Ellen Lorenzen

Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens.

It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non-specific anti-viral defence mechanisms.

DNA Vaccination of Rainbow Trout against Viral Hemorrhagic Septicemia Virus: A Dose–Response and Time–Course Study

Abstract Viral hemorrhagic septicemia (VHS) in rainbow trout Oncorhynchus mykiss is caused by VHS virus (VHSV), which belongs to the rhabdovirus family. Among the different strategies for immunizing fish with a recombinant vaccine, genetic immunization has recently proven to be highly effective. To further investigate the potential for protecting fish against VHS by DNA vaccination, experiments were conducted to determine the amount of plasmid DNA needed for induction of protective immunity. The time to onset of immunity and the duration of protection following administration of a protective vaccine dose were also analyzed. The dose–response analysis revealed that significant protection of rainbow trout fingerlings was obtained following intramuscular injection of only 0.01 μg of plasmid DNA encoding the VHSV glycoprotein gene. In addition, higher doses of DNA induced immunity to a virus isolate serologically different from the isolate used for vaccine development. Following administration of 1 μg of a DN...

A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot.

A DNA vaccine encoding the envelope glycoprotein from a fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), has previously been shown to induce both early and long time protection against the virus in rainbow trout. Challenge experiments have revealed that the immunity established shortly after vaccination is cross-protective against heterologous fish rhabdoviruses. In this study, we show that the DNA vaccine encoding the VHSV glycoprotein also induces early protection against a non-enveloped, positive-sense RNA virus belonging to the Nodavirus family, the Atlantic halibut nodavirus (AHNV). In a vaccine efficacy test using juvenile turbot as model fish, the fish injected with the VHSV vaccine were completely protected against a nodavirus challenge performed 8 days post vaccination, while the cumulative mortality in the control group reached 54%. A DNA vaccine carrying the gene encoding the capsid protein of AHNV revealed no protective properties against the nodavirus challenge. Histological examination of muscle tissue sections from the vaccine injection site showed that the DNA vaccine against VHSV triggered a pronounced inflammatory response in turbot similar to what has earlier been observed in rainbow trout.

The protective mechanisms induced by a fish rhabdovirus DNA vaccine depend on temperature.

DNA vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) have proved highly efficient in rainbow trout (Oncorhynchus mykiss) under experimental conditions. Non-specific as well as specific immune mechanisms seem to be activated. Temperature is an important external parameter affecting the immune response in fish. The present study aimed at determining the effectiveness of a DNA vaccine against VHS at different temperatures. Rainbow trout fingerlings acclimated at 5 degrees C, 10 degrees C or 15 degrees C, were given an intramuscular injection of 1 microg purified plasmid DNA and challenged with virulent VHSV 8 or 36-40 days later. The vaccine protected the fish well at all three temperatures, but the involvement of innate and adaptive mechanisms differed: at low temperature, non-specific protection lasted longer and at 36 dpv fish kept at 5 degrees C had no detectable response of neutralizing antibodies while 67% of the fish kept at 15 degrees C had seroconverted. Induction of Mx as measured in liver samples was delayed at 5 degrees C with no detectable response 7 dpv whereas fish maintained at 10 degrees C had significantly elevated levels of Mx3-transcripts at that time point. Immunohistochemical studies of the injection site of vaccinated fish also showed a clear effect of temperature: in fish maintained at 15 degrees C the vhsG-protein appeared earlier on the surface of transfected myocytes and the inflammatory response clearing away these myocytes arose earlier compared to fish kept at the lower temperatures of 5 and 10 degrees C.

Dual DNA vaccination of rainbow trout (Oncorhynchus mykiss) against two different rhabdoviruses, VHSV and IHNV, induces specific divalent protection.

DNA vaccines encoding the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV are very efficient in eliciting protective immune responses against their respective diseases in rainbow trout (Oncorhynchus mykiss). The early anti-viral response (EAVR) provides protection by 4 days post vaccination and is non-specific and transient while the specific anti-viral response (SAVR) is long lasting and highly specific. Since both VHSV and IHNV are endemic in rainbow trout in several geographical regions of Europe and Atlantic salmon (Salmo salar) on the Pacific coast of North America, co-vaccination against the two diseases would be a preferable option. In the present study we demonstrated that a single injection of mixed DNA vaccines induced long-lasting protection against both individual and a simultaneous virus challenge 80 days post vaccination. Transfected muscle cells at the injection site expressed both G proteins. This study confirms the applied potential of using a combined DNA vaccination for protection of fish against two different rhabdoviral diseases.

Immunoprophylaxis in fish by injection of mouse antibody genes

Antibodies are a crucial part of the bodys specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals. The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA. Circulating recombinant antibodies could later be detected in the fish, and protective immunity to the viral disease was established.

Immersion exposure of rainbow trout (Oncorhynchus mykiss) fry to wildtype Flavobacterium psychrophilum induces no mortality, but protects against later intraperitoneal challenge

Flavobacterium psychrophilum, the causative agent of RTFS or rainbow trout fry syndrome, causes high mortality among hatchery reared rainbow trout (Oncorhynchus mykiss) fry in Europe and the USA. Despite several attempts, no efficient vaccines have yet been developed, the main obstacle being that the fry have to be vaccinated very early, i.e. around 0.2-0.5 g, where RTFS usually starts to give problems in the fish farms. Consequently, only oral or bath vaccines are relevant. Immersion of fry in inactivated or attenuated bacteria has resulted in RPS values of less than 50%. However, the results are biased by the fact that the fish have been challenged by intraperitoneal (ip) or subcutaneous (sc) injection against which an immersion/oral vaccine may not protect. Therefore, the present study was undertaken in order to investigate whether the presumably most potent immersion immunization, i.e. bathing in high titres of non-attenuated isolates of F. psychrophilum, was able to induce immunity to a subsequent ip challenge. Immersion in live bacteria for 30 or 50 min caused no mortality and protected a major fraction of the fry against challenges 26 and 47 days later with RPS values of 88.2 and 60.3%, respectively. Increased specific antibody titres suggested that adaptive immune mechanisms were involved in the protection.

General and family-specific gene expression responses to viral hemorrhagic septicaemia virus infection in rainbow trout (Oncorhynchus mykiss)

The ability of rainbow trout (Oncorhynchus mykiss) to respond successfully to infection by viral hemorrhagic septicaemia virus (VHSV) is expected to involve a large number of biochemical processes. We hypothesized that this would be reflected at the gene expression level in infected fish, and we tested it by examining gene expression levels in the head kidney of trout at a genome-wide scale with a 16K cDNA microarray for salmonids. Expression levels were recorded during 16 days following bath challenge. The challenge experiment included a relatively low susceptibility (32% survival following challenge) and a relatively high susceptibility (18% survival following challenge) trout family that were both split into a group exposed to virus and a non-exposed control group. In total, 939 genes were differentially expressed between infected and non-infected fish (FDR p=0.05). Five groups of Gene Ontology categories were involved in immune-related processes and over-represented in infected fish: (i) stress and defense response, (ii) NFkappaB signal transduction, (iii) response to non-self, (iv) antigen processing and presentation, and (v) proteasome complexes. The first four categories were also over-represented among the 642 differentially expressed genes in the low-susceptibility trout family but not among the 556 differentially expressed genes in the high-susceptibility trout family. Expression profiles for most immune genes discussed showed increased transcription from day 3 post-challenge. The results suggest that the innate immune system may play an important role in the successful response to VHSV in rainbow trout. In addition, the results indicate that a superior regulation of the transcription of several key innate immune-related genes contribute to the increased survival in resistant fish.

Ichthyotoxicity of the microalga Pseudochattonella farcimen under laboratory and field conditions in Danish waters.

Blooms of the marine dictyochophyte Pseudochattonella farcimen have been associated with fish kills, but attempts to verify ichthyotoxicity of this microalga under experimental conditions have not been successful. In the early spring of 2009 and 2011, P. farcimen bloomed in the inner Danish waters. The blooms occurred at a seawater temperature of ~2°C and correlated with extensive kills of farmed salmonid fish (2009) and wild populations (2011). Several strains of P. farcimen were isolated from the 2009 bloom. However, exposure of rainbow trout Oncorhynchus mykiss to laboratory-grown P. farcimen cultures did not reveal any toxic effects. During the 2011 bloom, fish were exposed to bloom water under both laboratory and field conditions. While no clinical effect was observed on fish incubated in bloom water in the laboratory trial, a remarkable difference was seen in the field trial between rainbow trout kept in tanks supplied with a continuous flow of filtered versus non-filtered bloom water. Histological examination of the gill tissue revealed karyorrhexis and epithelial loosening in the affected fish. Microscopy analysis of algal cell morphology suggested that mucocysts detected on the cell surface only in freshly sampled bloom water might be associated with ichtyotoxicity.

Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.