- Email: [email protected]
Haemorrhagic kidney syndrome may not be a variation of infectious salmon anaemia
Journal Pre-proof Haemorrhagic kidney syndrome may not be a variation of infectious salmon anaemia
Hugh W. Ferguson, Emiliano Di Cicco, Carlos Sandoval, Daniel D. MacPhee, Kristina M. Miller PII:
S0044-8486(19)31412-7
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734498
Article Number:
734498
Reference:
AQUA 734498
To appear in:
Aquaculture
Received Date:
03 June 2019
Accepted Date:
09 September 2019
Please cite this article as: Hugh W. Ferguson, Emiliano Di Cicco, Carlos Sandoval, Daniel D. MacPhee, Kristina M. Miller, Haemorrhagic kidney syndrome may not be a variation of infectious salmon anaemia, Aquaculture (0), https://doi.org/10.1016/j.aquaculture.2019.734498
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Haemorrhagic kidney syndrome may not be a variation of Infectious salmon anaemia.
Hugh W. Ferguson1*, Emiliano Di Cicco2, 3, Carlos Sandoval5, Daniel D. MacPhee4 & Kristina M. Miller2
1 Marine
Medicine Program, Pathobiology, School of Veterinary Medicine, St George’s University,
Grenada, West Indies. [email protected] Tel: (+44) 7732 596733 2
Pacific Biological Station, Fisheries and Oceans Canada, 3190 Hammond Bay Rd, Nanaimo BC V9T
6N7, Canada. [email protected] & [email protected] 3
Pacific Salmon Foundation, 1682 W. 7th Ave., Vancouver, BC V6J 4S6, Canada.
4 Maritime
5
Veterinary Services, St. Andrews, New Brunswick, Canada. [email protected]
VeHiCe, Libertad 590, Puerto Montt, Chile. [email protected]
*To whom correspondence should be addressed.
Highlights: 1. Haemorrhagic kidney syndrome (HKS) was a disease that devastated farmed Atlantic salmon in Canada in the late 1990’s. 2. The original archived material from which HKS was described and defined was re-examined using in-situ hybridization techniques. 3. This approach showed that, associated with the renal lesions typical of HKS were 2 viruses, namely infectious salmon anaemia virus (ISAV) and piscine orthoreovirus (PRV). 4. The findings suggest that HKS is not simply a pathological variation of ISA, as was previously thought.
1
Journal Pre-proof Abstract. Haemorrhagic kidney syndrome (HKS) of Atlantic salmon is a condition of unknown aetiology that was responsible in the late 1990’s for large scale losses in Eastern Canada in sea-caged fish. Lesions included renal interstitial haemorrhage, plus acute renal tubular necrosis with eosinophilic casts. Affected fish were shown to have erythrocyte inclusion body virus (EIBS) but the significance of this infection was unknown at the time. Following the initial report describing this syndrome, data were published showing that infectious salmon anaemia virus (ISAV) was involved with HKS, that lesions typical of HKS could be reproduced using extracts of kidney from fish with HKS, and that these extracts contained ISAV. Even though no liver lesions were ever seen (the hallmark of ISA) the conclusion was reached that HKS was a pathological variation of ISA. Piscine orthoreovirus (PRV) is an emerging viral pathogen of both Atlantic and Pacific salmon. In the former, it is causally linked to heart and skeletal muscle inflammation (HSMI), while in Pacific salmon, a range of jaundice syndromes and distinctive renal tubular necrosis are reported. The similarity of the renal lesions in Pacific salmon to those seen in HKS prompted a re-evaluation of HKS, using in-situ hybridization to identify and localize both PRV and ISAV, using the archived material from which HKS was originally described. We show here the presence of both ISAV and PRV in affected tissues, concentrated in lesions. These findings show that fish with HKS had a dual viral infection, and that HKS was not, therefore, necessarily due to ISAV alone. Given the similarity between the renal lesions in HKS and those in chinook salmon with PRV, these findings also suggest that PRV was a more plausible aetiological candidate. It is just as likely, however, that both viruses were required, and that they acted in a synergistic fashion. The lesions in renal tubules in HKS, partly driven by haemoglobin and partly by one or both viruses, probably led to release of locally high levels of vasoactive factors that were able to target the peritubular capillaries of the renal portal system, leading to necrosis and interstitial haemorrhage. 2
Journal Pre-proof Key Words: HKS, ISA, orthoreovirus, EIBS, synergism, virus.
3
Journal Pre-proof 1. Introduction. Haemorrhagic kidney syndrome (HKS) is a disease of unknown aetiology that targeted Atlantic salmon (Salmo salar L.) in the mid-to-late 1990’s causing large scale losses in sea-caged fish in southwest New Brunswick, Canada (Byrne et al., 1998). The main pathological changes were by-andlarge restricted to the kidney in which there was pronounced interstitial haemorrhage plus acute renal tubular necrosis with prominent eosinophilic casts. Histopathological lesions elsewhere were minimal to non-existent, including in the liver. The epidemiology of the outbreaks strongly suggested an infectious aetiology, but initial attempts at virus or other pathogen isolation yielded nothing that was considered significant; the salmon head kidney cell line (SHK-1), capable of isolating infectious salmon anaemia virus (ISAV), was not used, as it was only just becoming available for diagnostic use. Blood smears from HKS fish showed the presence of numerous basophilic intracytoplasmic inclusions consistent with erythrocyte inclusion body syndrome (EIBS), and typical reoviral particles were confirmed by TEM. These were considered incidental to the renal lesions although their importance was unknown. Grossly, fish were marginally anaemic, and although this was thought to be probably due to the effect of the renal haemorrhage, samples were nevertheless submitted to government laboratories in Norway, due to concerns that HKS may be an unusual variation of ISA, despite there being no other pathological similarity between the two diseases. Initial testing of samples from New Brunswick for ISAV in Norway was limited to ISA indirect immunofluorescence antibody technique (IFAT) and early results were negative. After importation of the salmon head kidney (SHK-1) cell line into Canada for use in diagnostic laboratories, ISAV was eventually isolated approximately one year after the original HKS outbreak (Mullins et al., 1998). As this was the first diagnosis of ISAV from Canada, subsequent investigations inevitably centred on this finding. In a collaboration between Canadian and Norwegian scientists (Lovely et al., 1999), transmission studies showed that salmon injected with the virus cultured from fish with HKS (subsequently shown to be ISAV) did not reproduce the lesions of HKS. By contrast, filtered supernatant from the homogenised kidney of fish with HKS did reproduce typical HKS lesions in 4
Journal Pre-proof injected salmon, killing up to 100%, and ISA virus was recovered from these dead fish. No other virus was identified. It is worth noting here that no mention was made of EIBS in these studies. On the basis of these experiments, the authors concluded that HKS was an unusual pathological variation of ISA. The authors also reported that similar HKS-like lesions had historically been seen in Norway in association with ISA outbreaks (Poppe and Håstein, unpublished) and speculated that such changes may represent an early “primitive” form of ISA, a pathological presentation altered due to the strain of salmon infected, the strain of virus involved or possibly due to the environmental conditions found in the affected sites. Publication of the Lovely et al. findings reinforced the rationale behind the controls already instituted by the Canadian government (Province of New Brunswick). With extensive input from the aquaculture industry, these far-sighted measures, which largely remain in effect today, were put in place on the assumption that HKS was an infectious disease, even though no diagnosis had as yet been achieved. Piscine orthoreovirus (PRV) is an emerging viral pathogen of both Atlantic and Pacific salmon. In the former, it is causally linked to heart and skeletal muscle inflammation (HSMI) (Wessel et al., 2017; DiCicco et al., 2017) while in Pacific salmon, a range of jaundice syndromes and distinctive renal tubular necrosis are reported (DiCicco et al., 2018). Investigating this pathogen is hampered by the fact that it has not yet been cultured in vitro. The histopathological similarity of the renal lesions in Atlantic salmon with HKS and Pacific salmon with PRV infection, prompted us to revisit the archived pathological material that had originally been used to define HKS. In situ hybridization techniques enabled us to probe paraffin wax sections for the presence of both ISAV and PRV. Our findings show that renal (and other tissue) sections of fish with HKS are heavily stained for the presence of both ISAV and PRV. Given the fact that ISAV was isolated from fish with HKS (Lovely et al., 1999) and that PRV-like viruses (erythrocyte inclusion body
5
Journal Pre-proof syndrome - EIBS) were also seen in HKS (Byrne et al., 1998), these results came as no real surprise. It was the sheer extent of the staining that was notable, as were the localisations. These findings raise the likelihood that HKS was not simply a pathological (“primitive”) variation of ISA, as originally thought. Instead it would appear more likely that the lesions of HKS were a direct result of high loads of PRV, and therefore represent yet another example of haemoglobinuric nephrosis associated with PRV, albeit this time in a different species – Atlantic salmon instead of Pacific. It is a distinct possibility, however, that the two viruses acted synergistically, maybe to increase host susceptibility, in the same way that low pathogenicity avian influenza virus increases susceptibility of chickens to velogenic Newcastle disease virus, thereby altering the clinical outcome (Bonfante et al. 2017). 2. Materials and Methods. The archived paraffin wax blocks originally used to define HKS (Byrne et al., 1998) were retrieved from storage. These blocks represented tissues from 2 separate outbreaks of disease from 1996, from 3 and 8 fish respectively, and contained portions of liver, anterior and posterior kidney, spleen, heart, intestine, brain, gills and skeletal muscle with skin. All fish had lesions typical of HKS, namely renal interstitial haemorrhage and acute renal tubular necrosis with brightly eosinophilic intratubular casts. No other significant lesions were present. Sections from these wax blocks were prepared for routine histopathological evaluation as well as for in situ hybridization (ISH) for piscine orthoreovirus (PRV-1) and infectious salmon anaemia virus (ISAV), following the methods previously published (Di Cicco et al., 2018). Briefly, 3.5µm thick serial sections were obtained from each block; one section was stained with routine haematoxylin and eosin (H&E), while two consecutive serial sections were utilized for ISH. This was done using either BASEscope (RED) for PRV-1, or RNAscope (RED) for ISAV according to the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA). The sections were dewaxed by incubating for 60 min at 60°C and endogenous peroxidases were quenched with hydrogen peroxide for 10 min at room 6
Journal Pre-proof temperature. Slides were then boiled for 30 min in RNAscope target retrieval reagent and then incubated for 30 min either in RNAscope Protease III (PRV) or Protease Plus (ISAV) reagent prior to hybridization, as per the manufacturer’s instructions. For detecting PRV-1, the slides underwent hybridization with a BASEscope probe against a portion of PRV-1 genome segment L1 (that codes for PRV core shell; Advanced Cell Diagnostics, catalog #705151). This probe was designed with a sequence to prevent cross-reaction with other PRV strains (i.e. PRV-2 and PRV-3). A BASEscope probe against the bacterial gene dapB (Advanced Cell Diagnostics, catalog #701021) was used as negative control to confirm absence of background and/or non-specific cross-reactivity of the assay. Concurrently, PRV-1/HSMI (heart and skeletal muscle inflammation) positive (n = 2) and negative (n = 1) heart samples from Atlantic salmon collected during an experimental challenge for HSMI performed in Norway (Finstad et al., 2014) were used as positive and negative controls for PRV-1 presence and localization. For ISAV, the slides underwent hybridization with an RNAscope probe against a portion of ISAV genome segment 2 (that codes for viral polymerase; Advanced Cell Diagnostics, catalog #552371). An RNAscope probe against the bacterial gene dapB (Advanced Cell Diagnostics, catalog #310043) was used as negative control to confirm absence of background and/or of non-specific crossreactivity of the assay, while a probe against the housekeeping gene PPIB (Advanced Cell Diagnostic, catalog# 494421) was used to assess the quality of RNA present in the tissue sections. Concurrently, paraffin wax blocks of tissues collected from Atlantic salmon (n = 3) during an outbreak of ISA in New Brunswick in 1999 (confirmed positive by IFAT, PCR and tissue culture) were used as positive controls for ISAV presence, while one sample (n = 1) that had tested negative to ISAV from an experimental challenge (Finstad et al., 2014) was used as negative control. These sections were also stained for PRV-1. Signal amplification for both BASEscope and RNAscope probes was performed according to the manufacturer’s instructions, followed by counterstaining with Gill’s haematoxylin, and visualisation by bright field microscopy.
7
Journal Pre-proof 3. Results. Lesions typical for HKS were seen in all the H&E-stained sections, including renal interstitial haemorrhage and acute renal tubular necrosis associated with the presence of eosinophilic casts (Fig. 1a). Minimal changes in the spleen included congestion and haemosiderin accumulation while there were no obvious lesions in liver or heart. ISH staining for ISAV of the HKS samples revealed systemic involvement, although most of the staining occurred in the kidney and spleen. In the kidney, ISAV was localized in endothelial cells, renal portal macrophages and a few red blood cells (RBC’s), but was also present in the necrotic material contained within the damaged renal tubules and in the renal casts (Fig. 1c). The virus was also localized on the brush border of some apparently healthy renal tubules (Fig. 1d). In the spleen, ISAV was localized within macrophages and a few RBC’s, while most endothelial cells also appeared to be infected. Overall, ISAV appeared to involve primarily vascular endothelium within every organ tested, despite the lack of obvious lesions. ISH staining for PRV localized the virus to RBC’s in every organ. Several stained cells were also observed in the reticulo-endothelial system, particularly in the kidney (portal macrophages) and spleen (Fig. 1e), but no endothelial cells were stained. The staining in the kidney and spleen partially overlapped the staining for ISAV, including positive marking in the necrotic material contained within the damaged renal tubules and in the renal casts (Fig. 1e). PRV was also localized on the luminal brush border of apparently healthy renal tubules (Fig. 1f), the same ones as those staining for ISAV. No staining was present in the heart. By contrast, staining for PRV was detected in some hepatocytes, although this was minimal, especially by contrast with ISAV. The negative and positive controls worked as expected, although for the ISA positive samples, the spleen showed the strongest staining, notably in the centre of the section, suggesting that there had been some degradation during the 20+ years that the blocks had been in storage. In particular it should be noted that there was no staining for PRV in the kidney of any fish that did not have HKS. 8
Journal Pre-proof 4. Discussion. Re-examination of the archived material from which HKS was originally described and defined, has shown that sections of kidney (and other tissues) were heavily stained for the presence of both ISAV and PRV, and that the staining correlated with the typical lesions of HKS, namely renal interstitial haemorrhage plus nephrosis with eosinophilic casts. Staining for ISAV and PRV was seen at the brush border of some tubules, and this correlated with the staining densities seen in TEM (Byrne et al., 1998), thereby supporting the original suggestion that these electron densities are virus-related; a good location indeed for transmission to other fish! The significance of the presence of virus-induced erythrocytic inclusion bodies in the original description of HKS was unknown, and their presence was overlooked in subsequent work (Lovely et al., 1999). To date, all three strains of PRV have been shown to produce inclusion bodies in erythrocytes of various species, including Atlantic salmon (Wessel et al., 2014; Takano et al., 2016; Hauge et al., 2017). All three PRV strains have also been associated with various anaemia-related syndromes in Pacific salmon i.e. PRV-1 and jaundice/anaemia in chinook salmon (Di Cicco et al., 2018); PRV-2 and EIBS in coho salmon (Takano et al., 2016); and PRV-3 and HSMI-like disease in rainbow trout (Olsen et al., 2015) and coho salmon (Godoy et al., 2016). The high specificity of the probe used here shows that these fish with HKS were infected with at least PRV-1. The co-existence of other PRV sub-strains cannot be excluded/ruled out, although they are considered to be not present in Canada. This is a relatively small data set on which to base too many conclusions, but certain questions seem nevertheless appropriate. Was HKS a result of ISAV or PRV infection, or were both viruses necessary? The nephrotic lesions of HKS closely resemble those seen in Pacific salmon with PRV infection in which haemoglobinuria was confirmed (Di Cicco et al., 2018). The haemoglobinuria likely leads to oxidative damage to, and necrosis of, renal tubular epithelium, just as it does in mammals (Deuel et al., 2016). It is important to note that these changes with PRV infection in Pacific salmon occurred in 9
Journal Pre-proof the absence of any involvement of ISAV, although it is also important to appreciate that interstitial haemorrhage was not a feature either. So, if haemoglobinuric nephrosis in typical HKS fish can be explained by PRV alone, what role did ISAV play? Haemorrhage is the major histopathological feature of ISA, but in the liver, not the kidney (Evensen et al., 1991; Speilberg et al., 1995). The finding that the major target for ISAV are the cells of the vascular endothelium (or closely associated cells), partly explains the classical hepatic lesions in ISA. This could also explain the renal interstitial haemorrhage in HKS, but if ISAV is indeed directly involved in this haemorrhage, it should probably also be seen in anterior as well as posterior kidney (as well as in other organs). The lack of interstitial haemorrhage in the anterior kidney suggested originally that it was the tubular lesions driving the interstitial haemorrhage, rather than vice versa (Byrne et al., 1998). This is supported by the fact that the renal portal system is just as extensive in anterior kidney as in posterior, and that injected particulates are phagocytosed equally avidly by portal macrophages in both regions (Ferguson et al., 1982). ISA continues to be reported in Eastern Canada (Canadian Food Inspection Agency, 2019). By contrast, local diagnosticians in the Maritimes report that HKS is rarely seen these days. While there are data (testing by PCR) reporting the presence of PRV in farmed and wild salmon in Eastern Canada (ICES, 2015) we can find no published histopathological data or other evidence of disease. So if it can be accepted that HKS was caused primarily by PRV, or by a combination of PRV and ISAV, any subsequent decline in incidence of HKS might simply be due to reduced viral loads. How would this occur? One of the major governmental measures introduced to try and control the spread of what was at the time an unknown (but presumed infectious) disease, was stopping the practice of emptying “untreated blood water” into the marine environment at harvesting. Even though infection may not necessarily lead to cell lysis, the main target cell for PRV is the erythrocyte (Finstad et al., 2014), but ISAV also binds to erythrocytes (Aamelfot et al., 2012). This control measure alone should, therefore, have had a profound impact on levels of both PRV and ISAV in the seawater. If
10
Journal Pre-proof there is any level of synergy between PRV and ISAV, reducing levels of either pathogen should have a more profound impact than otherwise expected. The ISH methodology in this paper does not co-localize antigens within the same section, although that approach is technically possible. Instead serial sections were used, as the interest centred mainly on correlating the presence of virus(es) with typical HKS lesions. Nevertheless, serial sections at 4 microns allowed us to see the same histological structures, e.g. a glomerulus, in sequential sections, and indeed often the same cells. Nor does the methodology attempt to quantify the viral loads within the tissue. In this context, however, it is interesting to look at the HKS transmission studies (Lovely et al., 1998). Using an ultrafiltrate of kidney tissue produced up to 100% mortality. The fish all had lesions typical of HKS and not ISA, even though both ISAV and PRV were almost certainly present in the injected material (EIBS was not considered, so was not assayed). This might suggest that PRV was present at higher loads than ISAV, that it was more virulent than the ISAV, that the fish were more susceptible to PRV/resistant to the strain of ISAV present, or that the fish died from HKS before they could develop typical ISA lesions. Unfortunately this study does not mention when fish started to die, only that there was up to 100% mortality by day 45 post-injection. The incubation period for ISA is usually 10-20 days (Rimstad & Mjaaland, 2002), so this should have been long enough for at least some of the fish to have died with lesions typical of ISA; but none did. Another possibility is that the 2 viruses acted together, and as both have an affinity for erythrocytes, this seems the most reasonable suggestion. The combined or synergistic effect of the viruses would probably be to increase the rate of lysis of erythrocytes, faster than would have happened with one virus alone, leading to haemoglobinuria, nephrosis and death before hepatic vascular damage. It is not uncommon for two or more viruses to infect (co-infection) a plant or animal at the same time (Murphy & Bowen, 2006). One recent report showed that chickens infected with low pathogenicity avian influenza virus had increased susceptibility to velogenic Newcastle disease virus, thereby altering clinical outcomes (Bonfante et al., 2017). Similarly, a superinfection (infection of a
11
Journal Pre-proof host by two viruses at different times) of chickens by Marek’s disease virus (MDV) and avian leucosis virus was recently shown to have a synergistic role, leading to increased pathogenicity (Zhou et al., 2018). There are a few reports of viral co-infections in fish (Kotob et al., 2016); in some cases there is synergism and in others, antagonism. It is unclear if the present case of HKS represents a coinfection (dual infection), or whether it might be a superinfection, as the timing of the infections is unknown. Nor is there any evidence in this case to suggest synergism, but it must remain a distinct possibility. A background (endemic?) level of a low pathogenicity virus may be well tolerated at a population level and lead to few clinical cases of full-blown disease. In such circumstances it is easy to understand why little effort would be made to screen populations of fish for its presence, even if its identity were known. Such was probably the case with ISAV in New Brunswick up until the late 1990’s. Similarly, even moderate levels of PRV can probably be well tolerated by the host (Løvoll et al., 2012) so long as nothing supervenes to cause an acute haemolytic crisis, thereby suddenly releasing large quantities of viral antigen. But a superinfection of PRV on top of a low-level infection of ISAV (or vice versa) might well lead to augmentation (synergism) and more severe clinical disease. As seen in the case of chickens with avian leucosis virus that become superinfected with Marek’s disease, increased severity of lesions and clinical disease may occur due to increased interleukin production, enhanced rate of viral protein production and more severe cell degeneration (Zhou et al., 2018). The lesions in renal tubules in HKS, driven partly by haemoglobin and partly by virus, probably led to release of locally high levels of vasoactive factors which were then able to target the peritubular capillaries of the renal portal system (Ferguson, 1984), leading to necrosis and interstitial haemorrhage.
12
Journal Pre-proof
5. References. Aamelfot, M., Dale, O.B., Weli, S.C., Koppang, E.O., Falk, K., 2012. Expression of the infectious salmon anemia virus receptor on Atlantic salmon endothelial cells correlates with the cell tropism of the virus. Journal of Virology 86, 10571-10578. Bonfante, F., Cattoli, G., Leardini, S., Salomoni, A., Mazzetto, E., Davidson, I., Haddas, R., Terregino, C., 2017. Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent. Avian Pathology 46(5), 488-496. Byrne, P.B., Ostland, V.E., Johnson, G., MacPhee, D.D., Ferguson, H.W., 1998. Haemorrhagic kidney syndrome, a new disease of sea-caged Atlantic salmon (Salmo salar L). Journal of Fish Diseases 21, 81-92. CFIA, 2019. http://www.ices.dk/community/groups/Pages/WGPDMO.aspx Di Cicco, E., Ferguson, H.W., Kaukinen, K., Schulze, A., Li, S., Vanderstichel, R., Laurin, E., Wessel, O., Rimstad, E., Gardner, I.A., Hammell, L., Miller, K.M., 2017. Longitudinal Farm Study into Heart and Skeletal Muscle Inflammation (HSMI) disease on a British Columbia salmon farm. http://dx.doi.org/10.1371/journal.pone.0171471 Di Cicco, E., Ferguson, H.W., Kaukinen, K.H., Schulze, A.D., Li, S., Tabata, A., Gunther, O., Mordecai, G., Suttle, C., Miller, K.M., 2018. The same strain of Piscine OrthoReovirus (PRV) is involved with the development of two different diseases in Atlantic and Pacific Salmon in British Columbia. Facets2018-0008.R1 Deuel, J.W., Schaer, C.A., Boretti, F.S., Opitz, L., Garcia-Rubio, I., Baek, J.H., et al., 2016. Hemoglobinuria related acute kidney injury is driven by intrarenal oxidative reactions triggering a heme toxicity response. Cell Death and Disease, 7(1): e2064. DOI: 10.1038/cddis.2015.392
13
Journal Pre-proof Evensen, Ø., Thorud, K.E., Olsen, Y.A., 1991. A morphological study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar). Research in Veterinary Science 51(2), 215222.
Ferguson, H.W., Claxton, M.J., Moccia, R.D., Wilkie, E.J., 1982. The quantitative clearance of bacteria from the bloodstream of rainbow trout (S. gairdneri). Veterinary Pathology 19, 687-699. Ferguson, H.W. 1984. Renal portal phagocytosis of bacteria in rainbow trout (Salmo gairdneri Richardson): ultrastructural observations. Canadian Journal of Zoology 62, 2505-2511. Finstad, Ø. W., Dahle, M. K., Lindholm, T. H., Nyman, I. B., Løvoll, M., Wallace, C., Olsen, C. M., Storset, A. K., Rimstad, E., 2014. Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes. Veterinary Research 45(35), 1-13. Godoy, M.G., Kibenge, M.J., Wang, Y., Suarez, R., Leiva, C., et al. 2016. First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV. Journal of Virology 13, 98. https://doi.org/10.1186/s12985-016-0554-y PMID: 27296722 Hauge, H., Vendramin, N., Taksdal, T., Olsen, A.B., Wessel, Ø., Mikkelsen, S.S., et al. 2017. Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids. PLoS ONE, 12(7), e0180293. https://doi.org/10.1371/journal.pone.0180293 ICES 2015. Working Group on Pathology and Diseases of Marine Organisms, Gloucester Point, Virginia, USA. Canada country report, February 16-20. Kotob, M.H., Menanteau-Ledouble, S., Kumar, G., Abdelzaher, M., El-Matbouli, M., 2017. The impact of co-infections on fish: a review. Veterinary Research, 48, 26. Lovely, J.E., Dannevig, B.H., Falk, K., Hutchin, L., MacKinnon, A.M., Melville, K.J., Rimstad, E., Griffiths, S.G., 1999. First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome. Diseases of Aquatic Organisms 35, 145-148. 14
Journal Pre-proof Løvoll, M., Alarcon, M., Bang, J.B., Taksdal, T., Kristoffersen, A.B., Tengs, T., 2012. Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production. Diseases of Aquatic Organisms, 99, 7-12. Mullins, J. E., Groman, D., Wadowska, D., 1998. Infectious salmon anaemia in salt water Atlantic salmon (Salmo salar L.) in New Brunswick, Canada. Bulletin European Association Fish Pathologists, 18, 110–114. Murphy, J. F., Bowen, K. L., 2006. Synergistic disease in pepper caused by the mixed infection of Cucumber mosaic virus and Pepper mottle virus. Phytopathology, 96, 240-247. Olsen, A.B. Hjortaas, M., Tengs, T., Hellberg,H., Johansen, R., 2015. First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV). PLoS ONE 10(7): e0131638. doi:10.1371/journal.pone.0131638 Rimstad, E., Mjaaland, S., 2002. Infectious salmon anaemia virus: An orthomyxovirus causing an emerging infection in Atlantic salmon. APMIS Acta pathologica, Microbiologica, Immunologica Scandinavica, 110, 273-282. Speilberg, L., Evensen, Ø., Dannevig, B.H., 1995. A sequential study of the light and electron microscopic liver lesions of infectious anemia in Atlantic salmon (Salmo salar L.). Veterinary Pathology, 32(5), 466-478. Takano T, Nawata A, Sakai T, Matsuyama T, Ito T, Kurita J, et al., 2016. Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus. PLoS ONE 11(10): e0165424. https://doi.org/10.1371/journal.pone.0165424 Wessel, Ø., Braaen, S., Alarcon, M., Haatveit, H., Roos, N., Markussen, T., et al. 2017. Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle 15
Journal Pre-proof inflammation in Atlantic salmon. PLoS ONE 12(8): e0183781. https://doi.org/10.1371/journal.pone.0183781 Zhou, J., Zhao, G.L., Wang, X.M., Du, X.S., Su, S., Li, C.G., Nair, V., Yao, Y.X., Cheng, Z.Q., 2018. Synergistic Viral Replication of Marek’s Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens. Viruses, 10, 271. 6. Captions. Figure 1. Histopathology and ISH of posterior kidney from Atlantic salmon with HKS. Matching fields of consecutive serial sections from two paraffin wax blocks (a, c, e, and b, d, f, respectively) are stained with H&E (a, b), ISH for ISAV (c, d) and PRV-1 (e, f). (a) Section of posterior kidney showing diffuse interstitial haemorrhage and congestion, plus acute renal tubular necrosis associated with the presence of eosinophilic renal casts (arrows). (b) Posterior kidney showing diffuse interstitial haemorrhage and apparently healthy renal tubules, some of which contain necrotic debris (arrows). (c) ISAV (red) was localized primarily in the necrotic tubules (arrows) and in the necrotic debris of the renal casts (open arrow). (d) ISAV (red) localized in the tubular necrotic debris (open arrow). Some apparently healthy renal tubules also show ISAV staining on the luminal side of the epithelium (arrows). (e) PRV-1 (red) was localized primarily in the necrotic tubules and in the necrotic debris of the renal casts (arrows). (f) PRV-1 (red) localized in the tubular necrotic debris (open arrow). Some apparently healthy renal tubules also show PRV-1 particles on the luminal side of the epithelium (arrows). The scale bar in all figures is 100μm.
7. Funding. This work was partially supported by the Pacific Salmon Foundation.
16
Journal Pre-proof
17
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Hugh W. Ferguson, Emiliano Di Cicco, Carlos Sandoval, Daniel D. MacPhee, Kristina M. Miller PII:
S0044-8486(19)31412-7
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734498
Article Number:
734498
Reference:
AQUA 734498
To appear in:
Aquaculture
Received Date:
03 June 2019
Accepted Date:
09 September 2019
Please cite this article as: Hugh W. Ferguson, Emiliano Di Cicco, Carlos Sandoval, Daniel D. MacPhee, Kristina M. Miller, Haemorrhagic kidney syndrome may not be a variation of infectious salmon anaemia, Aquaculture (0), https://doi.org/10.1016/j.aquaculture.2019.734498
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Haemorrhagic kidney syndrome may not be a variation of Infectious salmon anaemia.
Hugh W. Ferguson1*, Emiliano Di Cicco2, 3, Carlos Sandoval5, Daniel D. MacPhee4 & Kristina M. Miller2
1 Marine
Medicine Program, Pathobiology, School of Veterinary Medicine, St George’s University,
Grenada, West Indies. [email protected] Tel: (+44) 7732 596733 2
Pacific Biological Station, Fisheries and Oceans Canada, 3190 Hammond Bay Rd, Nanaimo BC V9T
6N7, Canada. [email protected] & [email protected] 3
Pacific Salmon Foundation, 1682 W. 7th Ave., Vancouver, BC V6J 4S6, Canada.
4 Maritime
5
Veterinary Services, St. Andrews, New Brunswick, Canada. [email protected]
VeHiCe, Libertad 590, Puerto Montt, Chile. [email protected]
*To whom correspondence should be addressed.
Highlights: 1. Haemorrhagic kidney syndrome (HKS) was a disease that devastated farmed Atlantic salmon in Canada in the late 1990’s. 2. The original archived material from which HKS was described and defined was re-examined using in-situ hybridization techniques. 3. This approach showed that, associated with the renal lesions typical of HKS were 2 viruses, namely infectious salmon anaemia virus (ISAV) and piscine orthoreovirus (PRV). 4. The findings suggest that HKS is not simply a pathological variation of ISA, as was previously thought.
1
Journal Pre-proof Abstract. Haemorrhagic kidney syndrome (HKS) of Atlantic salmon is a condition of unknown aetiology that was responsible in the late 1990’s for large scale losses in Eastern Canada in sea-caged fish. Lesions included renal interstitial haemorrhage, plus acute renal tubular necrosis with eosinophilic casts. Affected fish were shown to have erythrocyte inclusion body virus (EIBS) but the significance of this infection was unknown at the time. Following the initial report describing this syndrome, data were published showing that infectious salmon anaemia virus (ISAV) was involved with HKS, that lesions typical of HKS could be reproduced using extracts of kidney from fish with HKS, and that these extracts contained ISAV. Even though no liver lesions were ever seen (the hallmark of ISA) the conclusion was reached that HKS was a pathological variation of ISA. Piscine orthoreovirus (PRV) is an emerging viral pathogen of both Atlantic and Pacific salmon. In the former, it is causally linked to heart and skeletal muscle inflammation (HSMI), while in Pacific salmon, a range of jaundice syndromes and distinctive renal tubular necrosis are reported. The similarity of the renal lesions in Pacific salmon to those seen in HKS prompted a re-evaluation of HKS, using in-situ hybridization to identify and localize both PRV and ISAV, using the archived material from which HKS was originally described. We show here the presence of both ISAV and PRV in affected tissues, concentrated in lesions. These findings show that fish with HKS had a dual viral infection, and that HKS was not, therefore, necessarily due to ISAV alone. Given the similarity between the renal lesions in HKS and those in chinook salmon with PRV, these findings also suggest that PRV was a more plausible aetiological candidate. It is just as likely, however, that both viruses were required, and that they acted in a synergistic fashion. The lesions in renal tubules in HKS, partly driven by haemoglobin and partly by one or both viruses, probably led to release of locally high levels of vasoactive factors that were able to target the peritubular capillaries of the renal portal system, leading to necrosis and interstitial haemorrhage. 2
Journal Pre-proof Key Words: HKS, ISA, orthoreovirus, EIBS, synergism, virus.
3
Journal Pre-proof 1. Introduction. Haemorrhagic kidney syndrome (HKS) is a disease of unknown aetiology that targeted Atlantic salmon (Salmo salar L.) in the mid-to-late 1990’s causing large scale losses in sea-caged fish in southwest New Brunswick, Canada (Byrne et al., 1998). The main pathological changes were by-andlarge restricted to the kidney in which there was pronounced interstitial haemorrhage plus acute renal tubular necrosis with prominent eosinophilic casts. Histopathological lesions elsewhere were minimal to non-existent, including in the liver. The epidemiology of the outbreaks strongly suggested an infectious aetiology, but initial attempts at virus or other pathogen isolation yielded nothing that was considered significant; the salmon head kidney cell line (SHK-1), capable of isolating infectious salmon anaemia virus (ISAV), was not used, as it was only just becoming available for diagnostic use. Blood smears from HKS fish showed the presence of numerous basophilic intracytoplasmic inclusions consistent with erythrocyte inclusion body syndrome (EIBS), and typical reoviral particles were confirmed by TEM. These were considered incidental to the renal lesions although their importance was unknown. Grossly, fish were marginally anaemic, and although this was thought to be probably due to the effect of the renal haemorrhage, samples were nevertheless submitted to government laboratories in Norway, due to concerns that HKS may be an unusual variation of ISA, despite there being no other pathological similarity between the two diseases. Initial testing of samples from New Brunswick for ISAV in Norway was limited to ISA indirect immunofluorescence antibody technique (IFAT) and early results were negative. After importation of the salmon head kidney (SHK-1) cell line into Canada for use in diagnostic laboratories, ISAV was eventually isolated approximately one year after the original HKS outbreak (Mullins et al., 1998). As this was the first diagnosis of ISAV from Canada, subsequent investigations inevitably centred on this finding. In a collaboration between Canadian and Norwegian scientists (Lovely et al., 1999), transmission studies showed that salmon injected with the virus cultured from fish with HKS (subsequently shown to be ISAV) did not reproduce the lesions of HKS. By contrast, filtered supernatant from the homogenised kidney of fish with HKS did reproduce typical HKS lesions in 4
Journal Pre-proof injected salmon, killing up to 100%, and ISA virus was recovered from these dead fish. No other virus was identified. It is worth noting here that no mention was made of EIBS in these studies. On the basis of these experiments, the authors concluded that HKS was an unusual pathological variation of ISA. The authors also reported that similar HKS-like lesions had historically been seen in Norway in association with ISA outbreaks (Poppe and Håstein, unpublished) and speculated that such changes may represent an early “primitive” form of ISA, a pathological presentation altered due to the strain of salmon infected, the strain of virus involved or possibly due to the environmental conditions found in the affected sites. Publication of the Lovely et al. findings reinforced the rationale behind the controls already instituted by the Canadian government (Province of New Brunswick). With extensive input from the aquaculture industry, these far-sighted measures, which largely remain in effect today, were put in place on the assumption that HKS was an infectious disease, even though no diagnosis had as yet been achieved. Piscine orthoreovirus (PRV) is an emerging viral pathogen of both Atlantic and Pacific salmon. In the former, it is causally linked to heart and skeletal muscle inflammation (HSMI) (Wessel et al., 2017; DiCicco et al., 2017) while in Pacific salmon, a range of jaundice syndromes and distinctive renal tubular necrosis are reported (DiCicco et al., 2018). Investigating this pathogen is hampered by the fact that it has not yet been cultured in vitro. The histopathological similarity of the renal lesions in Atlantic salmon with HKS and Pacific salmon with PRV infection, prompted us to revisit the archived pathological material that had originally been used to define HKS. In situ hybridization techniques enabled us to probe paraffin wax sections for the presence of both ISAV and PRV. Our findings show that renal (and other tissue) sections of fish with HKS are heavily stained for the presence of both ISAV and PRV. Given the fact that ISAV was isolated from fish with HKS (Lovely et al., 1999) and that PRV-like viruses (erythrocyte inclusion body
5
Journal Pre-proof syndrome - EIBS) were also seen in HKS (Byrne et al., 1998), these results came as no real surprise. It was the sheer extent of the staining that was notable, as were the localisations. These findings raise the likelihood that HKS was not simply a pathological (“primitive”) variation of ISA, as originally thought. Instead it would appear more likely that the lesions of HKS were a direct result of high loads of PRV, and therefore represent yet another example of haemoglobinuric nephrosis associated with PRV, albeit this time in a different species – Atlantic salmon instead of Pacific. It is a distinct possibility, however, that the two viruses acted synergistically, maybe to increase host susceptibility, in the same way that low pathogenicity avian influenza virus increases susceptibility of chickens to velogenic Newcastle disease virus, thereby altering the clinical outcome (Bonfante et al. 2017). 2. Materials and Methods. The archived paraffin wax blocks originally used to define HKS (Byrne et al., 1998) were retrieved from storage. These blocks represented tissues from 2 separate outbreaks of disease from 1996, from 3 and 8 fish respectively, and contained portions of liver, anterior and posterior kidney, spleen, heart, intestine, brain, gills and skeletal muscle with skin. All fish had lesions typical of HKS, namely renal interstitial haemorrhage and acute renal tubular necrosis with brightly eosinophilic intratubular casts. No other significant lesions were present. Sections from these wax blocks were prepared for routine histopathological evaluation as well as for in situ hybridization (ISH) for piscine orthoreovirus (PRV-1) and infectious salmon anaemia virus (ISAV), following the methods previously published (Di Cicco et al., 2018). Briefly, 3.5µm thick serial sections were obtained from each block; one section was stained with routine haematoxylin and eosin (H&E), while two consecutive serial sections were utilized for ISH. This was done using either BASEscope (RED) for PRV-1, or RNAscope (RED) for ISAV according to the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA). The sections were dewaxed by incubating for 60 min at 60°C and endogenous peroxidases were quenched with hydrogen peroxide for 10 min at room 6
Journal Pre-proof temperature. Slides were then boiled for 30 min in RNAscope target retrieval reagent and then incubated for 30 min either in RNAscope Protease III (PRV) or Protease Plus (ISAV) reagent prior to hybridization, as per the manufacturer’s instructions. For detecting PRV-1, the slides underwent hybridization with a BASEscope probe against a portion of PRV-1 genome segment L1 (that codes for PRV core shell; Advanced Cell Diagnostics, catalog #705151). This probe was designed with a sequence to prevent cross-reaction with other PRV strains (i.e. PRV-2 and PRV-3). A BASEscope probe against the bacterial gene dapB (Advanced Cell Diagnostics, catalog #701021) was used as negative control to confirm absence of background and/or non-specific cross-reactivity of the assay. Concurrently, PRV-1/HSMI (heart and skeletal muscle inflammation) positive (n = 2) and negative (n = 1) heart samples from Atlantic salmon collected during an experimental challenge for HSMI performed in Norway (Finstad et al., 2014) were used as positive and negative controls for PRV-1 presence and localization. For ISAV, the slides underwent hybridization with an RNAscope probe against a portion of ISAV genome segment 2 (that codes for viral polymerase; Advanced Cell Diagnostics, catalog #552371). An RNAscope probe against the bacterial gene dapB (Advanced Cell Diagnostics, catalog #310043) was used as negative control to confirm absence of background and/or of non-specific crossreactivity of the assay, while a probe against the housekeeping gene PPIB (Advanced Cell Diagnostic, catalog# 494421) was used to assess the quality of RNA present in the tissue sections. Concurrently, paraffin wax blocks of tissues collected from Atlantic salmon (n = 3) during an outbreak of ISA in New Brunswick in 1999 (confirmed positive by IFAT, PCR and tissue culture) were used as positive controls for ISAV presence, while one sample (n = 1) that had tested negative to ISAV from an experimental challenge (Finstad et al., 2014) was used as negative control. These sections were also stained for PRV-1. Signal amplification for both BASEscope and RNAscope probes was performed according to the manufacturer’s instructions, followed by counterstaining with Gill’s haematoxylin, and visualisation by bright field microscopy.
7
Journal Pre-proof 3. Results. Lesions typical for HKS were seen in all the H&E-stained sections, including renal interstitial haemorrhage and acute renal tubular necrosis associated with the presence of eosinophilic casts (Fig. 1a). Minimal changes in the spleen included congestion and haemosiderin accumulation while there were no obvious lesions in liver or heart. ISH staining for ISAV of the HKS samples revealed systemic involvement, although most of the staining occurred in the kidney and spleen. In the kidney, ISAV was localized in endothelial cells, renal portal macrophages and a few red blood cells (RBC’s), but was also present in the necrotic material contained within the damaged renal tubules and in the renal casts (Fig. 1c). The virus was also localized on the brush border of some apparently healthy renal tubules (Fig. 1d). In the spleen, ISAV was localized within macrophages and a few RBC’s, while most endothelial cells also appeared to be infected. Overall, ISAV appeared to involve primarily vascular endothelium within every organ tested, despite the lack of obvious lesions. ISH staining for PRV localized the virus to RBC’s in every organ. Several stained cells were also observed in the reticulo-endothelial system, particularly in the kidney (portal macrophages) and spleen (Fig. 1e), but no endothelial cells were stained. The staining in the kidney and spleen partially overlapped the staining for ISAV, including positive marking in the necrotic material contained within the damaged renal tubules and in the renal casts (Fig. 1e). PRV was also localized on the luminal brush border of apparently healthy renal tubules (Fig. 1f), the same ones as those staining for ISAV. No staining was present in the heart. By contrast, staining for PRV was detected in some hepatocytes, although this was minimal, especially by contrast with ISAV. The negative and positive controls worked as expected, although for the ISA positive samples, the spleen showed the strongest staining, notably in the centre of the section, suggesting that there had been some degradation during the 20+ years that the blocks had been in storage. In particular it should be noted that there was no staining for PRV in the kidney of any fish that did not have HKS. 8
Journal Pre-proof 4. Discussion. Re-examination of the archived material from which HKS was originally described and defined, has shown that sections of kidney (and other tissues) were heavily stained for the presence of both ISAV and PRV, and that the staining correlated with the typical lesions of HKS, namely renal interstitial haemorrhage plus nephrosis with eosinophilic casts. Staining for ISAV and PRV was seen at the brush border of some tubules, and this correlated with the staining densities seen in TEM (Byrne et al., 1998), thereby supporting the original suggestion that these electron densities are virus-related; a good location indeed for transmission to other fish! The significance of the presence of virus-induced erythrocytic inclusion bodies in the original description of HKS was unknown, and their presence was overlooked in subsequent work (Lovely et al., 1999). To date, all three strains of PRV have been shown to produce inclusion bodies in erythrocytes of various species, including Atlantic salmon (Wessel et al., 2014; Takano et al., 2016; Hauge et al., 2017). All three PRV strains have also been associated with various anaemia-related syndromes in Pacific salmon i.e. PRV-1 and jaundice/anaemia in chinook salmon (Di Cicco et al., 2018); PRV-2 and EIBS in coho salmon (Takano et al., 2016); and PRV-3 and HSMI-like disease in rainbow trout (Olsen et al., 2015) and coho salmon (Godoy et al., 2016). The high specificity of the probe used here shows that these fish with HKS were infected with at least PRV-1. The co-existence of other PRV sub-strains cannot be excluded/ruled out, although they are considered to be not present in Canada. This is a relatively small data set on which to base too many conclusions, but certain questions seem nevertheless appropriate. Was HKS a result of ISAV or PRV infection, or were both viruses necessary? The nephrotic lesions of HKS closely resemble those seen in Pacific salmon with PRV infection in which haemoglobinuria was confirmed (Di Cicco et al., 2018). The haemoglobinuria likely leads to oxidative damage to, and necrosis of, renal tubular epithelium, just as it does in mammals (Deuel et al., 2016). It is important to note that these changes with PRV infection in Pacific salmon occurred in 9
Journal Pre-proof the absence of any involvement of ISAV, although it is also important to appreciate that interstitial haemorrhage was not a feature either. So, if haemoglobinuric nephrosis in typical HKS fish can be explained by PRV alone, what role did ISAV play? Haemorrhage is the major histopathological feature of ISA, but in the liver, not the kidney (Evensen et al., 1991; Speilberg et al., 1995). The finding that the major target for ISAV are the cells of the vascular endothelium (or closely associated cells), partly explains the classical hepatic lesions in ISA. This could also explain the renal interstitial haemorrhage in HKS, but if ISAV is indeed directly involved in this haemorrhage, it should probably also be seen in anterior as well as posterior kidney (as well as in other organs). The lack of interstitial haemorrhage in the anterior kidney suggested originally that it was the tubular lesions driving the interstitial haemorrhage, rather than vice versa (Byrne et al., 1998). This is supported by the fact that the renal portal system is just as extensive in anterior kidney as in posterior, and that injected particulates are phagocytosed equally avidly by portal macrophages in both regions (Ferguson et al., 1982). ISA continues to be reported in Eastern Canada (Canadian Food Inspection Agency, 2019). By contrast, local diagnosticians in the Maritimes report that HKS is rarely seen these days. While there are data (testing by PCR) reporting the presence of PRV in farmed and wild salmon in Eastern Canada (ICES, 2015) we can find no published histopathological data or other evidence of disease. So if it can be accepted that HKS was caused primarily by PRV, or by a combination of PRV and ISAV, any subsequent decline in incidence of HKS might simply be due to reduced viral loads. How would this occur? One of the major governmental measures introduced to try and control the spread of what was at the time an unknown (but presumed infectious) disease, was stopping the practice of emptying “untreated blood water” into the marine environment at harvesting. Even though infection may not necessarily lead to cell lysis, the main target cell for PRV is the erythrocyte (Finstad et al., 2014), but ISAV also binds to erythrocytes (Aamelfot et al., 2012). This control measure alone should, therefore, have had a profound impact on levels of both PRV and ISAV in the seawater. If
10
Journal Pre-proof there is any level of synergy between PRV and ISAV, reducing levels of either pathogen should have a more profound impact than otherwise expected. The ISH methodology in this paper does not co-localize antigens within the same section, although that approach is technically possible. Instead serial sections were used, as the interest centred mainly on correlating the presence of virus(es) with typical HKS lesions. Nevertheless, serial sections at 4 microns allowed us to see the same histological structures, e.g. a glomerulus, in sequential sections, and indeed often the same cells. Nor does the methodology attempt to quantify the viral loads within the tissue. In this context, however, it is interesting to look at the HKS transmission studies (Lovely et al., 1998). Using an ultrafiltrate of kidney tissue produced up to 100% mortality. The fish all had lesions typical of HKS and not ISA, even though both ISAV and PRV were almost certainly present in the injected material (EIBS was not considered, so was not assayed). This might suggest that PRV was present at higher loads than ISAV, that it was more virulent than the ISAV, that the fish were more susceptible to PRV/resistant to the strain of ISAV present, or that the fish died from HKS before they could develop typical ISA lesions. Unfortunately this study does not mention when fish started to die, only that there was up to 100% mortality by day 45 post-injection. The incubation period for ISA is usually 10-20 days (Rimstad & Mjaaland, 2002), so this should have been long enough for at least some of the fish to have died with lesions typical of ISA; but none did. Another possibility is that the 2 viruses acted together, and as both have an affinity for erythrocytes, this seems the most reasonable suggestion. The combined or synergistic effect of the viruses would probably be to increase the rate of lysis of erythrocytes, faster than would have happened with one virus alone, leading to haemoglobinuria, nephrosis and death before hepatic vascular damage. It is not uncommon for two or more viruses to infect (co-infection) a plant or animal at the same time (Murphy & Bowen, 2006). One recent report showed that chickens infected with low pathogenicity avian influenza virus had increased susceptibility to velogenic Newcastle disease virus, thereby altering clinical outcomes (Bonfante et al., 2017). Similarly, a superinfection (infection of a
11
Journal Pre-proof host by two viruses at different times) of chickens by Marek’s disease virus (MDV) and avian leucosis virus was recently shown to have a synergistic role, leading to increased pathogenicity (Zhou et al., 2018). There are a few reports of viral co-infections in fish (Kotob et al., 2016); in some cases there is synergism and in others, antagonism. It is unclear if the present case of HKS represents a coinfection (dual infection), or whether it might be a superinfection, as the timing of the infections is unknown. Nor is there any evidence in this case to suggest synergism, but it must remain a distinct possibility. A background (endemic?) level of a low pathogenicity virus may be well tolerated at a population level and lead to few clinical cases of full-blown disease. In such circumstances it is easy to understand why little effort would be made to screen populations of fish for its presence, even if its identity were known. Such was probably the case with ISAV in New Brunswick up until the late 1990’s. Similarly, even moderate levels of PRV can probably be well tolerated by the host (Løvoll et al., 2012) so long as nothing supervenes to cause an acute haemolytic crisis, thereby suddenly releasing large quantities of viral antigen. But a superinfection of PRV on top of a low-level infection of ISAV (or vice versa) might well lead to augmentation (synergism) and more severe clinical disease. As seen in the case of chickens with avian leucosis virus that become superinfected with Marek’s disease, increased severity of lesions and clinical disease may occur due to increased interleukin production, enhanced rate of viral protein production and more severe cell degeneration (Zhou et al., 2018). The lesions in renal tubules in HKS, driven partly by haemoglobin and partly by virus, probably led to release of locally high levels of vasoactive factors which were then able to target the peritubular capillaries of the renal portal system (Ferguson, 1984), leading to necrosis and interstitial haemorrhage.
12
Journal Pre-proof
5. References. Aamelfot, M., Dale, O.B., Weli, S.C., Koppang, E.O., Falk, K., 2012. Expression of the infectious salmon anemia virus receptor on Atlantic salmon endothelial cells correlates with the cell tropism of the virus. Journal of Virology 86, 10571-10578. Bonfante, F., Cattoli, G., Leardini, S., Salomoni, A., Mazzetto, E., Davidson, I., Haddas, R., Terregino, C., 2017. Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent. Avian Pathology 46(5), 488-496. Byrne, P.B., Ostland, V.E., Johnson, G., MacPhee, D.D., Ferguson, H.W., 1998. Haemorrhagic kidney syndrome, a new disease of sea-caged Atlantic salmon (Salmo salar L). Journal of Fish Diseases 21, 81-92. CFIA, 2019. http://www.ices.dk/community/groups/Pages/WGPDMO.aspx Di Cicco, E., Ferguson, H.W., Kaukinen, K., Schulze, A., Li, S., Vanderstichel, R., Laurin, E., Wessel, O., Rimstad, E., Gardner, I.A., Hammell, L., Miller, K.M., 2017. Longitudinal Farm Study into Heart and Skeletal Muscle Inflammation (HSMI) disease on a British Columbia salmon farm. http://dx.doi.org/10.1371/journal.pone.0171471 Di Cicco, E., Ferguson, H.W., Kaukinen, K.H., Schulze, A.D., Li, S., Tabata, A., Gunther, O., Mordecai, G., Suttle, C., Miller, K.M., 2018. The same strain of Piscine OrthoReovirus (PRV) is involved with the development of two different diseases in Atlantic and Pacific Salmon in British Columbia. Facets2018-0008.R1 Deuel, J.W., Schaer, C.A., Boretti, F.S., Opitz, L., Garcia-Rubio, I., Baek, J.H., et al., 2016. Hemoglobinuria related acute kidney injury is driven by intrarenal oxidative reactions triggering a heme toxicity response. Cell Death and Disease, 7(1): e2064. DOI: 10.1038/cddis.2015.392
13
Journal Pre-proof Evensen, Ø., Thorud, K.E., Olsen, Y.A., 1991. A morphological study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar). Research in Veterinary Science 51(2), 215222.
Ferguson, H.W., Claxton, M.J., Moccia, R.D., Wilkie, E.J., 1982. The quantitative clearance of bacteria from the bloodstream of rainbow trout (S. gairdneri). Veterinary Pathology 19, 687-699. Ferguson, H.W. 1984. Renal portal phagocytosis of bacteria in rainbow trout (Salmo gairdneri Richardson): ultrastructural observations. Canadian Journal of Zoology 62, 2505-2511. Finstad, Ø. W., Dahle, M. K., Lindholm, T. H., Nyman, I. B., Løvoll, M., Wallace, C., Olsen, C. M., Storset, A. K., Rimstad, E., 2014. Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes. Veterinary Research 45(35), 1-13. Godoy, M.G., Kibenge, M.J., Wang, Y., Suarez, R., Leiva, C., et al. 2016. First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV. Journal of Virology 13, 98. https://doi.org/10.1186/s12985-016-0554-y PMID: 27296722 Hauge, H., Vendramin, N., Taksdal, T., Olsen, A.B., Wessel, Ø., Mikkelsen, S.S., et al. 2017. Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids. PLoS ONE, 12(7), e0180293. https://doi.org/10.1371/journal.pone.0180293 ICES 2015. Working Group on Pathology and Diseases of Marine Organisms, Gloucester Point, Virginia, USA. Canada country report, February 16-20. Kotob, M.H., Menanteau-Ledouble, S., Kumar, G., Abdelzaher, M., El-Matbouli, M., 2017. The impact of co-infections on fish: a review. Veterinary Research, 48, 26. Lovely, J.E., Dannevig, B.H., Falk, K., Hutchin, L., MacKinnon, A.M., Melville, K.J., Rimstad, E., Griffiths, S.G., 1999. First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome. Diseases of Aquatic Organisms 35, 145-148. 14
Journal Pre-proof Løvoll, M., Alarcon, M., Bang, J.B., Taksdal, T., Kristoffersen, A.B., Tengs, T., 2012. Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production. Diseases of Aquatic Organisms, 99, 7-12. Mullins, J. E., Groman, D., Wadowska, D., 1998. Infectious salmon anaemia in salt water Atlantic salmon (Salmo salar L.) in New Brunswick, Canada. Bulletin European Association Fish Pathologists, 18, 110–114. Murphy, J. F., Bowen, K. L., 2006. Synergistic disease in pepper caused by the mixed infection of Cucumber mosaic virus and Pepper mottle virus. Phytopathology, 96, 240-247. Olsen, A.B. Hjortaas, M., Tengs, T., Hellberg,H., Johansen, R., 2015. First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV). PLoS ONE 10(7): e0131638. doi:10.1371/journal.pone.0131638 Rimstad, E., Mjaaland, S., 2002. Infectious salmon anaemia virus: An orthomyxovirus causing an emerging infection in Atlantic salmon. APMIS Acta pathologica, Microbiologica, Immunologica Scandinavica, 110, 273-282. Speilberg, L., Evensen, Ø., Dannevig, B.H., 1995. A sequential study of the light and electron microscopic liver lesions of infectious anemia in Atlantic salmon (Salmo salar L.). Veterinary Pathology, 32(5), 466-478. Takano T, Nawata A, Sakai T, Matsuyama T, Ito T, Kurita J, et al., 2016. Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus. PLoS ONE 11(10): e0165424. https://doi.org/10.1371/journal.pone.0165424 Wessel, Ø., Braaen, S., Alarcon, M., Haatveit, H., Roos, N., Markussen, T., et al. 2017. Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle 15
Journal Pre-proof inflammation in Atlantic salmon. PLoS ONE 12(8): e0183781. https://doi.org/10.1371/journal.pone.0183781 Zhou, J., Zhao, G.L., Wang, X.M., Du, X.S., Su, S., Li, C.G., Nair, V., Yao, Y.X., Cheng, Z.Q., 2018. Synergistic Viral Replication of Marek’s Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens. Viruses, 10, 271. 6. Captions. Figure 1. Histopathology and ISH of posterior kidney from Atlantic salmon with HKS. Matching fields of consecutive serial sections from two paraffin wax blocks (a, c, e, and b, d, f, respectively) are stained with H&E (a, b), ISH for ISAV (c, d) and PRV-1 (e, f). (a) Section of posterior kidney showing diffuse interstitial haemorrhage and congestion, plus acute renal tubular necrosis associated with the presence of eosinophilic renal casts (arrows). (b) Posterior kidney showing diffuse interstitial haemorrhage and apparently healthy renal tubules, some of which contain necrotic debris (arrows). (c) ISAV (red) was localized primarily in the necrotic tubules (arrows) and in the necrotic debris of the renal casts (open arrow). (d) ISAV (red) localized in the tubular necrotic debris (open arrow). Some apparently healthy renal tubules also show ISAV staining on the luminal side of the epithelium (arrows). (e) PRV-1 (red) was localized primarily in the necrotic tubules and in the necrotic debris of the renal casts (arrows). (f) PRV-1 (red) localized in the tubular necrotic debris (open arrow). Some apparently healthy renal tubules also show PRV-1 particles on the luminal side of the epithelium (arrows). The scale bar in all figures is 100μm.
7. Funding. This work was partially supported by the Pacific Salmon Foundation.
16
Journal Pre-proof
17
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof
Journal Pre-proof