Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon

Heart‐ and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large‐scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV in vivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6 weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.     

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase^®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.     

Use of alternative disinfectants, individually and in combination, in aquacultural wastewater treatment

The inactivation effect of chlorine, iodine, ozone and UV irradiation was compared between phosphate buffered saline and waste water collected from a fish farm, with the fish-pathogenic bacterium Aeromonas salmonicida subsp. salmonicida as the model organism. In addition, the effect of combining chlorine, iodine or ozone with UV was evaluated. A tenfold increase in initial chlorine concentration, from 0.2 to 2.0 mg l^?1. had to be applied to maintain the same level of inactivation in waste water as in PBS. Iodine was less efficient than chlorine in PBS. by demanding a concentration of 1.0 mg l^?1 to obtain the same rate of inactivation as with 0.2 mg l^?1 chlorine. However, no difference between the two halogens was evident in the low-quality water. An initial ozone concentration of 0.1 mg l^?1 in PBS caused a rapid drop in bacterial viability for the first 20 s after which the curve levelled off. By continuous ozonation of waste water, a residual oxidant concentration of about 0.3 mg l^?1 had to be established before a rapid inactivation was observed. The UV inactivation profiles in PBS and waste water were almost identical, with a 99.9% reduction in viability after 48 and 50 s. respectively. When UV irradiation was combined with chlorine, a less than additive effect, as compared with the sum of individual death rates by the two treatments, was observed. The corresponding UV/iodine combination at least gave an additive effect in both water qualities. No increment in inactivation rate in PBS was observed when ozone was used in combination with UV, as compared with ozone alone.     

Infectious pancreatic necrosis virus in fish by‐products is inactivated with inorganic acid (pH 1) and base (pH 12)

The aquaculture industry needs a simple, inexpensive and safe method for the treatment of fish waste without heat. Microbial inactivation by inorganic acid (HCl) or base (KOH) was determined using infectious pancreatic necrosis virus (IPNV) as a model organism for fish pathogens. Salmonella and spores of Clostridium perfringens were general hygiene indicators in supplementary examinations. IPNV, which is considered to be among the most chemical‐ and heat‐resistant fish pathogens, was reduced by more than 3 log in 4 h at pH 1.0 and pH 12.0. Salmonella was rapidly inactivated by the same treatment, whereas spores of C. perfringens were hardly affected. The results indicate that low and high pH treatment could be particularly suitable for fish waste destined for biogas production. pH treatment at aquaculture production sites could reduce the spread of fish pathogens during storage and transportation without disturbing the anaerobic digestion process. The treatment could also be an alternative to the current energy‐intensive steam pressure sterilization of fish waste to be used by the bioenergy, fertilizer and soil improver industries.     

Effect of chemical and physical treatments on the inactivation of Macrobrachium rosenbergii nodavirus and extra small virus

White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.     

Studies on the effect of temperature and pH on the inactivation of fish viral and bacterial pathogens

Disposal of fish by‐products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by‐products according to risk, and specifies methods of disposal of by‐products according to that risk. There is provision under the regulation for composting or ensiling to be used for by‐products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24–48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by‐products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on‐farm processing of mortalities.     

Studies on the inactivation of selected viral and bacterial fish pathogens at high pH for waste disposal purposes

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24‐h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g^?1) that was reduced to approximately 2.2 × 10³ TCID₅₀ g^?1 after 24‐h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by‐products containing fish pathogens.     

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV‐1) in wild salmonids in Northern Europe and North Atlantic Ocean

Piscine orthoreovirus genotype 1 (PRV‐1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV‐1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV‐1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV‐1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV‐1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV‐1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV‐1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free‐living fish and provides rationale for screening wild broodfish used in restocking programmes.     

Piscine reovirus (PRV) in wild Atlantic salmon,Salmo salar L., and sea‐trout,Salmo trutta L., in Norway

This is the first comprehensive study on the occurrence and distribution of piscine reovirus (PRV) in Atlantic salmon, Salmo salar L., caught in Norwegian rivers. PRV is a newly discovered reovirus associated with heart and skeletal muscle inflammation (HSMI), a serious and commercially important disease affecting farmed Atlantic salmon in Norway. A cross‐sectional survey based on real‐time RT‐PCR screening of head kidney samples from wild, cultivated and escaped farmed Atlantic salmon caught from 2007 to 2009 in Norwegian rivers has been conducted. In addition, anadromous trout (sea‐trout), Salmo trutta L., caught from 2007 to 2010, and anadromous Arctic char, Salvelinus alpinus (L.), caught from 2007 to 2009, were tested. PRV was detected in Atlantic salmon from all counties included in the study and in 31 of 36 examined rivers. PRV was also detected in sea‐trout but not in anadromous Arctic char. In this study, the mean proportion of PRV positives was 13.4% in wild Atlantic salmon, 24.0% in salmon released for stock enhancement purposes and 55.2% in escaped farmed salmon. Histopathological examination of hearts from 21 PRV‐positive wild and one cultivated salmon (Ct values ranging from 17.0 to 39.8) revealed no HSMI‐related lesions. Thus, it seems that PRV is widespread in Atlantic salmon returning to Norwegian rivers, and that the virus can be present in high titres without causing lesions traditionally associated with HSMI.     

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.‐approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA‐positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.     

Mortality outbreak by perch rhabdovirus in European perch (Perca fluviatilis) farmed in Italy: Clinical presentation and phylogenetic analysis

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004–2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine‐based solution helps prevent apparent vertical transmission of PRV.     

Melanized focal changes in skeletal muscle in farmed Atlantic salmon after natural infection with Piscine orthoreovirus (PRV)

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV‐negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV‐negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes.     

Development of an Indirect ELISA to Detect Humoral Response to Flavobacterium columnare Infection of Channel Catfish,Ictalurus punctatus

ABSTRACT

The humoral antibody response of channel catfish, Ictalurus punctatus, to experimental Flavobacterium columnare infection was measured in control (non-infected) and infected (intraperitoneal- and intramuscular-injected) fish by an indirect enzyme-linked immunoassay (ELISA). The antibody response of the experimentally infected fish was significantly higher (P<0.0001) than that of the non-infected channel catfish by both indirect ELISA and agglutination. The indirect ELISA utilized goat anti-channel catfish IgM and a commercially available rabbit anti-goat IgG enzyme conjugate. The antibody response was directed against a cell-free sonicated antigen preparation derived from live whole F. columnare cells. Indirect ELISA and agglutination results correlated (r² = 0.60) for anti-F. columnare antibody response in experimentally and non-infected fish. We were also able to correlate ELISA and agglutination results of 60 fish naturally infected with F. columnare(r² = 0.76). Indirect ELISA will allow for rapid monitoring of humoral antibody, following natural exposure or vaccination against F. columnare, with a small sample of serum.     

Virucidal effects of common disinfectants against tilapia lake virus

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H₂O₂), 80 ppm formalin and 5,000 ppm (0.5%) Virkon^®. Factors that affect the disinfectants’ efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log₁₀ TCID₅₀ ml^?1 was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon^® at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log₁₀ inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H₂O₂ when tested at 10 min and 28°C; however, Virkon^® still achieved more than 5 log₁₀ inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented.     

Induction of meiotic gynogenesis in the stinging catfish Heteropneustes fossilis (Bloch) and evidence for female homogamety

This study reports the results on induced meiotic diploid gynogenesis and female homogametic nature in the Indian catfish, Heteropneustes fossilis. The eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm mL⁻¹ in Hanks balanced salt solution. Sperm were irradiated under UV light, with the exposure time ranging from 15 to 360 s (7500 ergs mm⁻² for 60 s). The genetic inactivation of paternal chromosomes was confirmed by chromosome counting from the larval cells and the larvae also had a characteristic haploid syndrome. A typical ‘Hertwig effect’ in the yield of hatched larvae was observed with doses of UV exposure >75 s (9375 ergs mm²). Larvae resulting from sperm UV irradiated above 120 s (15 000 ergs mm²) were 100% haploids. Application of heat shock to the activated eggs was effective in suppressing the release of the second polar body (meiotic gynogenesis) and resulted in diploid gynogenetic larvae morphologically identical to those of the control. The best yield of diploid gynogens (49.3% with respect to the control) was found to be at 6 min after egg activation and the heat shock at 41 °C for a 1-min duration, at an ambient water temperature of 27 °C. A total of 113 diploid gynogenetic fry from seven different female fish were reared and subjected to sexing. All gynogenetic fish were female in contrast to the control, which had a mean sex ratio of 56.7% females (which was not significantly different from 50% female). From these results, the sex determination mechanism in H. fossilis was presumed to be female homogamety.     

Piscine orthoreovirus: Biology and distribution in farmed and wild fish

Piscine orthoreovirus (PRV) is a common and widely distributed virus of salmonids. Since its discovery in 2010, the virus has been detected in wild and farmed stocks from North America, South America, Europe and East Asia in both fresh and salt water environments. Phylogenetic analysis suggests three distinct genogroups of PRV with generally discrete host tropisms and/or regional patterns. PRV-1 is found mainly in Atlantic (Salmo salar), Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon of Europe and the Americas; PRV-2 has only been detected in Coho Salmon of Japan; and PRV-3 has been reported primarily in Rainbow Trout (Oncorhynchus mykiss) in Europe. All three genotypes can establish high-load systemic infections by targeting red blood cells for principal replication. Each genotype has also demonstrated potential to cause circulatory disease. At the same time, high-load PRV infections occur in non-diseased salmon and trout, indicating a complexity for defining PRV's role in disease aetiology. Here, we summarize the current body of knowledge regarding PRV following 10 years of study.     

Induction of Diploid Androgenesis in the Stinging Catfish,Heteropneustes fossilis

This study reports the results on induced diploid androgenesis in the Indian catfish, Heteropneustes fossilis. Ultraviolet (UV) irradiation was used to inactivate maternal genome of H. fossilis. Complete inactivation of maternal genome was recorded at 12,500 ergs/mm². These genome‐inactivated eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm ml/L in Hank's Balanced Salt Solution. Egg viability was assessed for different exposure durations at fertilization, hatching, haploidy, and diploidization. Majority of the larvae derived from irradiated eggs had abnormal appearance. Complete inactivation of maternal genome was detected by haploid syndrome and confirmed by chromosome counting (n = 29). These eggs activated with sperm were subjected to heat shock at 40 and 41 C for different postactivation times and durations. Diploid androgens had a normal appearance as controls and confirmed by chromosome counting (n = 58). A maximum of 21 and 14% of diploidization was recorded at 30 min after activation, at 40 and 41 C, which corresponds to the first cleavage suppression time.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

Piscine orthoreovirus‐3 is prevalent in wild seatrout (Salmo trutta L.) in Norway

In 2017, a PCR‐based survey for Piscine orthoreovirus‐3 (PRV‐3) was conducted in wild anadromous and non‐anadromous salmonids in Norway. In seatrout (anadromous Salmo trutta L.), the virus was present in 16.6% of the fish and in 15 of 21 investigated rivers. Four of 221 (1.8%) Atlantic salmon (Salmo salar L.) from three of 15 rivers were also PCR‐positive, with Ct‐values indicating low amounts of viral RNA. All anadromous Arctic char (Salvelinus alpinus L.) were PCR‐negative. Neither non‐anadromous trout (brown trout) nor landlocked salmon were PRV‐3 positive. Altogether, these findings suggest that in Norway PRV‐3 is more prevalent in the marine environment. In contrast, PRV‐3 is present in areas with intensive inland farming in continental Europe. PRV‐3 genome sequences from Norwegian seatrout grouped together with sequences from rainbow trout (Oncorhynchus mykiss Walbaum) in Norway and Coho salmon (Oncorhynchus kisutch Walbaum) in Chile. At present, the origin of the virus remains unknown. Nevertheless, the study highlights the value of safeguarding native fish by upholding natural and artificial barriers that hinder introduction and spread, on a local or national scale, of alien fish species and their pathogens. Accordingly, further investigations of freshwater reservoirs and interactions with farmed salmonids are warranted.