Kasper Rømer Villumsen

Potential Role of Specific Antibodies as Important Vaccine Induced Protective Mechanism against Aeromonas salmonicida in Rainbow Trout

Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990s, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar). However, rainbow trout (Oncorhynchus mykiss) are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively). Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.

3D visualization of the initial Yersinia ruckeri infection route in rainbow trout (Oncorhynchus mykiss) by optical projection tomography.

Despite the fact that enteric redmouth disease (ERM) in farmed rainbow trout is one of the most devastating disease problems, little is known about the initial route of infection and pathogenicity of the aetiological agent, Yersinia ruckeri. In order to determine the initially infected organs, optical projection tomography (OPT), a novel three-dimensional (3D) bio-imaging technique, was applied. OPT not only enables the visualization of Y. ruckeri on mucosal surfaces but also the 3D spatial distribution in whole organs, without sectioning. Rainbow trout were infected by bath challenge exposure to 1×108 CFU/ml of Y. ruckeri O1 for 1 hour. Three fish were sampled for OPT and immunohistochemistry (IHC) 1, 10 and 30 minutes, 1, 3, 6, 12 and 24 hours, as well as 2, 3, 7 and 21 days after the start of the infection period. Y. ruckeri was re-isolated from the blood of infected fish as early as 1 minute post infection. Both OPT and IHC analysis confirmed that the secondary gill lamellae were the only tissues infected at this early time point, indicating that Y. ruckeri initially infects gill epithelial cells. The experimentally induced infection caused septicemia, and Y. ruckeri was found in all examined organs 7 days post infection including the brain, which correlated with the peak in mortality. To the best of our knowledge this is the first description of Y. ruckeri infection in the brain, which is likely to cause encephalitis. This in part could explain the lethality of ERM in rainbow trout. Using OPT scanning it was possible to visualize the initial route of entry, as well as secondary infection routes along with the proliferation and spread of Y. ruckeri, ultimately causing significant mortality in the exposed rainbow trout. These results demonstrate that OPT is a state-of-the-art technique capable of visualizing pathogenesis at high resolution.

Oral and anal vaccination confers full protection against enteric redmouth disease (ERM) in rainbow trout.

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3×108 CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

Long-lasting protection induced by bath vaccination against Aeromonas salmonicida subsp. salmonicida in rainbow trout

For decades Aeromonas salmonicida subsp. salmonicida (from here referred to as A. salmonicida) has been recognized as the causative agent of typical furunculosis. This disease has had a major impact on aquaculture worldwide, making it a target for international research, particularly within the field of immunoprohylaxis. Initial studies attempted vaccination via oral route and immersion. However, these vaccination methods proved insufficient when compared to intraperitoneally (i.p.) injected vaccines. The focus of vaccine research regarding A. salmonicida shifted towards the i.p.-injected vaccines during the 1980s and -90s, resulting in oil-adjuvanted vaccines providing high levels of protection over longer periods of time. The majority of this research has been conducted using salmon, while rainbow trout, which is also a commercially important species, has played a much less central role. In this study, we have examined the effect of a bath vaccination using an experimental A. salmonicida bacterin. Rainbow trout were vaccinated by a 5 min bath in a formalin-inactivated bacterin. Half of these fish was booster vaccinated using 50% of the initial vaccine dose 10 weeks post primary immunization. Along with an un-vaccinated control group, the fish were challenged by waterborne infection 24 weeks post primary immunization. Both vaccinated groups showed a significantly increased survival (>93% survival) compared to a 70% survival in the un-vaccinated control group (P = 0.005 and P = 0.019 for single and dual immunizations, respectively). When comparing the survival of the single and dual immunization groups, there was no significant difference (P = 0.531). ELISA showed no significant induction of specific circulating antibodies in either vaccinated group. These results are interesting with regard to the protective mechanisms, seen in the light of previous results obtained using bath as well as i.p. vaccination against furunculosis in salmonid fishes.

Adverse and long-term protective effects following oil-adjuvanted vaccination against Aeromonas salmonicida in rainbow trout

Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.

Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.

Alternatives to mineral oil adjuvants in vaccines against Aeromonas salmonicida subsp. Salmonicida in rainbow trout offer reductions in adverse effects

In an effort to reduce the frequency and severity of adverse reactions seen from the use of mineral oil adjuvants in salmonid fish, the effects of two alternative adjuvants were assessed, focusing on the induction of adverse effects as well as protection. Using rainbow trout (Oncorhynchus mykiss) as recipients, injection vaccines based on formalin-inactivated Aeromonas salmonicida subspecies salmonicida were formulated with CpG oligodeoxynucleotides, the liposomal cationic adjuvant formulation 01 (CAF01) or with Freund’s incomplete adjuvant and administered intraperitoneally. Control groups of unvaccinated, Tris-buffered saline-injected or bacterin-injected individuals were included, and each group included in the study held a total number of 240 individuals. Subsequently, individuals from each group were examined for differences in Fulton’s condition factor, macro- and microscopic pathological changes, as well as protection against experimental infection with A. salmonicida. While adverse effects were not eliminated, reductions in microscopic and macroscopic adverse effects, in particular, were seen for both the nucleotide- and liposome-based vaccine formulations. Furthermore, the induced protection appears similar to that of the benchmark formulation, thus introducing viable, potential alternative types of adjuvants for use in future fish vaccines.