Chettri JK, Skov J, Jaafar RM. Krossøy B, Kania PW, Dalsgaard I & Buchmann K (2015). Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout. Fish & Shellfish Immunology 44(2): 485-495.
When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.
Bartkova S, Kokotovic B, Skall HF, Lorenzen N & Dalsgaard I (2016). Detection and quantiﬁcation of Aeromonas salmonicida in ﬁsh tissue by real-time PCR. Journal of Fish Diseases, doi:10.1111/jfd.12505 (Epub ahead of print).
Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicidasubsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real-time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real-time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real-time PCR compared to 30% by a culture approach). Also, no real-time PCR-negative samples were found positive by culturing. A. salmonicida was detected by real-time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real-time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg−1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real-time PCR assay provides a sensitive tool for the detection of A. salmonicida.
Bartkova S, Kokotovic B & Dalsgaard I (2016). Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging. Journal of Fish Diseases, doi:10.1111/jfd.12491 (Epub ahead of print).
Recent development of imaging tools has facilitated studies of pathogen infections in vivoin real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual-labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild-type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24-h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.
Marana MH, Skov J, Chettri JK, Krossøy B, Dalsgaard I, Kania PW & Buchmann K (2016). Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model. Journal of Fish Diseases, doi: 10.1111/jfd.12527 (Epub ahead of print).
Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine. Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.
Norske laksproducenter dominerer markedet for laksefisk, men også i Danmark produceres regnbueørreder, der leveres både som mindre portionsfisk fra dambrug og som store sølvskinnende "lakseørreder". Danske dyrlæger er med til at holde fiskene i god sundhedstilstand.
Bartkova S (2016). Aeromonas salmonicida. Epidemiology, whole genome sequencing, detection and in vivo imaging. Technical University of Denmark. Pages 142.
Møller OS, Buchmann K & Dalsgaard I (2013). A simple and low-toxic method of preparing small specimens of bacteria, flagellates and their likes for Scanning Electron Microscopy. Dafinet Workshop Abstract Book.
The preparation of samples of bacteria and other very small organisms (<50 µm) for Scanning Electron Microscopy is often complex and intricate, which typically involve the use of specialized filter systems, complex handling and toxic chemicals. Based on the methods described in the literature and our own tests, we have distilled a simpler (although slightly crude) method to prepare bacterial samples in a fast way. We only employ readily available chemicals requiring no more than a fume hood, and low-cost, standard lab equipment like single use filters. The method is excellent for achieving relatively quick results for illustration purposes and does not require handling of highly toxic substances like Osmium-tetraoxide, which typically necessitates skilled/trained lab personnel. Thus, this method is well-suited for testing different bacterial concentrations, biotypes, and other variables relatively quickly. So far, this method has yielded good results on several pathogenic bacteria and parasites; Aeromonas salmonicida, Yersinia ruckeri, Ichthyobodo necator and theronts of Ichthyophthirius multifiliis.
We have tested the efficacies of two different vaccines (a commercial versus an experimental vaccine – both being oil adjuvanted) for rainbow trout against furunculosis caused by Aeromonas salmonicida infections. However, when challenging fish with live bacteria in order to assess protection following vaccination, the administration of the pathogen is important for the outcome of the experiments. We have therefore also compared injection challenge with cohabitation challenge. In addition, when doing so we also investigated the influence of environmental conditions such as salinity and temperature on the protection recorded. Thus, challenge studies were conducted at two temperatures (12 and 19°C) and at two salinities (0 and 15 ppt). Mortalities following challenge were recorded and RPS calculated for each group. Side effects of the vaccines were evaluated by using the Speilberg scale. Results from the challenge studies will be presented.
Despite vaccination with oil-adjuvanted vaccines against vibriosis and furunculosis, sea reared rainbow trout in Denmark often encounter outbreaks of furunculosis caused by Aeromonas salmonicida during warm summer periods. This implies an excessive use of antibiotics and has also decreased the fish farmers confidence in the commercially available vaccines. To address this issue two successive collaborative research projects have been established. Initially (MarinVac project), it was anticipated, based on the experience from Norway where furunculosis vaccines efficiently eliminated the disease problem in Atlantic salmon, that the commercially available vaccines could provide sufficient protection when used optimally. Field trials were therefore conducted with the aim of determining the optimal vaccination strategy for the sea reared rainbow trout in DK, particular in terms of time of vaccination before transfer of the fish to seacages. The results revealed that there were equal effects of early and late vaccination (1 year vs 3 month before transfer to sea). And needs for antibiotic treatment against furunculosis occurred in both groups during warm Summer periods. An experimental vaccination trial was then conducted under controlled lab conditions. Two groups of rainbow trout were vaccinated by intraperitoneal (i.p.) injection with two different commercial vaccines, both comprising Vibrio anguillarum (Va) serotype O1 and O2, and Aeromonas salmonicida (As) based on cultures of bacteria originally isolated from Atlantic salmon. The experiment also included a third group of non-vaccinated controls. All fish were individually chip-tagged. Challenge was performed as a combination of injection- and cohabitation challenge. Six months after vaccination at 10⁰C, half of the fish were challenged by ip. injection of A. salmonicida bacteria. While the non-vaccinated fish all died within 3 weeks, a certain level of protection was evident among the two vaccinated groups although high mortality also occurred here. No mortality/clinical disease was evident among the noninjected cohabitants at this stage. However, when the water temperature was subsequently raised to 17⁰C, the cohabitants started to die. Some variability was evident between replicate tanks, but the overall outcome was that non vaccinated fish performed at least as well as the vaccinated ones. The results demonstrate the importance of the challenge procedure for evaluation of vaccine efficacy under experimental conditions. Although it may be anticipated that the available commercial vaccines can confer some protection against furunculosis in rainbow trout, the results also indicate that there is a need for tailoring the vaccines to the needs of sea reared rainbow trout in Denmark. A new research project (ProFish) has recently been launched in which we will use antigens based on As isolates derived from disease outbreaks in sea reared rainbow trout in DK. Also, alternative adjuvants to the traditional mineral oil will be tested, and prime-boost vaccination strategies will be developed. Both projects received funding from the Danish Council for Strategic Research.
Enteric red mouth (ERM) disease and furunculosis are important bacterial diseases in rainbow trout aquaculture. While ERM is caused by Yersinia ruckeri the causative agent of furunculosis is Aeromonas salmonicida. While a number of publications have clearly shown humoral immune responses to bacterial diseases in fish, little is known about the impact of cellular immunity. Cellmediated cytotoxicity is one of the options the immune system can mount against intracellular infections particularly in viral diseases. However, a facultative intracellular lifestyle has also been suggested for Y. ruckeri and A. salmonicida.
Here we have used two approaches to investigate the role of cellular immunity in ERM and furunculosis: (1) adoptive transfer of immune cells from vaccinated donors followed by challenge of the recipients and (2) in vitro cell-mediated cytotoxicity of immune cells from vaccinated donors against bacterial infected target cells, respectively. Further, bacterin uptake by the gills has been studied for A. salmonicida.
As for ERM we have isolated PBL from bath vaccinated clonal trout donors and transferred to naïve genetically identical clonal fish recipients followed by challenge of the latter with live Y. ruckeri bacteria. All fish that have received PBL from vaccinated donors survived the challenge infection while control fish injected with leukocytes from non-vaccinated trout exhibited high mortality. Flow cytometry analysis of recipient leukocytes suggest that CD8+ and IgT+ cells from donors have a preference to home to or to proliferate in the recipient gills. Transfer of memory B cells has been concluded from the fact that IgM titres were higher in Y. ruckeri infected recipients injected with immune cells from vaccinated donors when compared to control fish. While this is the first report on adoptive cellular transfer of immunity in a bacterial infection in fish, our data further suggest that immunity in ERM is rather conferred by IgM+ B cells.
Concerning furunculosis, a new cell line matched in its MHC class I to our clonal trout has been established and intracellularly infected along with an MHC class I mismatched cell line with A. salmonicida bacteria. Cell-mediated cytotoxicity of leukocytes isolated from vaccinated donors was rather moderate and NK-like.
In addition, we have performed A. salmonicida bacterin uptake studies in the gills. Our flow cytometry and histology data have shown that killed A. salmonicida bacteria are taken up by M-like cells based on their lectin staining pattern and that antigen uptake took place in the direct neighbourhood of the interbranchial lymphoid tissue (ILT).
Skall HF, Lorenzen E, Kjær TE, Henriksen NH, Dalsgaard I, Madsen SB, Buchmann K, Krossøy B & Lorenzen N (2015). Danish sea reared rainbow trout suffer from furunculosis despite vaccination - How can applied research help to solve the problem? Dafinet & ProFish Workshop Abstract Book, p. 9.
Despite vaccination by intraperitoneal injection with oil-adjuvanted vaccines against vibriosis and furunculosis, sea reared rainbow trout in Denmark often develop furunculosis and occasionally vibriosis during warm summer periods. This implies an excessive use of antibiotics and has also decreased some fish farmer’s confidence in the commercially available vaccine. This vaccine comprises Aeromonas salmonicida subspecies salmonicida and Vibrio anguillarum serotypes O1 and O2a bacterins emulsified with mineral oil. The bacterin antigens are based on bacteria isolated from Atlantic salmon cultured outside Denmark.
Vaccination and challenge trials performed under experimental conditions suggest that the commercial vaccine provides good protection against challenge with Aeromonas salmonicida when the fish are exposed to the bacteria by injection. However, the protection is far less significant when the fish are challenged by cohabitation with infected donor fish followed by elevation of the water temperature. The results demonstrate the importance of optimizing the challenge procedure for evaluation of vaccine efficacy under experimental conditions. In terms of Vibrio anguillarum the commercial vaccine failed to protect the fish against an serotype O2 isolate from diseased fish, suggesting that tailoring the antigen composition to Danish/local bacterial variants is needed. Serological examination of the vaccine induced antibody response in the fish is in progress aiming at evaluating the contribution of the humoral immune response to protection as well as at development of an in vitro tool for evaluation of vaccine potency.
Rainbow trout, Oncorhynchus mykiss were vaccinated by intraperitoneal (i.p.) injection with vaccines containing bacterin alone or bacterin supplemented with experimental vegetable oilbased adjuvant or a paraffin oil adjuvant. The bacterin consisted of formalin killed bacteria of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a. Fish were challenged with the same bacterial strain of A. salmonicida either by i.p. or by applying a novel challenge method. Skin lesions were made by puncturing the caudal fin epidermis followed by layering the bacterial culture of A. salmonicida on the puncture site for 60 seconds. This challenge method mimics that rainbow trout in fish farms might be infected with A. salmonicida through injured epidermis (fin biting) and our new method resembles closely the natural infection route where bacteria gain access to fish through the lesions. The protection, antibody production and side-effects were examined during the trial. The bacterin administered with adjuvant elicited protection and antibody production that was dependent on the antigen concentration in the vaccine. The new infection model proved to be efficient in inducing a more natural disease progression in fish and a stable mortality. The method can differentiate efficacies of different vaccines with regard to adjuvant formulations and content of antigen.
Aeromonas salmonicida susbp. salmonicida, the causative agent of the disease furunculosis, causes great problems in Danish sea reared rainbow trout (Oncorhynchus mykiss) production. Outbreaks occur repeatedly during elevated temperatures and fish harboring A. salmonicida can also be covertly infected. A crucial factor for preventing spread of furunculosis is therefore gaining more knowledge of the covert stage, which includes knowing the route of entry and dissemination of the pathogen in the fish. To determine this, one could trace the bacterium using in vivo bioluminescence imaging (BLI) that enables visualization and quantification of infections within the same animal over several time points. In this research BLI was used to follow the infection of a virulent Danish A. salmonicida isolate transformed with pAKgfplux1; a plasmid vector containing the gfpmut3a gene from Aequorea victoria and the luxCDABE operon from Photorhabdus luminescens. Since A. salmonicida pAKgfplux1 displayed similar growth and pathogenicity as the wild type A. salmonicida, the transformed bacterium could be used to illustrate a progression of an A. salmonicida infection. Fish were infected with A. salmonicida pAKgfplux1 through an immersion bath and followed over a 24 h period using an IVIS® Spectrum system. Results showed a pattern of favourable pathogen attachment sites, internal passageway and main exit site that will be shown and discussed in the presentation.
Villumsen KR, Christensen D, Koppang EO, Bojesen AM (2015) Can the choice of adjuvant direct T-cell responses, minimize adverse effects and still confer protection? Dafinet & ProFish Workshop Abstract Book, p. 15.
For the past two decades, mineral oils have been utilized as a potent adjuvant when formulating injection furunculosis vaccines for intraperitoneal administration in aquaculture of salmonid fishes. Over this period, several field, as well as laboratory studies have demonstrated that these adjuvants improve protection against furunculosis, caused by Aeromonas salmonicida subsp. salmonicida. However, studies have also demonstrated a range of severe adverse effects associated with the use of mineral oils in fish vaccine formulations, a consequence that warrants further exploration of alternatives to the current status quo. Working with injection vaccines against furunculosis in rainbow trout, this current project aims to shed light on two main questions: 1. Considering a vaccine formulated with mineral oil as the benchmark, would it be possible to obtain a level of protection equal to, or higher than, that benchmark, while at the same time reducing the adverse effects, through the use of adjuvants with a higher expected safety profile? 2. Could the use of specific adjuvants help to direct the T-cell response towards specific Th-profiles, potentially proving one to be superior with regards to protection? Three formulations of A. salmonicida bacterin with either CpG oligodeoxynucleotides, Freund’s incomplete adjuvant or cationic adjuvant formulation 01 (CAF01) were prepared to approach these questions. Along with relevant control groups, the vaccine groups are now subjected to an experimental protocol assessing the nature of the respective leukocyte responses, adverse effects, antibody responses, as well as protection from infection of each group. Hopefully, the results will provide valuable insight into the optimal induction of the immune response of rainbow trout, and help to identify safer alternatives to the current adjuvants, paving the way to the formulation of the next generation of furunculosis vaccines for commercially farmed rainbow trout.
Two candidate adjuvants were tested with a commercial ERM dip vaccine (AquaVac™ Relera, MSD Animal Health) for rainbow trout in an experimental design compatible with common vaccination practices at farm level, i.e. immersion of fish in vaccine (±adjuvant) for 30 s. The adjuvants were the commercial product Montanide™ IMS 1312 VG PR (SEPPIC), and a soluble and ≥98% pure β- glucan from yeast (Saccharomyces cerevisiae) (Sigma-Aldrich). Hence, five experimental groups in duplicate were established and exposed to vaccine and adjuvants in the following combinations: AquaVac™ Relera (alone); AquaVac™ Relera + Montanide™; AquaVac™ Relera + β-glucan; Montanide™ (alone); and β-glucan (alone). Approximately 450 degree days post-vaccination, the fish were bath-challenged with live Yersinia ruckeri to produce survival curves. Blood, skin and gills were sampled at selected time points during the course of the experiment to test for plasma Ab levels and lysozyme activity, and the regulation of immune relevant genes and cells in external, mucosal tissues. Preliminary results show 96% to 100% survival of vaccinated fish with and without any of the two adjuvants, whereas unvaccinated controls and fish exposed to β-glucan alone experienced 58% and 60% survival, respectively (calculated at day 24 post-challenge). Montanide™ alone gave rise to an intermediate level of 72% survival. Lysozyme activity levels in plasma were markedly elevated at day 3 and day 24 post-challenge in fish exposed to Montanide™ alone or β-glucan alone compared to fish from any of the three vaccinated groups.
Bartkova SS, Kokotovic B & Dalsgaard I (2015) Detection of Aeromonas salmonicida in fish tissue by real-time PCR. Abstract Book, P-135 (side 359).
Disease outbreaks of furunculosis in rainbow trout freshwater farms do not occur as often in Denmark today as has otherwise been the case ever since the 1950s when the causative agent of furunculosis, Aeromonas salmonicida subsp. salmonicida, was first discovered in Denmark. However, even though the trout are vaccinated before transfer out to the sea, outbreaks of furunculosis continue to occur at sea during elevated temperatures. It has thus been speculated that fish could be carrying the bacterium as a latent infection from the freshwater to the sea. Several PCR and qPCR assays for detection of A. salmonicida have been developed in the past. Nevertheless, most of these assays either lack high sensitivity in tissue or have only been tested on pure culture and/or few tissue types. Moreover, in cases where carrier fish were investigated, laborious and slow enrichment steps were needed in order to acquire a positive result. A sensitive, specific, rapid and cost-effective assay for detecting A. salmonicida in carrier fish is thus still needed. In this study, a highly sensitive and specific real-time PCR has been developed, based on previous research by Balcazar et al. (2007). The assay uses a self-quenched fluorogenic primer set designed from a DNA probe sequence for A. salmonicida, which is the most frequently used target for species-specific A. salmonicida molecular methods to date. Thus far the assay has been tested on five different rainbow trout tissue samples (gills, kidney, brain, intestine, spleen) spiked with various A. salmonicida dilutions, without showing signs of inhibition. Preliminary results show that the assay has a higher sensitivity compared to traditional bacterial methods and both methods are currently being compared in fish from natural occurring outbreaks and fish without clinical signs of infection. Key results from both methods will be presented and discussed.