A novel high-throughput qPCR chip for solving co-infections in RAS farmed rainbow trout.

Recirculating aquaculture systems (RAS) have become more attractive due to reduced water consumption and effluent discharge. However, intensification of production increases the risk of introducing pathogens at farming sites. The emergence of uncultivable pathogens and RAS pathobiome diversity shifts the traditional disease paradigm from "one pathogen, one disease" to complex multiple-pathogen disease cases. Piscine orthoreovirus genotype 3 (PRV-3) is an excellent example, as it is capable of inducing anemia and heart pathology resembling heart and skeletal muscle inflammation under experimental conditions, and is associated with increased mortality in association with other pathogens in the field. The aim of this study was to develop a method for detection of multiple pathogens and putative pathogens, as co-infections are common in aquaculture. To do this, in the pilot study, we mapped the pathobiome of RAS-farmed rainbow trout (Oncorhynchus mykiss) (commercial RAS, farm A) using both standard diagnostic methods and metabarcording (16S rRNA) to investigate the gill microbiome. During this study, we observed infections with multiple pathogens, and detected two putative gill pathogens Candidatus Branchiomonas cysticola and Candidatus Piscichlamydia salmonis, both of which have been linked with complex gill disease in Atlantic salmon (Salmo salar). Based on the pilot study, we developed and tested a high throughput qPCR (HT-qPCR) chip targeting 22 viral and bacterial pathogens and putative pathogens, followed by a surveillance of a fish cohort in a commercial RAS farm during production (farm B). Co-infection with PRV-3 and Ca. B. cysticola combined with stress inducing management practices may explain the severe disease outbreak observed (37% mortality). The time course study sets the base for a future screening scheme for disease prediction and addresses limitations of the method when testing environmental DNA/RNA.

Characterization of a Novel Infectious Pancreatic Necrosis Virus (IPNV) from Genogroup 6 Identified in Sea Trout (Salmo trutta) from Lake Vänern, Sweden.

In November 2016, infectious pancreatic necrosis virus (IPNV) was isolated from a broodstock female of landlocked sea trout (Salmo trutta) in Lake Vänern in Sweden. VP2 gene sequencing placed the IPNV isolate in genogroup 6, for which pathogenicity is largely unknown. Lake Vänern hosts landlocked sea trout and salmon populations that are endangered, and thus the introduction of new pathogens poses a major threat. In this study we characterized the novel isolate by conducting an infection trial on three salmonid species present in Lake Vänern, whole genome sequencing of the isolate, and prevalence studies in the wild sea trout and salmon in Lake Vänern. During the infection trial, the pathogenicity of the Swedish isolate was compared to that of a pathogenic genogroup 5 isolate. Dead or moribund fish were collected, pooled, and analyzed by cell culture to identify infected individuals. In the trial, the Swedish isolate was detected in fewer sample pools in all three species compared to the genogroup 5 isolate. In addition, the prevalence studies showed a low prevalence (0.2-0.5%) of the virus in the feral salmonids in Lake Vänern. Together the data suggest that the novel Swedish IPNV genogroup 6 isolate is only mildly pathogenic to salmonids.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

Skin immune response of rainbow trout (Oncorhynchus mykiss) experimentally exposed to the disease Red Mark Syndrome.

Red Mark Syndrome (RMS) is a skin disease reported from farmed rainbow trout. Since the turn of the millennium it has been spreading through Europe. RMS is probably a bacterial disease caused by a Midichloria-like organism (MLO). It is non-lethal and causes little obvious changes in appetite or behavior but results in red hyperaemic skin lesions, which may lead to economic losses due to downgrading. Here we transfer RMS to naïve specific pathogen free (SPF) fish by cohabitation with RMS-affected seeder fish. During disease development we characterize local cellular immune responses and regulations of immunologically relevant genes in skin of the cohabitants by immunohistochemistry and qPCR. Skin samples from SPF controls and cohabitants (areas with and without lesions) were taken at 18, 61, 82 and 97 days post-cohabitation. Gene expression results showed that lesions had a Th1-type profile, but with concurrent high expression levels of all three classes of immunoglobulins (IgD, IgM and IgT). The marked local infiltration of IgD + cells in the skin lesions as well as a highly up-regulated expression of the genes encoding sIgD and mIgD indicate that this immunoglobulin class plays an important role in skin immunity in general and in RMS pathology in particular. The co-occurrence of an apparent B cell dominated immune reaction with a Th1-type profile suggests that the local production of antibodies is independent of the classical Th2 pathway.

Zebrafish (Danio rerio) as a model to visualize infection dynamics of Vibrio anguillarum following intraperitoneal injection and bath exposure.

Vaccine development is important for sustainable fish farming and novel vaccines need to be efficacy tested before release to the market. Challenge of fish with the pathogen towards which the vaccine has been produced can be conducted either by external exposure though bathing or cohabitation, or by bypassing the mucosa through injection. The latter approach is often preferred since it is easier to control than the former. However, injection is not a very natural route of infection, and the bypass of the mucosa may result in a different efficacy profile of experimental fish compared to farmed fish, for which the vaccines are targeted. The zebrafish is by now a well established practical vertebrate model species due in part to its size and ease of maintenance and genetic manipulation. Here we use zebrafish as a model to visualize and compare the development of infection of Vibrio anguillarum on and in the fish following injection or bathing. Injection of 103 bacteria per fish resulted in approximately 50% mortality by day 4 post-injection. Similar mortality levels were reached in the other group by bathing in 1.25 × 109 bacteria for 1 min. The spreading of bacteria was followed for the first 24 h after injection/bathing by immunohistochemistry and optical projection tomography. The tissues and organs where bacteria were detected differed significantly as a result of time as well as treatment. In the bath group, bacteria were initially found on external surfaces including gut. After 24 h V. anguillarum still persisted in gut but had now also spread to the blood. In the injection group bacteria were found in the blood throughout all sampling times, as well as in the hypodermis and body cavity at most sampling times.