Atlantic Salmon (Salmo salar L.): Infectious diseases - Molecular diagnosis (PCR, RT-PCR and Nucleic Acid sequencing)
Atlantic salmon (Salmo salar L.) is one of the most important commercial aquaculture fish species. Atlantic salmon is susceptible to a number of disease agents including viruses, bacteria, fungi, protozoa and helminths. We summarize some of the most frequent infectious in the Atlantic salmon that we can diagnosis by molecular methods. In case you do not find the answer you need please contact with us for further comments.
Pancreas disease (PD) (Salmon Alphavirus subtypes)
Pancreas disease (PD) is an economically important disease in European salmonid aquaculture affecting Atlantic salmon (Salmo salar L.) that affects farmed salmonids in the seawater phase in Ireland, Norway and Scotland. The disease is characterized by degeneration and necrosis of cardiomyocytes with subsequent inflammation, pancreatic acinar cell loss and subsequent skeletal muscle degeneration. PD is caused by salmon pancreas disease virus (SPDV) also referred to as salmonid alphavirus (SAV). This virus belongs to the genus Alphavirus within the family Togaviridae. The first alphavirus to be isolated from fish was recorded in 1995 with the isolation of salmon pancreas disease virus from Atlantic salmon, Salmo salar L., in Ireland. The alphaviruses are small, spherical, enveloped viruses that are important pathogens of animals and humans worldwide. Most alphaviruses are transmitted by highly specific arthropod vectors, of which mosquitoes are the most common, but others exist such as lice and mites. This together with specific environmental conditions and reservoir hosts might lead to restricted geographical distribution. Although SAV3 has been detected from the salmon louse Lepeophtheirus salmonis collected from diseased fish, the role of lice or other invertebrate vectors is yet to be determined as SAV infections can be transmitted without the aid of an arthropod, an this virus is shed via the feces and skin mucus from salmon.
SAV genomes consist of a single stranded, positive-sense RNA (+ss RNA) genome of 11–12 kb. The genome contains two open reading frames with the 5´ two-thirds of the genome encoding the four nonstructural proteins (nsP1–4), while the four capsid glycoproteins (E1, E2, E3 and 6K) are encoded in the 3´ end of the genome. Six SAV subtypes have been identified based on the nucleic acid sequences encoding proteins E2 and nsP3, namely SAV subtypes 1–6. The six genetically distinct subtypes, SAV1–6, are separated geographically. The SAV3 subtype was only been shown to cause pancreas disease outbreaks in Norway in seawater phase of the salmon life-cycle. Recently, a comparative experimental study in Atlantic salmon, conducted as a freshwater cohabitation trial, showed that all subtypes caused pathological changes typical of PD, although the relative virulence of the strains varied SAV3 represents a subtype that so far has been detected only in Norway where it causes PD in Atlantic salmon. However, following the first detection of a SAV2-related virus in 2011, this subtype is now frequently found in PD outbreaks in mid-Norway. The SAV subtype 2 is divided into two subgroups: the freshwater variant, SAV2 FW, and the marine variant, marine SAV2. SAV2 FW typically causes sleeping disease (SD) in freshwater-reared rainbow trout (Oncorhynchus mykiss in England, Scotland and a number of countries on the European mainland. Marine SAV2 typically causes PD in seawater-reared Atlantic salmon (Salmo salar L.). SAV 1, 4, and 6 have been identified in conjunction with Irish PD outbreaks, while SAV 1, 4, 5 and marine SAV2 have been associated with Scottish outbreaks SAV 3 has so far only been detected in Norway, where outbreaks caused by marine SAV2 are also seen.
Fish affected by the PD often show reduced appetite and abnormal swimming behaviour prior to the onset of mortality. Post-mortem inspection commonly finds yellow mucoid gut contents or empty intestines, faecal casts and signs of circulatory disturbance, while petechial haemorrhages in the periacinar fat may be observed. Histological examination usually reveals complete loss of exocrine pancreatic tissue, cardiac myocytic necrosis and inflammation and degeneration and/or inflammation of skeletal muscle. Clinical signs and macroscopic changes are not pathognomonic, and histopathological examination and additional diagnostic tests to detect the virus, such as real-time RT-PCR, are required in order to confirm a diagnosis of PD.
Infectious Salmon Anaemia (ISA) (Orthomyxoviridae)
Infectious Salmon Anaemia (ISA) is caused by ISA virus, an RNA virus of the family Orthomyxoviridae. Some infected fish will show signs of anaemia, such as pale gills, while others remain asymptomatic until death. The virus is transmitted horizontally as the virus is shed in epidermal mucus, urine, feces and gonadal fluids, and taken up through the gills or orally
Infectious salmon anaemia was first reported in 1984 in Norwegian farmed Atlantic salmon with the causal viral agent isolated 1n 1995. Farmed Atlantic salmon can be affected by clinical disease and other salmonid species can be ISA virus (ISAV) carriers. The first outbreak of ISA in Scottish farmed Atlantic salmon was confirmed in 1998 and was geographically wide spread. There is evidence that ISAV can be carried by wild salmonids.
Infectious pancreatic necrosis (IPN) virus (IPNV) (Birnaviridae, Aquabirnavirus)
Infectious Pacreatic Necrosis (IPN) is a highly contagious disease of young salmonids worldwide. Infectious pancreatic necrosis was initially reported to affect first-feeding fry, but in industrial culture of Atlantic salmon, severe outbreaks can also occur in post molts shortly after seawater entry. Infection with IPNV can result in clinical disease or subclinical infection. In both cases, survivors could become life-long carriers. In carriers approaching sexual maturity, variable levels of virus are found in the ovarian and seminal fluids.
This disease was first described in the 1950s and the causative virus (IPNV) was isolated in 1960. The first report in Europe was in 1965. Birnaviridae are small, non-membraned double-stranded RNA (dsRNA) virus (IPNV). IPN remains a significant cause of high mortality in first feeding fry and fingerlings of most salmonids and, as it can be vertically transmitted via the egg, broodstock carrier fish are an important facet of its pathogenesis. Although some of the earliest descriptions of the disease related to losses of Atlantic salmon fry, it is only in the past 20 years, as Atlantic salmon culture has dramatically expanded as a marine culture technology, it is now recognized to be the most important disease in its impact on salmon production in the European Union and in Norway. In many salmon producing countries, because of the risk of major losses in first-feeding fry infected from the parent via vertical transmission through the egg, broodstock are routinely tested for the presence of virus from the kidney or gonadal fluids. Eggs are also routinely disinfected with buffered iodophore disinfectants at the same time, but any eggs from parents that subsequently test positive are destroyed, as there are considerable doubts as to the efficacy of iodophore disinfection. Atlantic salmon hatcheries are also, where possible, maintained on IPNV-free fresh water. Molt stocks in fresh water may also be tested to confirm the overall disease status of the producing farm. The outbreaks were generally associated with a specific N subtype of the main Sp serotype of the virus and a subtype mutation of an Sp strain which has been designated the Sh subtype.
In Atlantic salmon the clinical features and pathology are with fry, darker in colour, showing in the surface water film or at outflows, making distinctive shimmering movements, whirling or lying on their side and hyperventilating. Outbreaks normally occur within 3 or 4 weeks of first feeding and may result from vertical transmission, poor biosecurity in the production system or contaminated water supplies. Mortality level is often related to stocking density and can be as high as 90% in very young fry. Survivors generally progress normally. The histopathological picture in young fry is one of severe necrosis of the pancreatic acinar cells, while endocrine and most of the limited fatty peripancreatic tissue is normal apart from some lipid necrosis. There is necrosis of intestinal mucosa, which is variable in intensity. The liver consistently shows areas of severe focal or generalized necrosis. Until the advent of intensive marine salmon culture, IPN of salmon was only an occasional problem of fry in fresh water.
Infectious Hematopoietic Necrosis virus (IHNV) (Rhaddoviridae, Novirhabdovirus)
IHNV is the causative agent of infectious hematopoietic necrosis (IHN), a widespread disease mainly found in salmonid fish species in western North-America, continental Europe and Asia. Clinically affected fish externally show the skin darkening, exophthalmia and pale gills. Common necropsy findings are pale internal organs with petechial hemorrhages, and intestines often filled with mucus-like fluid.
Infectious hematopoietic necrosis virus (IHNV) is a member of the Rhabdoviridae family, genus Novirhabdovirus, which are bullet shaped viruses with non-segmented, negative single stranded RNA genome (-ssRNA). The IHNV virion is bullet shaped and contains a single stranded, non-segmented, negative sense RNA genome of approximately 11 000 bases which encodes six proteins in the order nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV) and polymerase (L). The NV protein is unique and its presence has resulted in the establishment of a separate genus, Novirhabdovirus, within the Rhabdoviridae, with IHNV. Phylogenetic analyses of the genetically diverse G gene of IHNV define five major genogroups (U, M, L, E, J), which broadly refer to the geographical distribution of the genogroups.
IHNV is one of the three three rhabdovirus of finfish listed by the OIE (World Organization of Animal Heath). The presence of IHNV in Europe was first confirmed in 1987, with at least two different introductions in Italy and France, with viruses originating from the M genogroup. Since then, IHNV has spread in different European countries, evolving separately and constituting the E genogroup which is the youngest within IHNV genogroups. IHNV is currently endemic in continental Europe, while countries producing Atlantic salmon in northern Europe are declared officially free from the virus according to European legislation. The virus is widespread in western North-America, including seawater areas with Atlantic salmon farming, where a DNA vaccine is used to control the disease. In the seawater phase both Pacific salmonid species and Atlantic salmon are susceptible to Infection.
There appears to be one serotype in comparisons using polyclonal antisera, although sub-types/variants have been reported using monoclonal antibodies. Different electropherotypes have been described, but the method currently most widely used for strain differentiation is through sequence analysis.
Viral haemorrhagic septicaemia (VHS) (VHSV, Rhabdoviridae, Novirhabdovirus)
Viral haemorrhagic septicaemia is caused by VHSV (Rhabdoviridae, Novirhabdovirus). It is geographically wide spread and the The World Organisation for Animal Health (2014) list over 70 species of fish as susceptible including Atlantic salmon. It was first described in Europe in 1938 and the first isolation in wild marine fish was made 1979. To date, there have been two VHS outbreaks in Scottish farmed fish. The first was in 1994 in turbot and caused mortality, with the disease being contained within a single fish farm on the island of Gigha. The second outbreak in 2013 affected species of wrasse used as cleaner fish on marine Atlantic salmon farms. There are also reports of VHSV being found in wild marine fish caught in European waters.
Heart and Skeletal Muscle Inflammation (HSMI) (Piscine Orthoreovirus –PRV)
Piscine Orthoreovirus (PRV) is ubiquitous in farmed salmon in Norway during the sea water phase, and has emerged in recent years as a relevant threat for Atlantic salmon aquaculture being the etiological agent of heart and skeletal muscle inflammation (HSMI).
HSMI had been reported within the Norwegian farming industry since 1999. Even though the disease was infectious, disease challenge studies using viral filtrates from tissue homogenates of Atlantic salmon with HSMI replicated the patterns of inflammation in heart and skeletal tissue that are diagnostic of this disease. However, only recently was a cause-and-effect relationship established through a challenge based on an isolate of Piscine orthoreovirus purified from blood, the primary target for the virus. Since its discovery in 1999, the prevalence of HSMI has expanded from < 30 farms in central Norway to hundreds of farms throughout the country. While HSMI became a reportable disease in 2004, it is no longer reportable due both to the ubiquitous distribution of Piscine orthoreovirus (strains of which are collectively referred to as PRV) and the large-scale distribution of HSMI outbreaks. There are recent reports of HSMI outbreaks occurring in Norwegian freshwater hatcheries. HSMI has also been reported in farmed Atlantic salmon in Scotland and the United Kingdom, Chile and Canada.
While HSMI has been formally diagnosed only in Atlantic salmon, other salmonids and some marine fish are also susceptible to infection by PRV. In the last few years, there has been a surge of PRV-related diseases reported in Pacific salmon in Norway, Chile, Japan, and Canada. The same strain of PRV, and possibly PRV-1, has also been implicated in outbreaks in farmed Chilean Coho salmon (Oncorhynchus kisutch), in which major hepatic necrosis and erythrophagocytosis in the kidney and spleen were prominent pathological features commonly reported.
PRV is a non-enveloped virus with a segmented double stranded RNA genome (dsRNA) enclosed in a capsid with two concentric protein layers. The gross pathological findings of HSMI point towards circulatory failure, and characteristic histopathological findings are epi-, endo- and myocarditis, myocardial necrosis, red skeletal myositis and necrosis. PRV infection in Atlantic salmon induces a strong innate antiviral immune response in its major target cell, the erythrocyte, and thus this response can be measured in any vascularized organ, and has been described in various organs such as spleen, head kidney and heart tissue.
Viral Gill Diseases (Atlantic salmon Paramyxoviridae-Respirovirus –ASPV- and salmonid gill Poxvirus –SGPV-).
Two viruses to date have been associated with gill disease: Atlantic salmon paramyxovirus (ASPV) and salmonid gill Poxvirus (SGPV). In both cases, the mechanism by which they negatively affect the host and their contribution to gill pathology is unclear.
ASPV was first isolated in 1995 from farmed Atlantic salmon in Norway suffering from PGI. To investigate the significance of the virus, an experimental challenge with the isolate was performed but did not result in any mortality or pathology. Nevertheless, a synergistic role of this virus in combination with other gill pathogens was suggested. Another study attempting to uncover the aetiological significance of ASPV used immunofluorescent staining and immunohistochemistry. The virus was found associated with diseased tissue on three different farms affected with PGI, suggesting it may have a role in the disease. Through molecular studies, the virus was assigned to the genus Respirovirus within the family Paramyxoviridae. Paramyxoviruses have been associated with respiratory diseases in mammals and birds, and primary replication occurs mainly in the respiratory tract. A common finding in pneumonia in terrestrial animals is that a virus or viruses are involved as trigger pathogens that then allow secondary bacterial infections to become established. Although the biological model of virus/bacterial interaction for respiratory disease has been strongly documented in mammalian species, the role, if any, of ASPV or other viruses in diseases of fish is not clear. Furthermore, ASPV is not consistently detected in cases of PGI in Norway.
In 2006, Salmonid Gill Poxvirus (SGPV), a DNA virus, was first observed by electron microscopy in Norway. Gill epithelial cells infected with the Poxvirus exhibited extreme hypertrophy and degeneration of the nucleus. The virus was observed in the gills of Atlantic salmon suffering from a proliferative gill disease which had resulted in 20% mortality at a freshwater facility in northern Norway. Later in 2006, the virus was also observed in Atlantic salmon at two marine sites in western Norway where mortalities close to 80% were recorded. In both these marine sites, concurrent infections of the gills with the bacterium Piscichlamydia salmonis and the amoeba N. perurans may have contributed to the high mortalities. As with ASPV, the significance of this virus for salmonids as a gill pathogen remains to be determined, and again, multifactorial interactions may be a prerequisite for the expression of disease.
With advances in molecular and other techniques in recent years, it is becoming easier to detect viruses. The role these have played in gill disease may have been underestimated. The first report to describe epitheliocystis in bluegill, Lepomis macrochirus, includes an electron micrograph of affected gill tissue in which a virus was also observed. This virus had similarities to a Paramyxovirus, perhaps provideng the first support that the interaction of viruses with other infectious agents may be key to understanding their role in gill disease as a whole. The relationship, if any, between some of the systemic viral diseases, such as the salmonid alphavirus (SAV) and the infectious salmon anaemia virus (ISA), and gill disease also deserves further attention. The routes of entry of SAV and ISAV remain to be established fully; however, gills are a likely entry point and compromised or diseased gills may allow easier entry of the viruses into the hosts.
Francisellosis (Francisella noatunensis subsp. noatunensis y F. noatunensis subsp. orientalis)
In recent years bacteria belonging to the genus Francisella have “emerged” as serious pathogens of various fish species, both farmed and wild, from various geographical regions worldwide. Francisella infections in fish are serious and more widely distributed than previously thought just a few years ago. Nowadays, two subspecies are admitted, Francisella noatunensis subsp. noatunensis y Francisella noatunensis subsp. orientalis. All described incidences of francisellosis in fish manifest in a similar fashion which can be summarised as systemic, chronic, granulomatous infections resulting in varying degrees of mortality. A granulomatous condition is also reported in association with Francisella infections in Atlantic salmon followed by 5-20% in Atlantic salmon.
Francisella noatunensis is the causal agent of the Franciselosis of fish. In recent years, bacteria belonging to the species Francisella noatunensis have emerged as serious pathogens of various fish species, both wild and from fish farms, in various geographical regions around the world. Strains of F. noatunensis are highly pathogenic for fish and can cause high mortality and losses, causing epidemics throughout the world, especially in fish farms. Although the species F. tularensis has been associated with infections in fish since 1970, this bacterium has not been associated with fish disease in recent years. In light of the recent description of pathogenic Francisella species of fish, which share several phenotypic traits with F. tularensis, it is thought that these previous descriptions may be due to erroneous identifications. Similarly, it is believed that franciselosis is not a recent disease, but that the usual diagnostic methods have allowed its identification in the epidemics of recent years. Two subspecies of F. noatunensis have been described. F. noatunensis subsp. noatunensis that affects commercially important cold water fish such as Atlantic cod and Atlantic salmon. Meanwhile, F. noatunensis subsp. orientalis is the causative agent of francisellosis in warm-water fish, including tilapia, striped water bass, perch trilineata and ornamental fish.
All reported incidences of francisellosis in fish are manifested in a similar manner, as systemic, chronic, and granulomatous infections, which result in varying degrees of mortality. Externally, diseased fish do not show specific clinical signs. Common manifestations include the observation of a large number of protruding white nodules of various sizes, called granulomas, in the spleen, kidney and liver. However, practically any type of tissue can be affected, and the associated pathological changes in the gills, heart, testicles, muscles, brain and eye have also been described. Factors such as temperature and coinfection with other pathogenic bacteria seem to affect the mortality rate.
Furunculosis (tail-rot) (Aeromonas salmonicida)
Furunculosis is a bacterial infection caused by Aeromonas salmonicida, a gram-negative, rod shaped facultative anaerobe whose virulence is attributed to an array of proteins, called the A-layer, that protects the bacterium. This disease, found mostly in Atlantic salmon, is characterized by boils on the sides of the fish and is highly contagious through open sores, resulting in its spread through salmon farms.
Furunculosis was first described on fish farms in Germany in the late 1800s and the causative bacterial agent then named Bacillus der Forellenseuche was isolated in 1894 from diseased brown trout. The bacterium was subsequently referred to as Bacillus salmonicida and has been known as Aeromonas salmonicida since the 1950s. In the 1980s, furunculosis was devastating to the growing Scottish farmed Atlantic salmon industry. During the epidemics peak in 1989, estimated losses of 15%–20%. Following the development of successful A. salmonicida vaccines mortality due to furunculosis in Scottish farmed fish is now uncommon.
Bacteria Gill Diseases
Aeromonas salmonicida, the causative agent of furunculosis is generally a septicaemic condition, but is included here as one of the features of the disease is bacterial colonies in the gills of affected fish. The gill lamellae are a common site for such bacterial colonies. Since the widespread use of A. salmonicida vaccines and other management changes, clinical furunculosis is rarely observed in farmed salmonids, although it is still frequently observed in wild fish. In a study investigating the relationship between different salmonid gill bacteria and AGD (Amoebid Gill Disease), bacteria from the genus Psychroserpens were found in increased numbers in fish affected with AGD.
A survey of gill-associated bacteria in farmed salmon with PGI in Norway observed a wide distribution of c-proteobacteria. Bacterial communities of individual fish gills were generally quite simple and often dominated by one of two phylotypes, most commonly Burkholderia-like bacteria (previously pseudomonads) and Psychrobacter spp. Fish spoilage organisms and pathogenic bacteria identified in this study included members of the genera Photobacterium, Flavobacterium, Aliivibrio, Shewanella, Tenacibaculum and at least one species closely related to Francisella. Bacterial communities appeared relatively consistent in clinically healthy fish in individual fish farms; however, a higher degree of variation was observed in fish affected by PGI. This information is useful in the light of recent evidence that some of the microorganisms capable of causing gill disease do so through a quantitative effect and may be present at low numbers in healthy fish.
Bacterial Kidney Disease (BKD) (Renibacterium salmoninarum)
The first report of BKD was in Scottish wild Atlantic salmon in 1933 with the disease first being reported in reared fish in 1935. The causative agent was isolated in 1956 and named Renibacterium salmoninarum in 1980. Renibacterium salmoninarum can cause BKD, primarily in wild and farmed salmonids and can infect some species of non-salmonids. Wild marine fish are not reported to be susceptible although there are reports of BKD in association with salmonid aquaculture and R. salmoninarum infection. Bacterial kidney disease was first reported in Scottish farmed Atlantic salmon in 1980. Since then, the prevalence of R. salmoninarum in Scottish aquaculture sites has varied year on year being generally more prevalent in rainbow trout. In recent years, R. salmoninarum has been infrequently isolated from farmed Atlantic salmon, disease progression tends to be slow, and for farmed Atlantic salmon, mortalities tend to occur in larger sea grown fish, which are more valuable, with only occasional disease outbreaks.
Tenacibaculosis (Flexibacteriosis) (Tenacibaculum maritimum)
Tenacibaculum maritimum (formerly Cytophaga marina, Flexibacter marinus and F. maritimus) is the causative agent of tenacibaculosis (or flexibacteriosis) in marine fish. It is a Gram-negative, filamentous bacterium, first described in 1986. A number of different names have been used to describe tenacibaculosis based on the diversity of clinical presentations that occur, including gliding bacterial diseases of sea fish, eroded mouth syndrome and gill rot. The first report of gill lesions associated with T. maritimum was in 1995 on chinook salmon on the Pacific coast of North America. The disease is usually more severe in juvenile fish when water temperatures are above 15ºC.
Clinical signs of tenacibaculosis are variable and related to the type of infection. Fish with gill infections may be moribund, lethargic and display increased respiratory rate. Sometimes yellow or brown mats can be seen on the gills, emerging beneath the opercula or following exposure to harmful zooplankton on the gill rakers. On clinical examination, gills may have increased mucus, be pale and frank patches of severe necrosis may be visible on the lamellae. Skin lesions may also be present. When explored the relative susceptibility of a number of different species of fish to T. maritimum it was found that Atlantic salmon is particularly susceptible, whereas other fish species seemed to be more resistant to infection.
The mode of transmission and the route of infection of T. maritimum are not well understood. Transmission through sea water and direct transmission from host to host have been proposed as possible routes, in addition to ingestion along with food. An unusual mode of transmission has been suggested when described a marine salmon farm in Scotland with high numbers of the jellyfish, Philalella quadrata, in close proximity to fish with significant gill disease and tenacibaculosis. Jellyfish were carrying high loads of T. maritimum and was suggested that the jellyfish act as vectors for the bacteria, the gills were initially damaged by nematocyst-derived toxins from the jellyfish and then the pathology was compounded by secondary bacterial infection with T. maritimum.
Investigations into the effects of environmental factors on T. maritimum show it has a fastidious requirement for salt and it will only grow in media with salinities greater than 7 g/L. Its optimum pH range is between 6 and 8 and optimum growth temperatures appear to be dependent on the strain of bacteria. Salinity or temperature modifications could potentially be used to control disease in closed systems.
Epitheliocystis and proliferative gill inflammation (PGI) (Piscichlamydia salmonis and Clavochlamydia salmonicola)
Epitheliocystis is an infectious condition that affects the gills and less commonly the skin of fish. It has been reported from more than 50 freshwater and marine species. The disease was first described in Germany in 1920 in common carp, Cyprinus carpio L., and named Mucophilus cyprini as it was attributed at that time to unicellular algae. When investigating disease and mortalities in bluegills, which initially was considered to be because of protozoans, eventually confirmed the cause of the problem as being because of Bedsonia (now termed Chlamydia) and the condition was termed epitheliocystis. Epitheliocystis in fish is considered to be caused by a cosmopolitan group of intracellular gramnegative bacteria, many of which remain to be characterized. To date, all have been members of the phylum Chlamydiae. In general, it is widely suggested that these bacteria are more frequently opportunistic rather than primary pathogens of vertebrates. Epitheliocystis has long been a focus of attention in farmed Atlantic salmon and debate persists as to its significance. The presence of the condition has been considered to be little more than an incidental finding however, have been observed epitheliocystis associated with cases of gill-related mortality and disease. Mortalities of up to 80% in some farm sites in Norway in addition to the loss of production in terms of growth have been attributed to PGI, which is frequently associated with epitheliocystis. Proliferative gill inflammation is a disease that causes significant losses in farmed Atlantic salmon in Norway. It has also been recently diagnosed in Scotland, and similar pathologies have been observed in Ireland. The most severe losses occur in spring salmon (S1s) following the first few months after sea transfer. The disease has been a challenge for Norwegian aquaculture since the 1980s. In the 1998–1999 production cycle, 18.8% of farms in Norway were affected by PGI. In 2002–2003, that figure rose to approximately 35% or 250 sites. The number of cases of PGI appears to be declining again, with 182 outbreaks recorded in 2008–2009. The apparent recent decrease in the number of PGI cases may also be as a result of decreased reporting or a result of more specific categorization for the identification of the syndrome.
In salmonids, at least two different bacterial species from the phylum Chlamydiae have been associated with epitheliocystis. These are Candidatus Piscichlamydia salmonis from marine stage Atlantic salmon in Norway and Ireland and Candidatus Clavochlamydia salmonicola from freshwater Atlantic salmon. Epitheliocystis caused by C. salmonicola has been observed in freshwater salmonids with mild gill inflammation and respiratory problems in Norway. Studies in salmon infected with Ca. C. salmonicola from fresh water to sea water, showed on microscopy that the epitheliocystis disappeared within 6 weeks of seawater transfer, in addition to all traces of the bacteria by PCR. A positive association existed between the presence of Ca. P. salmonis and the disease in Epitheliocystis infections are frequently described without any associated pathology or clinical signs. In infections where there is a proliferative host response to the cysts, affected fish have been described as lethargic and displaying signs of respiratory distress such as increased ventilation and gasping at the water surface. Deformation of the opercular cover, increased mucus production and distortion of the lamellar structure have also been described. The gills of moribund or dead fish may be densely covered in white or mucoid spheres. Under light microscopy, large cysts can sometimes be seen in the wet preparations of gill tissue, but more frequently the epitheliocysts are only visible on histopathology. Infected gill lamellar epithelial cells become spherical basophilic cysts surrounded by an eosinophilic hyaline capsule which are proposed to be the remnants of the host cell membrane and cytoplasm. The cysts contain basophilic granular material, which represents the bacteria. A complete developmental cycle has been described for the agents of epitheliocystis from a range of fish species using electron microscopy. The bacteria proceed from small rigid infectious forms to larger pleomorphic non-infectious forms that divide by fission to produce daughter cells.
Vibriosis (Vibrio anguillarum and Vibrio salmonicida)
Another bacterial infection, is caused by Vibrio species such as Vibrio anguillarum and Vibrio salmonicida which are gram-negative, curved rod-shaped facultative anaerobes with one and at least nine flagella respectively. Infecting fish residing in shallow salt and brackish water, this disease is characterized by damage to and discolouration of the skin and fins.
Columnaris (Flavobacterium columnare)
Columnaris is an infection caused by Flavobacterium columnare, a gram-negative, rod-shaped aerobe. Signs of columnaris include damaged fins, ulcers on the skin and discolouration of the gills as a result of acquiring other infections through the lesions
Amoebic Gill Disease (AGD) (Neoparamoeba pemaquidensis; Neoparamoeba branchiphila; Neoparamoeba perurans –NCGD-;)
The most significant disease caused by gill parasites, in terms of economic impact in marine salmonid aquaculture, is amoebic gill disease (AGD). AGD has been recognized as a significant problem of marine-farmed salmonids since 1984 in Tasmania where it has been estimated to account for 14% of production costs a year in terms of treatments and lost productivity, affecting fish growth as well as causing direct mortality. Outbreaks have also been reported in farmed Atlantic salmon, Salmo salar L., in Ireland, France, Scotland, Chile, Spain, New Zealand and most recently in Norway. The first outbreak in North America was in marine coho salmon, Oncorhynchus kisutch, where it resulted in significant losses. Atlantic salmon appear to be the most susceptible salmonid species affected by AGD. Outbreaks in Pacific salmon have generally been minor and sporadic, suggesting they may have inherent resistance to the disease. AGD has been recorded in chinook salmon, Oncorhynchus tshawytscha, farmed in New Zealand but is insignificant in terms of mortalities and rarely warrants treatment.
Neoparamoeba pemaquidensis, a free-living amoeboid protozoan found in the marine environment, was, for some time, regarded as the only aetiological agent of AGD as it had been consistently isolated from diseased fish. However, attempts to elicit experimental AGD using cultured gill-derived N. pemaquidensis failed to elicit AGD experimentally in Atlantic salmon. The true aetiology of AGD remained elusive until non-cultured, gill-derived (NCGD) amoebae with those of both N. pemaquidensis and another potential parasitic amoeba, Neoparamoeba branchiphila. Resulting phylogenetic analyses found the NCGD amoebae were separate from these other members of the genus Neoparamoeba. Species-specific oligonucleotide probes used on gill tissue from AGD-affected Atlantic salmon indicated that this NCGD amoeba was a new species, now known as Neoparamoeba perurans. Besides, the study confirmed that it has been the predominant agent of epizootics of AGD in Tasmania as well as cases in Ireland, North America, Scotland, New Zealand and NW Spain. It has been suggested that a number of other amoebae may be involved in AGD in addition to Neoparamoeba sp. They isolated a number of other genera that have been associated with disease in other teleosts. Platyamoeba, Flabellula and Vexillifera spp. have all been recorded on the gills of Atlantic salmon with AGD from both Ireland and Tasmania. The pathogenic potential of these genera and their role as potential agents of AGD are not established. They may not have a primary role in precipitating an outbreak of AGD but it is plausible they can play a part once the gills are compromised.
Parasites Gill Disease –Trichinodinosis- (Trichodina spp.; Ichthyobodo necator)
Trichodinids are important protozoan parasites of both freshwater and marine fish. By colonizing both the skin and gills, they can cause significant pathological effects. Hyperplasia of the epidermis and destruction of the normal gill structure are the most common effects of infestation. The freshwater species are commonly found on the skin, while marine trichodinids more frequently affect the gills. Reports of serious mortalities in salmonids caused by trichodinids are sparse in the marine environment but it has been reported substantial mortalities in salmon reared at sea in Ireland in the summers of 1982 and 1983. Fish were observed to be lethargic and incapable of maintaining their position in the water column. Flared opercula and a bluish film caused by excess mucus were observed. The gills of the fish were pale and eroded. On microscopy of fresh gill mounts, massive numbers of trichodinids were visible. Severe hyperplasia of the distal extremity of the primary and secondary lamellae, oedema and severe extensive erosion of some of the filaments were observed on histopathology. Bath treatments using formalin (30 min, 1:4000) were partially effective in controlling the infestation. Trichodina sp. and another protozoan, Ichthyobodo necator, have occasionally been observed in fish with PGI, but a significant role for either in this disease is considered doubtful.
Freshwater bathing of marine Atlantic salmon has recently been attempted in Ireland in fish with severe gill pathology and high levels of trichodinids and marine Ichthyobodo (= Costia). The treatment appeared to be effective in the short term substantially reducing the numbers of both parasites on the gills. The efficacy of hydrogen peroxide has also been tested as a treatment for trichodinosis and has been found that while the hydrogen peroxide appeared to be effective against some other external parasites, high levels of Trichodina sp. remained on the gills of the treated fish. The high levels of mucus production associated with trichodinosis may have protected the parasites from the hydrogen peroxide. Use of a salt bath prior to the hydrogen peroxide was suggested to reduce the mucus load on the affected fish
Microsporidial Gill Disease (Loma salmonae)
Microsporidial Gill Disease of Salmon (MGDS), caused by the microsporidian Loma salmonae, once considered an emerging disease in Canadian aquaculture, is now best considered as an endemic disease with a strong seasonal trend favoring late summer and early fall of each year. The first reported case of MGDS in British Columbia, Canada was in coho salmon (Oncorhynchus kisutch) smolts from a hatchery on Vancouver Island in 1987. Since its initial discovery in Canada, L. salmonae has been identified as an important salmonid pathogen for the British Columbia Pacific salmon industry; MGDS is characterized as a severe inflammatory gill disease, with variable (generally lesser) degrees of systemic organ involvement, high mortality rates, and prolonged recovery periods during which production efficiency is severely affected. Until recently, little pertinent information on the transmission of this pathogen was available. Accordingly, management techniques to minimize MGDS on fish farms were limited and were not linked to specific attributes of the causative agent. The earlier reports of L. salmonae from freshwater hatcheries initially led to the concept that the pathogen infected young salmon while they were in their juvenile freshwater production phase and that clinical expression of the disease (MGDS) arising later in the saltwater production phase of salmon stemmed from recrudescence following prolonged latency of the pathogen within an infected host. Strategies to screen juvenile salmon for L. salmonae, prior to the transfer of these salmon to marine net-pen sites, were therefore considered a key management tool. Current findings reverse this early assumption and demonstrate the ease with which this parasite transmits horizontally within environments of widely different salinities. Understanding the extracorporeal and corporeal persistence of the spore stages of this pathogen, and developing models to better evaluate the efficiency of horizontal transmission provide a strategic basis to limit the effect of MGDS on high host density salmon farms.
Cryptobiosis (Cryptobia [T.] salmositica)
Cryptobia (T.) salmositica infects all species of salmon on the Pacific coast. Cryptobiosis is caused by a bifagellated protozoa (Cryptobia, Kinetoplastida). The parasite is elongated and has two flagella which originate from the anterior end; the anterior flagellum is free while the recurrent flagellum is attached to the body and ends as a posterior free flagellum. Its kinetoplasts large and it is anterior to the nucleus which is located at the anterior part of the organism. There are at least 52 nominal species; most species are not well studied and as far as we know are not known to cause disease. Species that inhabit the blood (haemotozoic) are normally transmitted indirectly by blood sucking leeches, while those in the digestive system and body surface have direct transmission.
Proliferative Kidney Disease (PKD) (Myxozoa: PKX -Sphaerospora oncorhynchi, Kudoa, Chloromyxum spp.)
Myxozoans are well known for their distinctive propagules which are found within the fish’s tissues, often in conspicuous cysts. Seven species infect Atlantic salmon. Proliferative kidney disease (PKD), which affects most farmed freshwater salmonids, including S. salar, is caused by an unidentified myxozoan usually referred to as “PKX.” This pathogen has, in retrospect, been known for 75 years but was only recognized in the 1980s. The similarity between the observed sporogonic stages of PKX and corresponding stages in Sphaerospora species has been stressed and various Sphaerospora spp. infecting teleosts have been identified with the causative agent of PKD. Although implicated Sphaerospora oncorhynchi, a parasite of mature sockeye salmon in northwest North America, as the PKX organism, it has been suggested that PKX is not closely related to Sphaerospora. On the other hand, the complex life cycle of myxozoans, which may involve a tubificid intermediate host, makes them unlikely to sustain persistent disease epidemics in the wild, although infections from farmed to wild fishes may be possible. The epidemiology of marine myxozoans such as Kudoa and Chloromyxum species is so poorly understood that their effect on salmon survival or fitness is entirely unknown.
Salmon lice - Crustacea (Lepeophtheirus salmonis and Caligus elongatus)
Salmon may also be infected with sea lice, particularly Lepeophtheirus salmonis, which causes lesions at the site of attachment, making the fish more susceptible to other infections. Lepeophtheirus salmonis lives part of its life cycle as an obligate parasite but its free swimming stage also allows for direct transmission from fish to fish.
Sea lice (Lepeophtheirus salmonis and Caligus elongatus) represent one of the greatest threats to the salmon marine ranching industry, with the possibility that they may become major pathogens of wild salmonids also. Although known to be occasionally pathogenic, sea lice have generally been regarded as relatively harmless in wild populations. However, their importance within sea pens has been increasing since the mid-1960s, while reports of disease outbreaks in wild salmonids have also increased. Sea lice are epithelial browsers and do little damage to this constantly renewed tissue in light infections. Within sea pens however, heavy infections build up, with significant erosion of the epidermis, especially around the head. Death then follows, from osmotic shock or secondary infections. In heavy infections of wild fishes, death results from the same epidermal erosion, white head of infected fishes, as skin sloughs off leaving unpigmented tissue. In wild fishes the disease is associated with delays within river mouths, waiting for water levels to rise, and there seems little doubt that sea louse transmission takes place most efficiently in warm shallow coastal waters, although these parasites also survive in the open sea. It is particularly unfortunate, therefore, that the estuarine environment is also that used for salmon ranching, with release of enormous numbers of sea louse larvae into the environment. It remains controversial whether sea lice from farmed salmon have a significant effect on wild salmonid populations.
These comprise the Digenea and Cestoda (both Platyhelminthes), the Nematoda, and the Acanthocephala. All have complex life cycles involving at least one, and usually several, intermediate hosts which are links in the food web of the salmon. This complexity, and the need to coordinate with at least two hosts, means low transmission rates and epidemics rarely occur. Because of the short summers at high latitudes, many helminths become entrained to seasonal patterns of infection. Although the individual host may carry an impressive burden of large helminths, the impact on the host population remains small. Some helminths do raise concerns for wild salmon populations, the tapeworm Eubothrium crassum has sublethal effects on the salmon in sea pens and is moderately common in wild salmonids and the nematode may also be pathogenic in freshwater and may infect fry in large numbers. These helminth groups are the most species-rich yet to be considered.
Atlantic salmon is also host to species living extraintestinally including exotic marine species such as Hepatoxylon squali. The final host of this tapeworm is the shark, the salmon being an intermediate host which must be eaten for completion of the life cycle. Among the nematodes, the air bladder nematode Cystidicola farionis may be pathogenic in freshwater and Anisakis may have public health significance, as infections of humans can result from consumption of under cooked salmon. As this pathogen is associated with pelagic existence, this is peculiarly a risk associated with wild salmon caught during or after their sojourn at sea. Over 40 species of digenean infect salmon and one is suggested to cause an effect upon the hosts´ migratory behavior. The adult digenean Phyllodistomum umblae in the ureters of Arctic char and other salmonids may suppress anadromous migration, although this hypothesis has been criticized. One important digenean group in freshwater, are the eye flukes in the genera Diplostomum and Tylodelphus. The metacercariae stages of these flukes may blind the infected fish, making it vulnerable to predation, and are important in farms.
Fungi (Saprolegnia spp., Phialophora spp., Exophiala spp., Ichthyophonus hoferi)
A range of fungi have been collected from Atlantic salmon but their impact in wild populations is poorly understood and probably small. The best known is the oomycete Saprolegnia spp, frequently recorded from adults and kelts, in association with other pathogens, although it can initiate primary infections. This fungus is also well known for its effects on eggs and immature salmonids in culture. It may preferentially infect stressed and hence immunosuppressed salmonids. As an egg pathogen, Saprolegnia is also generally a secondary opportunist infecting moribund eggs and record infections in the presence of preexisting Aeromonas salmonicida.
Several fungi of the genera Phialophora and Exophialia also infect salmon; however, only Ichthyophonus hoferi is a significant pathogen of farmed salmon and has a major impact on marine fish populations (principally herring) in the northwest Atlantic. Nevertheless, there is no evidence that this fungus has a significant impact on marine salmon stocks.
Test performed in IVAMI (alphabetic order):
- Bacterial Kidney Disease (Renibacterium salmoninarum) - Molecular diagnosis (PCR).
- Columnaris (Flavobacterium columnare) - Molecular diagnosis (PCR).
- Cryptobiosis (Cryptobia [Trypanoplasma] salmositica) - Molecular diagnosis (PCR).
- Epitheliocystis and Proliferative Gill Inflammation (PGI) (Piscichlamidia salmonis; Clavochlamydia salmonicola) - Molecular diagnosis (PCR).
- Francisellosis (Francisella noatunensis subsp. noatunensis y F. noatunensis subsp. orientalis) – Molecular diagnosis (PCR).
- Fungi-Saprolegniosis (Saprolegnia parasítica; Saprolegnia diclina, others - Culture; Molecular identification (PCR and sequencing).
- Fungi Phialophora spp.; Exophiala spp.;) – Culture; Molecular identification (PCR and sequencing).
- Furunculosis (tail-rot) (Aeromonas salmonicida) - Molecular diagnosis (PCR).
- Gill Amoebic Disease (Neoparamoeba pemaquidensis; Neoparamoeba bronchiphila; Neoparamoeba perurans –NCGD-) - Molecular diagnosis (PCR).
- Gill Bacterial Diseases - Molecular diagnosis (PCR).
- Gill Microsporidial Disease (Loma salmonae) – Molecular diagnosis (PCR).
- Gill Parasitic disease - Dermocystidiosis (Spironucleus salmonicida) – Molecular diagnosis (PCR).
- Gill Viral Diseases (Atlantic salmon Paramyxoviridae –ASPV- and salmonid gill Poxvirus –SGPV-) - Molecular diagnosis (RT-PCR and PCR, respectively).
- Heart and Skeletal Muscle Inflammation (HSMI) virus (Piscine Orthoreovirus –PRV) Molecular diagnosis (RT-PCR).
- Helminths – Microscopic exam; Molecular identification (PCR and sequencing).
- Ichthyophonosis (Ichthyophorus hoferi) – Molecular diagnosis (PCR).
- Infectious Hematopoietic Necrosis virus (IHNV) (Rhabdoviridae, Novirhabdovirus) – Molecular diagnosis (RT-PCR).
- Infectious Pancreatic Necrosis (IPN) virus (Birnaviridae, Aquabirnavirus) – Molecular diagnosis (RT-PCR).
- Infectious Salmon Anemia (ISA) (Orthomyxoviridae) - Molecular diagnosis (RT-PCR).
- Pancreatic Disease (PD) (Alphavirus subtypes) – Molecular diagnosis (RT-PCR)
- Pancreatic Disease (Alphavirus subtypes) – Molecular subtyping SAV 1 to 6 (RT-PCR and sequencing)
- Parasites Gill Disease (Trichinodinosis) (Trichodina spp.; Ichthyobodo necator, Ichthyobodo salmonis) - Molecular diagnosis (PCR).
- Proliferative Kidney Disease (Myxozoa: Sphaerospora oncorhynchi) - Molecular diagnosis (PCR).
- Sea lice/Salmon lice – Crustacea, Copepoda- (Lepeophtheirus salmonis y Caligus elongatus) – Macroscopic and microscopic exam; Molecular identification (PCR and sequencing).
- Tenacibaculosis (syn. Flexibacteriosis) (Tenacibaculum maritimum) - Molecular diagnosis (PCR).
- Vibriosis (Vibrio anguillarum and Vibrio salmonicida) - Molecular diagnosis (PCR).
- Viral hemorrhagic septicaemia virus (VHSV) (Rhabdoviridae, Novirhabdovirus) – Molecular diagnosis (RT-PCR).
Conservation and shipment of samples:
- Up to 48 hours: refrigerated.
- More than 48 hours: frozen.
Delivery of results:
- Molecular diagnosis 24 to 48 hours during working days after reception of samples.
- Molecular typing or species identification (PCR or RT-PCR and sequencing) (72 to 96 hours, working days).
Cost of tests:
- Molecular diagnosis for bacteria, viruses, fungi, parasites (protozoa, helminths, arthropods (lice, etc.) DNA (PCR): Consult to email@example.com.
- Molecular diagnosis for RNA virus (RT-PCR): Consult to firstname.lastname@example.org.
- Molecular identification for species identification when more than one species is included in the genus, or subtyping, if requested (PCR or RT-PCR followed by sequencing): Consult to email@example.com.