Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes

Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.     

Immunohistochemical detection of piscine reovirus (PRV) in hearts of Atlantic salmon coincide with the course of heart and skeletal muscle inflammation (HSMI)

ABSTRACT: Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins μ1C and σ1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes.     

Effect of Pollution on Fish Diseases: Potential Impacts on Salmonid Populations

Abstract

Anthropogenic factors have contributed to the precipitous decline of wild Pacific salmon stocks, although the mechanisms and processes at work are largely unknown. Pollution may be one of these factors. Sediments in estuaries are known to act as repositories for contaminants, and estuaries are important habitats for ocean- and river-migrating salmon. We have shown that juvenile salmon Oncorhynchus spp. and their prey bioaccumulate chlorinated hydrocarbons and aromatic hydrocarbons—important classes of toxic xenobiotics. Furthermore, we have shown that exposure to these pollutants can lead to immunosuppression and increased disease susceptibility in juvenile salmon. Whether pollution influences natural disease outbreaks in host populations, including salmon, is currently unknown. It is postulated that the occurrence of disease depends on the interaction of the host, the environment, and the pathogen. Absence of pathogens would reduce the potential for adverse environments to influence disease outbreaks. However, a recent reconnaissance survey of juvenile chinook salmon Oncorhynchus tshawytscha from Oregon coastal rivers revealed that pathogens were an integral component in all systems studied, although the prevalence of the pathogens varied. Furthermore, recent studies of natural fish populations have demonstrated that infectious-disease-induced mortality can significantly reduce the size of the host population. By creating adverse environments (e.g., polluted estuaries) which alter the susceptibility of the host to pathogens that are integral and ubiquitous components of the habitat, pollution increases the probability of disease-related impacts on fish populations.     

Communications: The Occurrence of Myxobolus neurobius (Myxosporea) in Wild Young Atlantic Salmon and Arctic Char in Newfoundland

Abstract

Spores identified as Myxobolus neurobius were found in head homogenates of both wild young Atlantic salmon Salmo salar and Arctic char Salvelinus alpinus. Formalin-preserved spores from Atlantic salmon measured an average of 12.9 × 9.4 μm and contained two polar capsules that measured 6.4 × 2.9 μm. The spores from the Arctic char appeared morphologically identical by bright-field microscopy to those from the Atlantic salmon except they measured 13.7 × 9.6 μm and had polar capsules that measured 6.9 × 3.4 μm. Myxobolus neurobius has not previously been reported from Atlantic salmon or Arctic char.     

Molecular detection of Mycoplasma suis in captive white-lipped peccaries (Tayassu pecari) and wild boars (Sus scrofa) in Brazil

Mycoplasma suis, the etiological agent of swine hemoplasmosis, is an epicellular bacterium that adheres to the surface of pig erythrocytes leading to deformations of the target cells. Little is known about the occurrence of M. suis in wild swine populations around the world, its economic impact on swine herds, and the risk of human infection. The aim of this study was to investigate, by quantitative real-time PCR (qPCR) based on the 16S rRNA gene, the occurrence of M. suis in a captive population of white-lipped peccaries (100 Tayassu pecari) and in free-living wild boars (14 Sus scrofa) in Brazil. None of the white-lipped peccaries were positive for M. suis, whereas seven (50%) wild boars were positive in qPCR assays. The quantification of M. suis-16S rRNA copies/μL ranged from 1.42 × 10° to 3.906 × 10¹ in positive animals, indicating a low bacteremia and a chronic carrier status in free-living wild boars. In conclusion, M. suis might be a non-frequent pathogen in wild suids maintained in captivity. Despite the low bacteremia, the prevalence of M. suis in wild boar population in Brazil seems to be high.     

Transmission of Loma salmonae (Microsporea) to chinook salmon in sea water.

Transmission studies were conducted to determine if Loma salmonae was transmissible in sea water. Transmission of L. salmonae to chinook salmon (Oncorhynchus tshawytscha) held in sea water was achieved by exposing fish to macerated, infected gill tissue. Fish were exposed in seawater in a flow-through aquarium, and the infection was detected as soon as 5 wk after exposure. Heavily infected fish exhibited numerous xenomas in the branchial arteries, central venous sinusoids, and within the blood channels of the lamellae. The pathological changes were similar to those seen in pen-reared salmon with L. salmonae infections. The infection was not observed in Atlantic salmon (Salmo salar), Pacific herring (Clupea pallasi, family Clupeidae), or shiner perch (Cymatogaster aggregata, family Embiotocidae), experimentally exposed using identical methods. This study suggests that L. salmonae is transmissible to chinook salmon in seawater netpens. Fish farmers and fish health specialists should consider this possibility when developing and implementing strategies to control the infection.     

The efficacy of a single intraperitoneal injection of oxolinic acid in the treatment of bacterial infections in goldsinny wrasse (Ctenolabrus rupestris) and corkwing wrasse (Symphodus melops) studied under field and laboratory conditions

The efficacy of a single intraperitoneal injection of oxolinic acid to control an outbreak of atypical Aeromonas salmonicida infection in goldsinny wrasse (Ctenolabrus rupestris) and in the treatment of systemic vibriosis in corkwing wrasse (Symphodus melops) was examined. In addition a field study was performed to examine the effect of medication on the survival rate of goldsinny wrasse in Atlantic salmon cages. Four groups of wild caught goldsinny wrasse, each of 50 fish, were treated with an intraperitoneal injection of propylene glycol:saline (50:50) (control) or 50 mg/kg oxolinic acid at a concentration of 50 mg/mL. Three days after medication the fish in all groups were treated by an intraperitoneal injection of prednisolone acetate and an increase in seawater temperature from 9.0 to 11.5 degrees C. Cumulative mortalities were 18% in the two groups treated with oxolinic acid and 94 and 100% in the unmedicated control groups, giving a 'relative percentage survival' (RPS) value of 82%. A laboratory maintained population of originally wild caught corkwing wrasse experiencing high daily mortality was treated with oxolinic acid (50 mg/kg) or propylene glycol:saline (control). Cumulative mortalities were 84% (control) and 42% (oxolinic acid medicated group) giving an RPS value of 50%. In a field investigation using goldsinny wrasse approximately 30% were medicated with oxolinic acid (50 mg/kg) prior to stocking in cages with Atlantic salmon. In two of three cages the cumulative mortality was significantly lower (P = 0.025 and P < 0.001) in the medicated groups.     

Fish Processing Facilities: New Challenge to Marine Biosecurity in Canada

Abstract

The transmission of pathogens is a common consequence of animal food production. Marine salmon farms and their processing facilities can serve as sources of virulent fish pathogens; our study is the first to confirm the broadcast of a live fish pathogen from a farmed salmon processing facility into the marine waters of Canada's Pacific coast. We found live salmon lice Lepeophtheirus salmonis, mucus, and fish tissue in effluent from the processing facility. Sea lice transmitted from this source may pose a threat to wild salmon populations, and the release of untreated offal, including blood water, is of considerable concern. Further research is needed to quantify the extent to which processing facilities release sea lice and to determine whether more virulent fish pathogens are present in effluent. These data underscore the need for fish farming nations to develop mandatory biosecurity programs to ensure that farmed salmon processing facilities will prevent the broadcast of infectious fish pathogens into wild fish habitat.

Received April 26, 2012; accepted September 7, 2013     

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens – dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) – administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.     

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar)

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.     

Infectious Salmon Anemia Virus (ISAV) in Brown Trout

Abstract

Infectious salmon anemia (ISA) is a viral disease of Atlantic salmon Salmo salar that have been exposed to seawater in fish farms or hatcheries. This disease was previously believed to be exclusively one of salmon. However, it has been shown that anadromous brown trout Salmo trutta may carry the ISA virus (ISAV). Propagation of the ISAV in brown trout without the trout's showing any gross clinical signs of disease could be a result of a longstanding host-pathogen relationship between the virus and brown trout. A brown trout population isolated from the sea during the last 5,000 years and expected to be naive to the virus was challenged. These fish did not develop any gross signs of disease, but a few ISAVs were present as late as 46 d postchallenge. It was also shown that the ISA virus was present in brown trout as late as 7 months after challenge.     

Metabolism and residue depletion of albendazole and its metabolites in rainbow trout, tilapia and Atlantic salmon after oral administration

Metabolic and residue depletion profiles of albendazole (ABZ) and its major metabolites in three fish species, rainbow trout, tilapia and Atlantic salmon are reported. Based on these profiles, similarities (or dissimilarities) between species will determine the potential to group fish species. ABZ at 10 mg/kg body weight was incorporated into fish food formulated in a gelatin base or in gel capsule and fed as a single dose to six fish from each species. Rainbow trout were held three each in a partitioned 600-L tank. Tilapia and Atlantic salmon were housed in separate 20-L tanks. Samples of muscle with adhering skin were collected at 8, 12, 18, 24, 48, 72, and 96 h postdose from trout kept at 12 degrees C, at 4, 8, 12, 24, 48, 72, 96, 120, and 144 h postdose from tilapia kept at 25 degrees C and at 8, 14, 24, 48, 72, and 96 h postdose from Atlantic salmon kept at 15 degrees C. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final extracts were analyzed for parent drug ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO2) and albendazole aminosulfone using high-performance liquid chromatography with fluorescence detection. ABZ was depleted by 24 h in trout and tilapia and by 48 h in salmon; ABZ-SO, a pharmacologically active metabolite, was depleted by 48 h in tilapia, by 72 h in rainbow trout and was present until 96 h in salmon; and low levels of ABZ-SO2 and albendazole aminosulfone, both inactive metabolites, were detectable at least till 96 h in all three fish species.     

Survey of pathogens in hatchery Chinook salmon with different out-migration histories through the Snake and Columbia rivers

The operation of the Federal Columbia River Power System (FCRPS) has negatively affected threatened and endangered salmonid populations in the Pacific Northwest. Barging Snake River spring Chinook salmon Oncorhynchus tshawytscha through the FCRPS is one effort to mitigate the effect of the hydrosystem on juvenile salmon out-migration. However, little is known about the occurrence and transmission of infectious agents in barged juvenile salmon relative to juvenile salmon that remain in-river to navigate to the ocean. We conducted a survey of hatchery-reared spring Chinook salmon at various points along their out-migration path as they left their natal hatcheries and either migrated in-river or were barged through the FCRPS. Salmon kidneys were screened by polymerase chain reaction for nine pathogens and one family of water molds. Eight pathogens were detected; the most prevalent were Renibacterium salmoninarum and infectious hematopoietic necrosis virus. Species in the family Saprolegniaceae were also commonly detected. Pathogen prevalence was significantly greater in fish that were barged through the FCRPS than in fish left to out-migrate in-river. These results suggest that the transmission of infectious agents to susceptible juvenile salmon occurs during the barging process. Therefore, management activities that reduce pathogen exposure during barging may increase the survival of juvenile Chinook salmon after they are released.     

Evaluation of the dorsal aorta cannulation technique for pharmacokinetic studies in Atlantic salmon (Salmo salar) in sea water

The antimicrobial drug flumequine was given intravascularly and orally to cannulated and non-cannulated Atlantic salmon (Salmo salar) in sea water at 11°. The cannulated fish were divided into two groups, which were given flumequine (25 mg/kg) intravenously into the caudal vein (n = 8) and orally via a stomach tube down the oesophagus (n= 8). After a washout period of 2 days, the intravenously administered fish were given the drug orally, and the orally administered fish were given the drug intravenously. Blood samples were taken at different time points after drug administration through a cannula inserted into the dorsal aorta. The fish in the non-cannulated group were either given flumequine intravenously or orally, and blood samples were collected by killing five fish at predetermined time points after administration. The haematocrit values were measured in all the fish daily for 4 days after drug administration and thereafter, in all the collected blood samples throughout the whole experiment. The haematocrit values differed significantly between the cannulated and the non-cannulated fish. We found low haematocrit values and slow drug elimination in the cannulated groups, compared with higher haematocrit values and faster drug elimination in the non-cannulated groups, but further investigations are needed to prove any causal relations of this observation. The volume of distribution (V_d(ss)) was twice as large in the cannulated groups compared with the non-cannulated group, in the fish administered the drug intravenously. In the last part of the elimination phase, the half-lives differed considerably between the cannulated and the noncannulated groups both after oral and intravenous administration. The slower depletion of the drug concentration in the plasma of the cannulated fish is due to the large V_d(ss) as there are only small differences in clearance (Cl_T) between the groups. In this study the elimination of flumequine in cannulated Atlantic salmon differed from the elimination of flumequine in non-cannulated Atlantic salmon.     

Morphological, histochemical and morphometric study of the myotomal muscle tissue of the pacu (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei)

Histochemical, ultrastructural and morphometric methods were used to study growth patterns of red, pink and white muscle fibres and their relation to body weight and total length in the fast-growing freshwater fish Piaractus mesopotamicus Holmberg. The correlations amongst body weight, body length and diameter of red, pink and white fibres were low. From 10-15 to 40-50 cm, body weight increased 102.7 times, while the diameter of each type of fibre increased by factors of 0.94, 0.74 and 0.70, respectively. Muscle fibres revealed different morphological and histochemical stages of maturation. The frequencies of < 20 microns fibres of red, pink and white muscle tissue in the youngest and oldest classes were 64.5 and 11.0, 38.2 and 7.7 and 24.0 and 1.4%, respectively. In 30-40 cm fish, the frequency of < 20 microns fibres in the red and pink tissue was 24.5 and 25.5%, while in the white tissue it was 11.5%. During sexual maturity (40-50 cm), the recruitment of < 20 microns fibres in white muscle was 1.4%. Muscle fibres of this species showed continuous growth by both hyperplastic and hypertrophic mechanisms, and hyperplasia was particularly active in the juvenile phase.     

Report of Trichinella spiralis in a red fox (Vulpes vulpes) in Northern Ireland

No systematic studies of the occurrence of Trichinella in wildlife have been carried out in Northern Ireland (NI) in recent years, and the last reports of trichinellosis in livestock and human outbreaks in NI date back to 1979 and 1945, respectively. In this study, covering the period 2003/2004 and 2007/2008, a total of 443 red foxes (Vulpes vulpes) were collected throughout the country and screened for trichinellosis using a modified muscle digest method. One examined animal was found to be infected with larvae from Trichinella spiralis, indicating a national prevalence in NI of Trichinella in foxes of 0.2%. This prevalence compares well to the findings reported from the bordering Republic of Ireland [Rafter, P., Marucci, G., Brangan, P., Pozio, E., 2005. Rediscovery of Trichinella spiralis in red foxes (Vulpes vulpes) in Ireland after 30 years of oblivion. J. Infect. 50, 61–65] and could be a further indication for a sylvatic Trichinella life cycle existing independently from the domestic cycle.     

Survey of Pathogens in Hatchery Chinook Salmon with Different Out-Migration Histories through the Snake and Columbia Rivers

The operation of the Federal Columbia River Power System (FCRPS) has negatively affected threatened and endangered salmonid populations in the Pacific Northwest. Barging Snake River spring Chinook salmon Oncorhynchus tshawytscha through the FCRPS is one effort to mitigate the effect of the hydrosystem on juvenile salmon out-migration. However, little is known about the occurrence and transmission of infectious agents in barged juvenile salmon relative to juvenile salmon that remain in-river to navigate to the ocean. We conducted a survey of hatchery-reared spring Chinook salmon at various points along their out-migration path as they left their natal hatcheries and either migrated in-river or were barged through the FCRPS. Salmon kidneys were screened by polymerase chain reaction for nine pathogens and one family of water molds. Eight pathogens were detected; the most prevalent were Renibacterium salmoninarum and infectious hematopoietic necrosis virus. Species in the family Saprolegniaceae were also commonly detected. Pathogen prevalence was significantly greater in fish that were barged through the FCRPS than in fish left to out-migrate in-river. These results suggest that the transmission of infectious agents to susceptible juvenile salmon occurs during the barging process. Therefore, management activities that reduce pathogen exposure during barging may increase the survival of juvenile Chinook salmon after they are released.

Received May 27, 2010; accepted January 17, 2011     

Infectious Hematopoietic Necrosis Virus (IHNV) in Coho Salmon

Abstract

Infectious hematopoietic necrosis virus (IHNV) causes important losses of chinook salmon Oncorhynchus tshawytscha, sockeye salmon Oncorhynchus nerka, and rainbow trout and steelhead Oncorhynchus mykiss on the west coast of North America. Although coho salmon Oncorhynchus kisutch are considered resistant to IHNV infection, the virus was detected in numerous adult coho salmon returning to Trinity River Hatchery, California, in 1985 and 1986. The virus was isolated from internal organs and ovarian fluids of these fish. Antigenic and structural polypeptides of the viruses were identical in adult coho and chinook salmon collected at the same location. Chinook salmon and rainbow trout alevins exhibited high degrees of susceptibility to IHNV obtained from adult coho and chinook salmon. Coho salmon alevins were resistant to both virus isolants.     

Anisakid nematodes in beaked redfish (Sebastes mentella) from three fishing grounds in the North Atlantic,with special notes on distribution in the fish musculature

Parasitic anisakid nematodes commonly occur in the musculature and visceral organs of many fish species from the North Atlantic. In this respect, the presence of the third stage larvae of Anisakis spp. in the fish musculature may pose a potential consumer hazard due to the parasite's ability to cause anisakidosis. Thus, knowledge on the occurrence and distribution of these potentially zoonotic parasites in the commercially important North Atlantic fish species is crucial in order to evaluate and consequently prevent human infections.In the present study, 300 Sebastes mentella from three North Atlantic fishing grounds (Northern North Sea: Tampen; Barents Sea: off Bear Island; Irminger Sea: off SE Greenland) were examined for anisakid nematodes, with emphasis on occurrence and distribution in the musculature. Overall larval prevalence and mean intensity were significantly higher in redfish from Tampen (94%; 13.5 ± 20.0) and Bear Island (94%; 14.5 ± 19.4) than in fish from SE Greenland (75%; 6.0 ± 5.8; p < 0.01). The same trend was observed for larval infection in the musculature showing prevalence and mean intensities of 79%, 73%, and 55%, and 5.9 ± 6.6, 5.8 ± 6.5, and 3.2 ± 2.4, in the musculature of redfish from Tampen, Bear Island, and Greenland, respectively. Conventional microscopy and rDNA ITS-gene sequencing of various subsamples of muscle-dwelling nematode larvae of redfish from every catching locality revealed the presence of two anisakid species; Anisakis simplex sensu stricto and the non-zoonotic Hysterothylacium aduncum. Since the larvae of H. aduncum typically occur in or on the viscera of fish, our findings of two specimens in the belly flaps of redfish were unusual. Additionally, more than 92% of the muscle-dwelling larvae occurred in the belly flaps, i.e. the hypaxial part of the musculature surrounding the visceral organs on either fish side. Thus, trimming the fillets of beaked redfish by removal of most of the belly flaps would significantly reduce the probability of anisakid nematode larvae to be present in the final product.