Paul J. Midtlyng

Non-native aquatic animals introductions have driven disease emergence in Europe

In this paper it is argued, using examples of disease emergence in aquatic animals in Europe, that the introduction of non-native species drives disease emergence by both extending the geographic range of parasites and pathogens and facilitating host-switching. Enteric red mouth disease and infectious haematopoietic necrosis of salmonids have extended their geographic range from North America to Europe with the import of live fish (Pimephales promelas) and rainbow trout eggs, respectively. Host-switching results in disease emergence when previously unidentified commensal organisms or known pathogen switch to new naïve hosts. The most serious endemic diseases of wild aquatic animals in Europe in recent years can be traced to the introduction of non-native species. Across Europe dramatic populations declines have occurred in native crayfish (e.g. Astacus astacus), oysters (Ostrea edulis) and eels (Anguilla anguilla), all which can be attributed, in varying degrees, to diseases (crayfish plague, Bonamia ostreae and Anguillicoloides crassus, respectively) introduced with non-native species. The severe adverse effects at a population level can be attributed to the lack of immunity in the new hosts. The impact of parasites more recently introduced to Europe, Sphaerothecum destruens (the rosette agent), and Batrachochytrium dendrobatidis, have yet to be fully determined. Both are generalists, with wide host ranges, and may present serious threats to native species. Aquaculture is the key driver for the introduction of non-native species. Most farming systems allow pathogen exchange between farmed and wild populations which underpins host-switching. Subsequently movements of animals between farms may result in the spread of newly emerged diseases. The introduction of non-native aquatic animals drives disease emergence, thus the ex-ante assessment of these hazards is severely limited. Generic risk mitigation measures (e.g. movement of disinfected eggs in place of live animals) and improved methods for rapid detection of new diseases are vital.

Stochastic modelling of direct costs of pancreas disease (PD) in Norwegian farmed Atlantic salmon (Salmo salar L.).

An economic model for estimating the direct costs of disease in industrial aquaculture was developed to include the following areas: biological losses, extraordinary costs, costs of treatment, costs of prevention and insurance pay-out. Direct costs of a pancreas disease (PD) outbreak in Norwegian farmed Atlantic salmon were estimated in the model, using probability distributions for the biological losses and expenditures associated with the disease. The biological effects of PD on mortality, growth, feed conversion and carcass quality and their correlations, together with costs of prevention were established using elicited data from an expert panel, and combined with basal losses in a control model. Extraordinary costs and costs associated with treatment were collected through a questionnaire sent to staff managing disease outbreaks. Norwegian national statistics for 2007 were used for prices and production costs in the model. Direct costs associated with a PD-outbreak in a site stocked with 500,000 smolts (vs. a similar site without the disease) were estimated to NOK (Norwegian kroner) 14.4 million (5% and 95% percentile: 10.5 and 17.8) (NOK=euro0.12 or

Detection of Infectious pancreatic necrosis virus in subclinically infected Atlantic salmon by virus isolation in cell culture or real-time reverse transcription polymerase chain reaction: influence of sample preservation and storage

0.17 for 2007). Production was reduced to 70% (5% and 95% percentile: 57% and 81%) saleable biomass, and at an increased production cost of NOK 6.0 per kg (5% and 95% percentile: 3.5 and 8.7).

Impact of antibiotic treatments on the expression of the R plasmid tra genes and on the host innate immune activity during pRAS1 bearing Aeromonas hydrophila infection in zebrafish (Danio rerio)

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at −80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at −80°C after homogenization in TM.

Development of an antibody ELISA for potency testing of furunculosis (Aeromonas salmonicida subsp salmonicida) vaccines in Atlantic salmon (Salmo salar L)

BackgroundThe transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model.ResultsThree days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1β and IL-8) and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish.ConclusionsTreatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR) A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may stimulate innate defenses against bacterial infections.

Three Rs Approaches in the Production and Quality Control of Fish Vaccines

The study was conducted in Atlantic salmon to establish the initial and basic scientific documentation for an alternative batch potency test for salmon furuculosis vaccines. We assessed the antibody response development for Aeromonas salmonicida vaccines at different immunisation temperatures (3, 12 and 18 °C), by an enzyme-linked-immunosorbent assay (ELISA) 3, 6, 9 and 12 weeks post vaccination, and the correlation between antibody response and protection in cohabitation challenge experiments performed 6 and 12 weeks post vaccination. Fish immunised with a vaccine containing full antigen dose had a significant increase in antibody response after 252 day degrees and the measured values correlated well with protection after 500 day degrees. Fish vaccinated with a reduced antigen dose showed a significant lower antibody response than fish vaccinated with the full dose vaccine at all samplings, and showed a similar low relative percent survival (RPS) in the challenges. The results from this study indicate that an antibody ELISA can discriminate between vaccines of different antigen content and the method may replace challenge tests in batch potency testing of furunculosis vaccines in Atlantic salmon. An immunisation temperature of 12 °C and sampling after 6-9 weeks, seemed to be the most appropriate time for using antibody responses to confirm batch potency.

Preliminary experiments on use of zebrafish as a laboratory model for Giardia duodenalis infection

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.

16. Vaccination against Furunculosis

Although Giardia duodenalis is considered a parasite of mammals, different genotypes have been identified as infecting several species of freshwater and marine fish in Australia. Establishment of G. duodenalis infection in common laboratory zebrafish (Danio rerio), could provide an excellent tool for a range of studies on Giardia. We conducted preliminary experiments to investigate this possibility. Zebrafish were inoculated with viable G. duodenalis cysts from two different Assemblages (A and D) using a modified oro-gastric tube. Direct microscopy and immunofluorescent antibody test were used to check for Giardia cysts/trophozoites in the intestine, and histology was performed on intestinal mucosa to evaluate possible pathological changes. Giardia cysts were successfully deposited in the zebrafish alimentary tract using a modified oro-gastric tube, and were maintained in the fish gut for at least 8 days. Although a single trophozoite was observed in one fish three days post-exposure, we were unable to demonstrate established, propagative infection under the conditions tested.

Methods for Measuring Efficacy, Safety and Potency of Fish Vaccines

Publisher Summary This chapter focuses on vaccination as a preventive measure against the disease. It discusses some of the milestones of furunculosis vaccine research. Dr D. Cecil B. Duff was the first author to report a clinical trial on furunculosis vaccination. Dr Winslow Whitney Smith reported the successful induction of antibacterial antibodies in carp, trout, and even turtles after injection of an A. salmonicida bacterin. The success of inactivated bacterins used in mammals inspired scientists to test this approach for the prevention of diseases in fish. The efficacy, long-term antibody response, and protection from parenteral vaccination were demonstrated. The strong adjuvant effect of mineral oil emulsions—with or without immunostimulating bacteria, the so-called Freunds complete (FCA) and Freunds incomplete (FIA) adjuvant systems—were discovered during the Second World War. This technique for enhancing the immune response was rapidly adopted into vaccine development. With the aid of these adjuvants, George E. Krantz, Jack M. Reddecliff, and Caroll E. Heist were the first scientists to demonstrate the efficacy of intraperitoneal (i.p.) vaccination in salmonids. Avirulent or low virulent strains, both live and inactivated, were later confirmed to induce both humoral immune response and protection in brook trout. W.D. Klontz investigated the role of soluble toxins as potential vaccine antigens. Rocco C. Cipriano investigated the immunogenic properties of virulent and avirulent strains of A. salmonicida. The Wildlife vaccines that were named “ASB” (A. salmonicida bacterin), “ASB-VAB-2” ( A. salmonicida- Vibrio anguiUarum bacterin), and “ASB-ERB” (A. salmonicida-enteric redmouth bacterin) are the first commercial vaccines used against furunculosis.

Protection, immune responses and side effects in Atlantic salmon (Salmo salarL.) vaccinated against furunculosis by different procedures

This chapter summarises the common methods for demonstrating safety, potency and efficacy of fish vaccines, allowing them to be licensed and marketed as veterinary medicinal products. It comprises a brief overview of clinical method development since the beginning of fish vaccinology and discusses the current guidelines, requirements and recommendations for choice of study methods. Historically, the same methods have been used for routine product quality testing (batch safety and potency) and for generating scientific documentation of the vaccines’ clinical safety and efficacy that forms the basis for granting marketing authorisations. With the development and publication of new experimental disease models and methods, evaluation of fish vaccines is in the process of becoming more sophisticated, and further efforts to increase the diversity of clinical methods are being advocated. The essential role of field trials for confirmation of experimental efficacy results and for the assessment of long-term side effects of fish vaccines is acknowledged. In addition, recent work to reduce and refine the use of live fish in batch quality testing of fish vaccines is reviewed, representing a clear welfare benefit for the fish being used during vaccine manufacture. For future improvement of fish vaccine safety and efficacy assessment, emphasis is placed on the development of alternatives to current challenge assays for batch potency and for a broader characterisation of immune mechanisms, including the use of quantitative (real-time) PCR assays to demonstrate up- or downregulation of relevant cytokines and immunorelevant genes.