Amoebic gill disease (AGD) is an emerging disease in North European Atlantic salmon (Salmo salar Linnaeus 1758) aquaculture caused by the amoeba Paramoeba perurans. The host immune response to AGD infection is still not well understood despite past attempts to investigate hostpathogen interactions. With the significant increase in our knowledge of cytokine genes potentially involved in Th responses in recent years, we examined their involvement in this disease using Atlantic salmon post-smolts sampled 3 weeks after exposure to either 500 or 5000 cells/l P. perurans. Gene expression analysis of cytokines potentially involved in the different Th pathways was performed on the first gill arch including the interbranchial lymphoid tissue (ILT). Th1, Th17 and Treg pathways were found to be significantly down regulated, mainly in samples from fish given the higher dose. In contrast, the Th2 pathway was found to be significantly up regulated by both infection doses. Correlation analysis of the gene expression data and the P. perurans load, assessed by real time RT-PCR of the 18S rRNA, was also performed. In humans, Th2 driven responses are characterized by the production of IgE, which in the majority of worm infections results in the generation of a Th2-mediated response and directs the immune system away from a Th1 inflammatory response. The present results seen during late stage AGD suggest that either an immune evasion strategy, similar to the responses driven by helminthic parasites to avoid cell-mediated killing mechanisms, or an allergic reaction caused by the parasite is occurring. This has been done.2. In Figure legends, "mean + SEM" should be "mean ± SEM".The graphs show only the positive standard error and not both positive and negative standard errors, and for this reason we believe the figure legends are correct.3. Figure 9, the alphabets and arrows should be more clarified (making bigger).They have been made bigger, in terms of the letters, arrows, scale bars and the image itself.Reviewer #2: Dear authors I find your paper important, thorough and very well written. The parasite/host interaction is of great importance and knowledge on pathways and immune evasion strategies are very valuable. You have presented your data in an orderly and statistically solid way and I only have minor comments:1. Please write all abbreviations in full the first time you mention them. Corrected.2. Please rewrite the sentence from line 253 -257 on page 9.The sentence has been changed.3. Remember that the latin name (P. perurans) should not be in italics when it is mentioned in a headline, which is in italics.This has been corrected in the headline 3.3. You should go through your MS and add commas to improve reading of the text.More been added to improve the reading of the text. 6. I think you should change the maximum on your Y-axis to a lower number on some of your figures (3, 4, 5, 7). That would increase the visual understanding of the data.The Y-axis have been lowered for figures 3, 4, 5, 6, 7, as suggested. Can yo...

Highlights d IgD + IgM À B cells constitute the main non-IgT B cell subset in rainbow trout guts d Gut IgD responses establish a two-way interaction with the local microbiota d Mucosal but not splenic IgD undergoes clonal expansion and diversification d Despite the lack of germinal centers, mucosal IgD is mildly mutated in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.

Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.