Low prevalence of VHSV detected in round goby collected in offshore regions of Lake Ontario

Journal of Great Lakes Research 38 (2012) 575–579

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Low prevalence of VHSV detected in round goby collected in offshore regions of Lake Ontario Emily R. Cornwell a,⁎, Rodman G. Getchell a, Geoffrey H. Groocock a, Maureen G. Walsh b, 1, Paul R. Bowser a a b

Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 USA United States Geological Survey, Great Lakes Science Center, Lake Ontario Biological Station, 17 Lake Street, Oswego, NY 13126 USA

a r t i c l e

i n f o

Article history: Received 10 March 2012 Accepted 13 June 2012 Available online 13 July 2012 Communicated by T. Stewart Index words: Fish disease Lake Ontario Neogobius melanostomus Viral hemorrhagic septicemia virus

a b s t r a c t Since the first reports of mortalities due to viral hemorrhagic septicemia virus (VHSV) type IVb in the Laurentian Great Lakes basin during 2005 (Lake St. Clair, USA and Bay of Quinte, Lake Ontario, Canada), many groups have conducted surveillance efforts for the virus, primarily in nearshore areas. The round goby (Neogobius melanostomus) has been identified as a key species to target for surveillance, because they have a very high probability of infection at a given site. Our objective in this study was to document and quantify VHSV in round gobies in offshore waters of Lake Ontario using molecular techniques. We collected 139 round gobies from depths ranging from 55 to 150 m using bottom trawls during the early spring of 2011 and detected VHSV in 4 individuals (1/26 fish at 95 m, 2/12 fish at 105 m, and 1/24 fish at 135 m). These results expand the known depth range of VHSV in the Great Lakes. They also have implications on the management of the spread of VHSV within infected bodies of water related to the mixing of populations of fish that would remain distinct in their breeding habitats, but then have the opportunity to mix in their overwintering habitats, as well as to increase overlap of predator and prey species in overwintering habitats. © 2012 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction Viral hemorrhagic septicemia (VHS) is a devastating disease of finfish that affects fish worldwide (World Organisation for Animal Health, 2009). This disease is caused by a rhabdovirus, viral hemorrhagic septicemia virus (VHSV), which has recently invaded the Laurentian Great Lakes. The first reports of VHSV type IVb in the Great Lakes occurred in muskellunge (Esox masquinongy) in Lake St. Clair during 2003 and 2005 and in freshwater drum (Aplodinotus grunniens) in the Bay of Quinte during 2005 (Elsayed et al., 2006; Lumsden et al., 2007). Since these first detections, VHSV has been detected throughout the Great Lakes (Bain et al., 2010; Cornwell et al., 2011; Frattini et al., 2011). Clinical signs associated with VHSV include discoloration, lethargy, erratic swimming, exophthalmia, serous to serosanguineous abdominal ascites, splenomegaly, hepatic and gill pallor due to anemia, and petechial hemorrhages in some or all of the following locations: periocular, dermal, peritoneal, at the fin bases, and throughout the viscera (Rasmussen, 1965; Kim and Faisal, 2011). However, these signs are not specific to VHSV and naturally and experimentally infected fish can carry high levels of the virus (up to 107 VHSV nucleoprotein gene copies per 50 ng RNA ⁎ Corresponding author at: Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 USA. Tel.: +1 607 253 4028. E-mail addresses: [email protected] (E.R. Cornwell), [email protected] (R.G. Getchell), [email protected] (G.H. Groocock), [email protected] (M.G. Walsh), [email protected] (P.R. Bowser). 1 Tel.: +1 315 343 3951x6512.

measured by quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) without showing any gross clinical signs (Cornwell et al., 2012; Groocock et al., 2012). The susceptibility of fish to VHSV IVb varies across species (Kim and Faisal, 2010a; Groocock et al., 2012). Although 28 species within the Great Lakes are considered susceptible, some species are highly susceptible to the virus (e.g., tiger muskellunge ♂Esox lucius×♀E. masquinongy), while other species (e.g. walleye Sander vitreus), are not very susceptible under experimental conditions, but the virus has been isolated from dead fish in the wild (Groocock et al., 2012). Very low median lethal infectious doses have been demonstrated experimentally in juvenile muskellunge (E. masquinongy), suggesting that muskellunge are particularly susceptible to infection and disease (Kim and Faisal, 2010b). The susceptibility of many other Great Lakes fish species including round goby (Neogobius melanostomus) remains unstudied despite their presence in mortality events attributed to VHSV (Groocock et al., 2007). Government and academic institutions have led numerous surveillance efforts to determine the distribution and prevalence of VHSV in the Great Lakes and to evaluate the risk of this disease to Great Lakes fish populations. During 2006, a sample of apparently healthy fish in New York State showed a range of 25–100% in VHSV prevalence at sites where at least one fish tested positive by virus isolation in cell culture. This work also led to the inclusion of two additional fish species (Pimephales notatus and Notropis atherinoides) on the 2008 USDA Federal Order listing of VHSV-susceptible species (Frattini et al., 2011). Surveillance led by the United States Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services (USDA-APHIS-VS)

0380-1330/$ – see front matter © 2012 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jglr.2012.06.008

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during 2007 to 2009 included 18 states, 6 of which border the Great Lakes (USDA-APHIS-VS, 2009). During that period, VHSV was only detected in regions where it was previously known to occur. Additional surveillance efforts have detected VHSV throughout the Great Lakes in the absence of concurrent mortality events and led to the earliest evidence for VHSV in Lake Superior by molecular methods (Bain et al., 2010; Cornwell et al., 2011, 2012). These surveillance efforts have expanded the known geographic and host range of VHSV as well as identified risk factors for VHSV infection in near-shore populations. Species was one of the main risk factors for infection with VHSV identified by these surveillance efforts, with round goby having a significantly higher risk of infection than other species tested. Round gobies are native to the Ponto-Caspian region (Ricciardi and MacIssac, 2000), but were introduced via shipping practices to the Great Lakes where they were first observed during 1990 (Jude et al., 1992). By 1998, round gobies were reported in western Lake Ontario (Hoyle et al., 2003), and they first appeared in the United States Geological Survey (USGS) offshore bottom trawl samples during 2002 (Walsh et al., 2007). In Lake Ontario, after thermal stratification breaks down in the fall, round gobies move offshore and they have been collected as deep as 170 m in spring (Walsh, unpublished data). Walsh et al. (2007) measured the abundance of round gobies in April from 2002 to 2005 in depths ranging from 35 to 150 m and found an increasing abundance over time, with the majority of fish collected during 2005 found at 75 m. Round gobies spend the summer in shallow waters, where they can be reproductively active from May to July (MacInnis and Corkum, 2000). Round gobies are highly abundant in nearshore waters of Lake Ontario during summer months (Pennuto et al., 2012); the presence of round gobies at high densities during early summer when they are reproductively active and temperatures are conducive to VHSV replication (5–18 °C) has been associated with multiple outbreaks of VHSV. The first detection of VHSV by virus isolation in New York State occurred in round goby (Groocock et al., 2007). Subsequent surveillance for VHSV has shown the round goby to have a consistently high prevalence of infection in New York waters (Cornwell et al., 2012). Most previous surveillance efforts for VHSV in the Great Lakes have focused on collections of fish from nearshore waters (defined as coastal waters less than 15 m deep; Hoyle et al., 2003) and collections of fish throughout the Great Lakes. Yellow perch (Perca flavescens) were collected from offshore populations in Lake Erie (Kane-Sutton et al., 2010) during one surveillance effort, but the greatest depth sampled in this study was 20 m. Additionally, the Michigan Department of Natural Resources has sampled fish from offshore Michigan waters of Lake Huron, Lake Michigan, and Lake Superior (Faisal et al., 2012). Our objective in this study was to document and quantify VHSV in round gobies in offshore waters using molecular techniques. Given the historic high prevalence of VHSV in round gobies (Cornwell et al., 2012) and their seasonal migration patterns, we hypothesized that some round gobies occupying deep waters of Lake Ontario are infected with VHSV in the spring.

The fin, gill, and pooled viscera were each placed in a separate homogenizing tube containing 200 μL RNAlater® (Ambion, Applied Biosystems, Carlsbad, California) and frozen on ice packs previously frozen overnight at −80 °C until transport to the laboratory where samples were kept at − 80 °C until extraction of RNA. To prevent cross-contamination between samples, all dissection instruments and surfaces were disinfected with a 10% solution of household bleach (3–6% sodium hypochlorite, Clorox, final solution approximately 10,000 mg/L active chlorine, contact time at least 30 s) then dried between each fish and a new, sterile scalpel blade was used to dissect each fish. Sex, total length, and any grossly visible internal or external abnormalities were recorded for all round goby collected. Immediately prior to extraction of RNA, 200 μL sterile cell culture media (Minimal Essential Medium with Hanks' salts prepared with 10% fetal bovine serum, penicillin [100 IU/mL], streptomycin [100 μg/mL] and HEPES buffer [1 M 0.015 mL/mL] [Gibco, Invitrogen, Carlsbad California]) and one 1.3 mm chrome steel bead (BioSpec Products, Bartlesville, Oklahoma) were added to each homogenizing tube containing either fin, gill, or pooled organs. All samples were kept on ice until loading onto the sample plate for extraction. Extraction of nucleic acids was performed using a MagMax magnetic bead extraction system and the MagMax-96 viral RNA isolation kit (Life Technologies, Carlsbad, California) using the protocols described in the kit and extraction program AM1836_DW_50_V2. Specifically, each well of the deep well sample plate contained a mixture of 130 μL of prepared lysis binding solution, 20 μL of a 1:1 solution of magnetic beads and binding enhancer, and 50 μL of homogenized sample. Two deep well plates containing 150 μL of Wash 1 solution per well and two deep well plates containing 150 μL of Wash 2 solution per well were prepared. Elution was performed in 75 μL of elution buffer. The extraction program we used is optimized for viral RNA extraction from large volumes and involves 10 min of agitated lysis binding, 3 min and 30 sec in each Wash 1 plate, 2 min and 40 sec in each Wash 2 plate, 1 min of drying, 3 min of heated elution, and 1 min of bead collection. Eluted RNA was immediately placed in sterile microcentrifuge tubes following extraction and frozen at −80 °C until use in the qRT-PCR assay. For VHSV nucleoprotein (N) gene detection by qRT-PCR, samples, a no template control, and at least three standards were loaded onto a 96-well PCR microplate (Axygen, Union City, California) in duplicate. Standards were prepared from RNA from a round goby infected with VHSV as described in Hope et al. (2010) and were between 1.5×102 and 1.5×106 VHSV N gene copies. The qRT-PCR assay was conducted as described by Hope et al. (2010) with modifications described in Cornwell et al. (2011). Nucleic acid quantity (ng/μL) and quality were assessed just prior to or just after samples were loaded onto the qRT-PCR plate using a NanoVue spectrophotometer (GE Healthcare, Piscataway, New Jersey). Copy numbers in unknown samples were determined by the calculation of a linear regression based on the standards using the qRT-PCR machine manufacturer-supplied software and standardized to 50 ng RNA using the concentration measured by the NanoVue spectrophotometer and the following formula:

Material and methods N gene copies per 50 ng RNA ¼ copy number=5 μL=RNA quantity  50 ng

Round gobies were collected using an 18-m headrope bottom trawl from two sites, Rochester and Fair Haven, in Lake Ontario during spring 2011 (Fig. 1). These sites were chosen because they are routinely sampled by USGS during their spring cruise and because the areas nearshore at these locations have a history of VHSV detection during prior surveillance (Cornwell et al., 2012). Details on trawling procedures and gear descriptions have been described in O'Gorman et al. (2000) and Walsh et al. (2007). Round goby collected for this study were euthanized (decapitation followed by pithing) and tested for infection with VHSV by molecular methods. Fin, gill, and a pooled sample of the kidney, spleen, and heart were collected separately for each fish. Fish were either dissected immediately upon collection on the R/V Kaho, or stored on ice and dissected immediately upon return to the laboratory, no more than 12 h after capture.

Samples that tested positive in only one out of two replicates were tested again in triplicate and only those samples where at least two reactions amplified the VHSV genetic material were considered positive samples. All statistical analyses were conducted in JMP v. 9.0.2. The relationship between the presence of abnormalities at necropsy and testing positive for VHSV RNA was tested using Fisher's exact test. Results We collected round gobies on 22 April, 23 April, 2 May, and 3 May 2011 from a total of 12 bottom trawl samples, eight at Rochester

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Canada

Lake Ontario

Fair Haven Rochester

U.S.A.

Lake Erie

Fig. 1. Map of sites sampled for round goby. Insets expand the areas sampled. Viral hemorrhagic septicemia virus was detected in fish collected at two sites off of Rochester (circle with x at the center) and no fish tested positive at 10 additional sampled sites (x without an inscribed circle).

(55–150 m) and four at Fair Haven (65–125 m) (Fig. 1). Bottom temperatures at these sites ranged from 2.8 to 3.4 °C. Other species collected in samples with round goby included alewife (Alosa pseudoharengus), rainbow smelt (Osmerus mordax), slimy sculpin (Cottus cognatus), and deepwater sculpin (Myoxocephalus thomsonii), a typical species mix for these depths and regions. Out of 139 round gobies examined and tested for VHSV RNA; four round gobies tested positive for VHSV RNA (Table 1). All four round gobies that tested positive were caught off of Rochester, at 95 m (1/26 fish; sampled on 23 April), 105 m (2/12 fish; sampled on 23 April), and 135 m (1/24 fish; sampled on 22 April) (Fig. 1, Table 1). The virus was detected from a fin (at 105 m) in one fish, a lethal pooled organ sample (at 135 m) in one fish, and a gill sample (at 95 and 105 m) in two fish (Table 1). Nine N gene copies per 50 ng RNA were detected in the gills from the round goby collected at 105 m, two N gene copies per 50 ng RNA were detected in the fin of a round goby collected at 105 m and in the gills of a round goby collected at 95 m, 0.5 N gene copies per 50 ng RNA were detected in the pooled viscera of a round goby collected at 135 m.

All of the round gobies collected had at least one abnormality on necropsy: 131 fish had hepatic pallor and swollen intestines, erythema on the gonads was observed in two fish, one fish had a swollen kidney, external hemorrhage was observed in 23 fish, and one fish had hemorrhage on its gill. The presence of these abnormalities was not associated with testing positive for VHSV (Fisher's Exact Test, p=1.00).

Discussion This study is the first to report VHSV viral RNA in round gobies collected from deep water in Lake Ontario and documents the deepest occurrence of VHSV-infected fish to date in the Great Lakes. Similar to the present study, yellow perch infected with VHSV were detected offshore in Lake Erie, however the depths of capture there were considerably shallower (≤ 20 m, Kane-Sutton et al., 2010). In our study, we sampled depths of up to 150 m and found VHSV RNA in fish collected at 95, 105, and 135 m.

Table 1 Characteristics of round goby sampled for viral hemorrhagic septicemia virus testing. Site

Depth Total number (m) of round goby collected

Number testing positive for VHSV

Rochester 1 2 3 4 5 6 7 8 Fair Haven 9 10 11 12

55 70 85 95 105 115 135 150 65 105 115 125

0 0 0 1 2 0 1 0 0 0 0 0

1 3 2 26 12 28 24 10 1 26 5 1

Tissue type testing positive

Gill Fin, gill Pool

Mean total length (mm) (standard deviation)

Number with hepatic pallor and intestinal swelling

Number with Number with erythema in swollen gonads kidneys

Number with external hemorrhage

Number of fish with hemorrhage in gills

115 71 (5) 102 (39) 99 (20) 107 (24) 103 (23) 128 (19) 129 (19) 86 108 (15) 107 (7) 106

1 2 2 22 9 28 24 10 1 26 5 1

0 0 0 0 0 0 1 1 0 0 0 0

0 3 2 13 2 2 0 0 0 1 0 0

0 0 0 0 0 1 0 0 0 0 0 0

0 0 0 0 0 0 1 0 0 0 0 0

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The time of year and temperature may have contributed to the low levels of the virus found in the round goby in this study (0.5–9 VHSV N gene copies per 50 ng RNA). These fish were collected prior to spawning, which for round gobies in the Great Lakes can start as early as May and continue through the summer in the Great Lakes (MacInnis and Corkum, 2000), being stimulated by temperatures between 9 and 26 °C (Kornis et al., 2012). Kane-Sutton et al. (2010) found that initiation and progression of VHSV infection is generally during or immediately after spawning in yellow perch. If round gobies are similar to other fish species, and spawning does contribute to the virulence of VHSV, then we would expect prevalence to increase from the observed levels later in May or in June. In vitro replication of VHSV type IVa has been shown to occur at temperatures as low as 10 °C, but lower temperatures (such as those experienced by fish in this study) have not been tested (Arkush et al., 2006). Despite the significance of detecting VHSV in offshore round gobies, our methods have some limitations. The qRT-PCR assay used in this study cannot determine whether VHSV is replicating and viral isolation in cell culture was not a part of this study. In addition to the low levels of the virus found in each infected fish, a low sample size and a restricted length range of round gobies collected may have influenced our results. Mean total length of round gobies examined at all sites was less than 130 mm, and although these sizes are still consistent with adult fish (Kornis et al., 2012), they do not fully represent the length range of round gobies in Lake Ontario (maximum size approximately 180 mm, M. Walsh, unpublished data). All of the round gobies collected in this study had one or more abnormalities noted on necropsy. Although many of the abnormalities we observed are clinical signs observed with VHSV infection, their presence was not associated with VHSV detection in this study. It is likely that some of these abnormalities (particularly hepatic pallor, swollen intestines, which were noted in almost all the fish) were associated with round goby physiology related to conditions of overwintering or collection methods and that erythema and hemorrhages were observed as a result of injuries obtained during the trawling process. Barotrauma could also explain the majority of the lesions noted. Fish in this study were collected at depths ranging from 55 to 150 m and evidence of barotrauma has been observed elsewhere when fish were rapidly brought to the surface from depths as shallow as 10 m (Parker et al., 2006). Moreover, our molecular assay only tested for VHSV so it is also possible that some of the erythema, hemorrhage, and pallor observed may be due to different disease processes. The detection of VHSV in round gobies sampled in deep water in the spring has serious implications for the role this species may play in the distribution, maintenance, and spread of VHSV in the Great Lakes. Round gobies live up to at least three years in Lake Ontario (Taraborelli et al., 2010), which affords multiple opportunities for visits to overwintering areas. More information is needed about the seasonal migration patterns of round gobies in the Great Lakes to more fully understand their effects on the distribution of VHSV. For example, it will be critical to know the degree of site fidelity to spawning locations and whether fish that spawn in different geographical areas congregate together in the winter. There is evidence for significant genetic structure in round goby populations within the Great Lakes, including within Lake Ontario (Brown and Stepien, 2009). Based on microsatellite analysis of round gobies collected from these locations, these authors proposed a possible barrier to gene flow between western and eastern Lake Ontario, suggesting at least some limitation to the degree of population mixing occurring in overwintering areas. In addition to the effects of mixing between round goby populations, seasonal variation in distribution overlaps between round gobies and other species may allow virus transport between species whose distributions do not overlap. For example, the offshore habitat of slimy and deepwater sculpin, in the absence of overwintering round gobies, in the past may have prevented a barrier to VHSV transmission from nearshore fishes known to harbor the virus. However, the habitats of sculpin and round gobies now

overlap, allowing for the exposure of sculpins to VHSV from nearshore fishes. In addition to their possible role in spreading VHSV via migration patterns, round goby may be important in transporting VHSV vertically within food webs as they have become important prey for many Great Lakes piscivores. Warm and cool water fish that are known to consume round gobies include the longnose gar (Lepisosteus osseus), bowfin (Amia calva), northern pike (E. lucius), brown bullhead (Ameiurus nebulosus), channel catfish (Ictalurus punctatus), smallmouth bass (Micropterus dolomieu), largemouth bass (Micropterus salmoides), yellow perch, walleye, and freshwater drum, (Taraborelli et al., 2010). They are also becoming important in the diet of lake trout, a native coldwater fish in the Great Lakes (Salvelinus namaycush; Bergstedt et al., 2012), and have been hypothesized to be the cause of lake trout spending more time in colder waters (an average of 2 °C colder across the year; Bergstedt et al., 2012). Transmission of VHSV can occur through predation (Meyers and Winton, 1995) so it seems likely that the increasing frequency of round gobies as a prey source for coldwater fish also plays a role in their ability to transmit VHSV during overwintering. This study provides evidence that VHSV RNA can be detected in late April in round gobies occupying deepwater habitats. The detection of low levels of VHSV in the spring in deepwater habitats may be important to the annual cycling of the virus in Lake Ontario. At the prevalence level detected in this study, large sample sizes are needed at each location to be confident in reporting the absence of VHSV. For example, at the overall prevalence level detected in this study (4/139), 103 fish would need to be tested at each depth to be 95% confident that infected fish were not present at a depth where no fish tested positive (Sergeant, 2009). Additional studies should be undertaken to test the site fidelity of round gobies moving back to shallow waters after overwintering, to see if the prevalence of VHSV increases during and after spawning in round gobies, and if aggregates of spawning fish can introduce VHSV to new locations and susceptible fish populations. More information is also needed on the spatial and temporal distribution of VHSV in round gobies. Acknowledgments We gratefully acknowledge the assistance of the USGS, especially the staff at the Great Lakes Science Center and the crew of the R/V Kaho for the collection of fish in this study. This work was funded in part by the Cooperative Agreement 10-9100-1294-GR to Cornell University from the USDA APHIS as part of an Interagency Agreement from the US EPA to USDA-APHIS entitled “Great Lakes Restoration Initiative (GLRI) Implementation—USDA-APHIS.” Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. government. This is contribution number 1700 of the USGS Great Lakes Science Center. References Arkush, K.D., Mendonca, H.L., McBride, A.M., Yun, S., McDowell, T.S., Hedrick, R.P., 2006. Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV). Dis. Aquat. Org. 69, 145–151. Bain, M.B., Cornwell, E.R., Hope, K.M., Eckerlin, G.E., Casey, R.N., Groocock, G.H., Getchell, R.G., Bowser, P.R., Winton, J.R., Batts, W.N., Cangelosi, A., Casey, J.W., 2010. Distribution of an invasive aquatic pathogen (viral hemorrhagic septicemia virus) in the Great Lakes and its relationship to shipping. PLoS One 5, e10156, http://dx.doi.org/10.1371/journal.pone.0010156. Bergstedt, R.A., Argyle, R.L., Krueger, C.C., Taylor, W.W., 2012. Bathythermal habitat use by strains of Great Lakes- and Finger Lakes-origin lake trout in Lake Huron after a change in prey fish abundance and composition. Trans. Am. Fish. Soc. 141, 263–274. Brown, J.E., Stepien, C.A., 2009. Invasion genetics of the Eurasian round goby in North America: tracing sources and spread patterns. Mol. Ecol. 18, 64–79. Cornwell, E.R., Eckerlin, G.E., Getchell, R.G., Groocock, G.H., Thompson, T.M., Batts, W.N., Casey, R.N., Kurath, G., Winton, J.R., Bowser, P.R., Bain, M.B., Casey, J.W., 2011. Detection of viral hemorrhagic septicemia virus by quantitative reverse transcriptase polymerase chain reaction from two host species at two sites in Lake Superior using a targeted surveillance approach. J. Aquat. Anim. Health 23, 207–217.

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