Quimby, 1962) for VHSV, in 150 cm2 tissue culture flasks. The collected viruses were subjected to titration by end point dilutions assays. Titres were calculated according to the SpearmanKarber formula (Finney, 1978) and expressed as TCID50/ml. 2.4. Challenge procedures Experimental fish were challenged with VHSV (80/V10) and IHNV (409/V06) by immersion. Rainbow trouts were used as positive controls. Each strain of marble trout (Adige, Brenta and Piave) was treated separately. Ninety fish groups of marble trout and rainbow trout were infected by adding 50 mL of virus solution (approximately 107.05 TCID50/ml VHSV and 107.55 TCID50/ml IHNV) to 10 L of water and adding the fish for 4 h in closed systems with aeration. Each group was then placed in a separate 200-L aquarium and maintained at a temperature of 12 ± 1 °C. An additional group of each fish species and strain was mock infected by immersion containing MEM-10 without virus to act as negative control. Fish were checked twice a day and dead fish were removed. Samples of spleen, head kidney and heart were collected individually from each dead fish and stored at −80 °C. The experiment was ended on day 30. All remaining fish were euthanized by an overdose of MS-222, sampled and stored at −80 °C until analyses. The experimental protocol was evaluated and approved by the Internal Ethic Commission and the Italian Ministry of Health. 2.5. Virological analysis Samples were examined by standard virological techniques, as described in Commission Decision 2001/183/EC. Briefly, spleen, heart and head kidney were homogenized in a mortar with a pestle and sterile sand. The samples were diluted 1:10 in transport medium (Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 2% of antibiotic/antimycotic solution–10.000 IU/ml penicillin G, 10 mg/ml streptomycin sulfate, 25 μg/ml amphotericin B–0.4% of 50 mg/ml kanamycin solution) and centrifuged at 4000 g at 4 °C for 15 min. Ten percent of antibiotic/antimycotic solution was added to the supernatant and the samples incubated at 4 °C overnight. The 24-wells plates with 24 h old EPC and BF-2 cell lines were inoculated with samples in final dilutions 1:100 and 1:1000. The inoculated cell cultures were incubated at 15 °C for 1 week, with regular examination for cytopathic effect (CPE). After 1 week, samples with no CPE were re-inoculated into a new culture of EPC cells. If no CPE was observed at the end of the 2nd week, the sample was considered as being negative for the virus. Cell cultures with positive CPE were stored at −80 °C. The rRT-PCR analyses were performed according to the published protocol (Jonstrup et al., 2013; Overturt et al., 2001) for VHS and IHN. 2.6. Statistical analysis Mortality data were reported as mean ± SD in term of percentage. Each fish in the study was followed over time and the event “death” was recorded and verified by PCR to make a distinction between “death because of infection” and “death for other causes.” Two different survival analyses were performed to evaluate the event “dead” and “ dead for infection.” In this second case and in accordance to the Cox model, the dead fish with positive PCR were considered as the “event,” while those with negative PCR were considered as “censured data.” To analyze the survival function from lifetime data, the Kaplan– Meier estimator was used, which allows drawing the survival curve as
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a step curve for each group, measuring the length of time the fish survives the infection. In the graph, the y-axis plots the cumulative probability of the surviving fish at each time (van Belle et al., 2004). The semi-parametric Cox model was used to compare the survival function in the case of verified proportional hazards, taking into account the possible effect of species, treatment and interaction between these two variables. In the case of overlapped curves, the nonparametric log-rank test was applied to verify the equality of survivor functions across the groups (van Belle et al., 2004). Statistical analyses were performed using STATA 12 software. 3. Results 3.1. Mitochondrial and nuclear DNA analyses Fig. 2. Kaplan–Meier survival function from lifetime data for VHS infected fish.
Of 90 samples of marble trout, 4 (3 hybrid trouts from the Adige strain and 1 from the Piave population) were discharged after molecular characterization, which resulted in an introgression with brown trout alleles either at the D-Loop mitochondrial region or in nuclear LDHC1. The remaining 86 samples underwent additional genotyping at 12 microsatellite loci and AFLPs to define population differentiation. The three groups were completely separated, as assessed by bioinformatics parameters. These fish were used as broodstock for the production of genetically known fingerlings to restock rivers of origin and to perform the experimental infection described in this paper (Fig. 1).
3.2.2. IHN Also in the group challenged with IHNV a low mortality was recorded for all the strains of marble trout (3.2 ± 2.1%), with no differences among strains (p N 0.05; Fig. 3). Challenge of rainbow trout resulted in 28.4% of mortality (Fig. 3). Mortality started 4 days post-challenge and continued until the termination of the trial, with similar but milder clinical signs than those noted for VHS. In all challenged fish, IHNV was confirmed by rRT-PCR, as reported in Table 1. IHNV was re-isolated in all the rainbow trout tested, whereas a mean of 30% occurred in marble trout.
3.2. Challenge with VHSV and IHNV
4. Discussion
Among all the marble trouts, a little higher mortality was recorded in the Piave strain of all groups, including the negative control. The Cox regression analysis showed that the Piave strain runs a significantly higher risk of mortality if compared to the other strains (p b 0.05), independently from the treatment. Therefore, this event may be considered as a physiological mortality, probably due to a higher susceptibility of that strain to husbandry, as recorded at the Centro Ittico Valdastico in the same period (personal communication). For this reason, we reported the mortality rate considering “death for infection” only the rRT-PCR positive dead fish.
Viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) are the two most economically important viral fish diseases, particularly in salmonid farming. Viruses with a broad hostrange may pose a risk for new species in fish farming; however, strains which are virulent in one species may have low pathogenicity in another. In aquaculture, the identification of disease resistant species of fish is an important step toward the development of healthier stocks. Marble trout are of great economic importance in northern Italy, considering the widespread release into the wild for conservation purposes. This species is also valued by anglers for its shiny and wary mood, which makes it difficult to fish. Due to the ecological and economic significance of this species, a preliminary study on the resistance of marble trout to viral diseases was carried out some years ago in Italy (Borghesan et al., 2004). This species appeared more resistant than rainbow trout to VHS infection, but as susceptible as this latter for IHN infection. Unfortunately, results were biased by testing dead fish in pool and only with viral isolation methods; therefore, the obtained data cannot be considered exhaustive and additional investigations are necessary. As a matter of fact, the present study demonstrated a lower susceptibility of marble trout to both VHSV and IHNV under simulated natural conditions if compared to the one registered in rainbow trout. The low
3.2.1. VHS In the group challenged with VHSV, a low mortality was recorded for all the tested strains of marble trout (7.1 ± 0.5%), with no differences among strains (p N 0.05; Fig. 2). Challenge of rainbow trout resulted in 82.5% of mortality (Fig. 2), which started 4 days post-challenge in those fish manifesting the typical clinical signs (i.e., darkening, pop eyes, abnormal swimming) and stopped at the termination of the trial. In all challenged fish, VHSV was confirmed by rRT-PCR as reported in Table 1. VHSV was re-isolated in all rainbow trout tested, whereas a mean of 40% occurred in marble trout.
Fig. 1. Specimen of a broodstock marble trout (Salmo marmoratus) farmed in Centro Ittico Valdastico (Veneto Agricoltura, Valdastico, Vicenza) showing the typical color pattern. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Table 1 Summary of mortality and positivity to rRT-PCR for VHSV and IHNV in rainbow trout and marble trout experimentally challenged. Data are reported as positive to rRT PCR/total fish. Species
Strain
Virus
Positive fish/dead fish
% positive fish/dead fish
Positive fish/survivors fish
% positive fish/survivors
Rainbow trout Marble trout Marble trout Marble trout Rainbow trout Marble trout Marble trout Marble trout Rainbow trout Marble trout Marble trout Marble trout
– Adige Brenta Piave Adige Brenta Piave – Adige Brenta Piave
IHN IHN IHN IHN VHS VHS VHS VHS Negative control Negative control Negative control Negative control
26/26 2/4 5/7 1/12 74/74 6/7 7/7 6/14 0/1 0/0 0/3 0/14
100.0 50.0 71.4 8.3 100.0 85.7 100.0 42.9 0 0 0 0
2/64 1/86 0/83 0/74 12/16 0/83 1/83 2/76 0/89 0/90 0/87 0/76
3.8 1.0 0.0 0.0 75.0 0.0 1.2 2.6 0 0 0 0
susceptibility was assessed by means of two independent laboratory tests (virus isolation and rRT-PCR) and supported by statistical analysis. Similar results were obtained in other species and other viruses (Follett et al., 1997; McAllister et al., 2000), where very low mortality rates were found in experimentally infected fish, which highlighted the importance of subclinical reservoir species in transferring viral diseases. Moreover, precautions should be taken to prevent possible adaptation of the viruses to this species, as occurred elsewhere (Follett et al., 1997). Marble trout is a species of great conservation concern due to the high risk of hybridization with the introduced brown trout, as highlighted by the mitochondrial and nuclear DNA analyses performed. As a matter of fact, 4.5% of the tested fish proved to be hybrids although a strict phenotype selection had been carried out. Despite the clear genetic differences observed among the three marble trout populations under study, no differences were detected for disease susceptibility, at least not for the two viruses tested in the present study. Unfortunately, the presence of a certain number, although small, of chronically infected marble trout confirms that some individuals can survive the infection and eventually act as carrier of the disease. Additional work could be required to confirm whether IHNV and VHSV can persist in marble trout for a period of time longer than the one considered in this work (30 day post infection) and to assess the potential risk of IHN and VHS survivors to act as a reservoir. It could be important, for example, to determine the ability of chronically infected fish to transmit the viruses to susceptible species through cohabitation trial, as suggested by St-Hilaire et al. (2001) for Atlantic salmon. Anyway, according to the definition given by EFSA (2007) and by the Aquatic Animal Health code (OIE 2014; Chapter 1.5), in which a susceptible species means any species in which infection by a disease agent has been demonstrated by natural cases or by experimental infection that mimics the natural pathways, marble trout should definitely be added to the list of species susceptible to VHS and IHN.
Fig. 3. Kaplan–Meier survival function from lifetime data for IHN infected fish.
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