Deteksjon av antistoffer i atlantisk laks med et kulebasert multipleks immunoassay

Viral diseases are among of the main challenges in aquaculture in Norway today. Heart- and skeletal muscle inflammation, caused by Piscine orthoreovirus (PRV), and pancreas disease (PD), caused by Salmonid alphavirus (SAV), both cause huge losses of farmed Atlantic salmon. Vaccination is a possible solution that is already contributing to the control of bacterial diseases. Unfortunately, making effective vaccines against viral diseases has been challenging and information about which immune mechanisms that are involved in protection is lacking. Antibodies can mediate complete protection against some diseases, and antibody levels after vaccination can correlate with protection, even if the protection is mediated by other mechanisms. In addition, antibody detection can be used in disease surveillance to determine if a fish population is or has been infected with a virus. Unfortunately, measuring antibody levels in salmon is not straightforward. Such measurements have not been widely used, and often show high levels of background binding. An important reason for this is that fish antibodies are of the IgM type. IgM is less specific than IgG, the dominating antibody in mammals. Therefore, other methods, like PCR and histology, are more used in diagnostics in Atlantic salmon. In this work, we have for the first time used an assay based on microscopic magnetic beads conjugated with antigen to measure antibody levels in Atlantic salmon. This method has a high sensitivity and can be used to measure antibodies against several proteins simultaneously in the same sample. By using this method, we have detected antibodies against PRV and SAV. In paper I and II, plasma from two PRV challenge trials was used to detect antibodies against the PRV proteins μ1c, μNS and σ1. We also detected antibodies against PRV1- σ1 in plasma from PRV-3-infected rainbow trout. There have been no previous publications detecting antibodies against PRV. The peak antibody level coincided with decreased pathology in the heart. In addition to showing virus-specific antibodies, our results show an increase in non-specific antibodies in PRV-infected salmon. This unspecific binding, but not the virus-specific binding, was decreased by heat treatment of samples. The nonspecific antibodies could be so-called polyreactive antibodies. Polyreactive antibodies could be crucial in protecting the salmon against infections before the adaptive immune system has had time to react, but their function, as well as the function of virus-specific antibodies during a PRV infection is unclear. In paper III, we detected antibodies against whole SAV particles disrupted with Triton-X. The antibody binding increased from between week three and week six after the introduction of SAVinjected shedder fish. The SAV particles also worked well with little background binding when analyzing plasma from a PD outbreak in the field. Antibodies were detected in most fish from four weeks after the start of the outbreak, and the antibody level stayed elevated until the last sampling point at 15 weeks. At this time point, most samples were negative for virus when analyzed with RTqPCR, showing that serology has a longer window of detection compared to detection of viral RNA by RT-qPCR. These results show that both virus-specific and non-specific antibodies can be produced after infection in Atlantic salmon. The method used is well suited for antibody detection in salmon but can be complicated by the presence of non-specific antibodies. It is therefore crucial to optimize the antigens for detection of specific antibodies.Virussykdommer er en av de store utfordringene i oppdrettsnæringa i Norge i dag. Hjerte- og skjelettmuskelbetennelse, forårsaket av Piscine orthoreovirus (PRV), og pankreas sykdom (PD), forårsaket av Salmonid alphavirus (SAV), fører begge til store tap av atlantisk laks i oppdrett. Vaksinering er en mulig løsning som allerede har bidratt til god kontroll på bakterielle infeksjoner hos laks. Dessverre er det utfordrende å lage vaksiner som virker godt mot virusinfeksjoner. Informasjon om hvilke immunfunksjoner som er involvert i beskyttelse er også mangelfull. Antistoffer kan gi full beskyttelse mot noen sykdommer, og antistoffnivået etter vaksinering kan korrelere med beskyttelsen, også om beskyttelsen skyldes andre immunmekanismer. I tillegg kan antistoffdeteksjon brukes i sykdomsovervåkning for å finne ut om fiskepopulasjoner er eller har vært infisert med et virus. Dessverre er det ikke rett fram å måle antistoffnivå hos laks. Antistoffmåling er lite brukt og det er ofte mye bakgrunnsbinding. En viktig grunn til dette er at fiskens antistoff er av typen IgM, som er mindre spesifikk enn IgG i pattedyr. Derfor er andre metoder, som PCR og histologi, mest brukt til påvisning av virussykdom hos laks. I dette arbeidet har vi for første gang brukt et assay basert på mikroskopiske magnetiske kuler konjugert med antigen til å måle antistoffnivå i laks. Denne metoden har høy sensitivitet og gir muligheten til å måle antistoffer mot mange proteiner i samme prøve samtidig

Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI

Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)

Abstract Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids.Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection AssaypublishedVersio

How to generate graded spinal cord injuries in swine – tools and procedures

ABSTRACT Spinal cord injury (SCI) is a medically, psychologically and socially disabling condition. A large body of our knowledge on the basic mechanisms of SCI has been gathered in rodents. For preclinical validation of promising therapies, the use of animal models that are closer to humans has several advantages. This has promoted the more-intensive development of large-animal models for SCI during the past decade. We recently developed a multimodal SCI apparatus for large animals that generated biomechanically reproducible impacts in vivo. It is composed of a spring-load impactor and support systems for the spinal cord and the vertebral column. We now present the functional outcome of farm pigs and minipigs injured with different lesion strengths. There was a correlation between the biomechanical characteristics of the impact, the functional outcome and the tissue damage observed several weeks after injury. We also provide a detailed description of the procedure to generate such a SCI in both farm pigs and minipigs, in the hope to ease the adoption of the swine model by other research groups

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay

International audienceSalmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture

Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI