No abstract

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444–2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ∼40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis typestrain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsialike organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.KEY WORDS: Piscirickettsia salmonis · European sea bass Dicentrarchus labrax · IFAT · Immunohistochemistry · rDNA · Phylogeny Resale or republication not permitted without written consent of the publisherDis Aquat Org 64: [107][108][109][110][111][112][113][114][115][116][117][118][119] 2005 was placed among the Gammaproteobacteria (Fryer et al. 1992). Subsequently, Mauel et al. (1999) used comparisons of 16S rRNA gene sequences and internal transcribed spaces (ITS) sequences to determine the relatedness of other P. salmonis isolates from Chile, Canada and Norway. They established that these strains formed a monophyletic group within the Gammaproteobacteria, although 1 Chilean isolate, EM-90, had diverged sufficiently to allow differentiation from the other isolates based on restriction fragment length polymorphism (RFLP) (Mauel et al. 1996). This group found only 1 ITS sequence in the isolates examined but, following polyacrylamide gel analysis of amplified ITS regions, Casanova et al. (2001) have suggested that P. salmonis may contain at least 2 rRNA (rrn) operons, as is commonly the case for other Gram-negative bacteria (Gürtler & Stanisich 1996, Crosby & Criddle 2003. More recently, have extended the information on regional variation of P. salmonis isolates through their comparison of 16S rDNA and ITS sequences from Scottish and Irish isolates.In this study, routine diagnostic histopathological examination was conducted on European sea bass presenting with clinical signs of nervous disease. In paraffin sections an RLO was seen to be associated with the encephalitic lesions. Serological analyses, indirect fluorescent (IFAT) and immunohistochemistry (IHC) were then used to confirm the tentative diagnosis and to confirm whether or not the organism was antigenically related to the Piscirickettsia salmonis type-strain, LF-89. The DNA sequences of the 16S rDNA and ITS region were then compared with those of published P. salmonis strains to establish whether or not the sea bass piscirickettsia-like organism (SBPLO) might be another strain of P. salmonis and how closely genetically related it was to the salmonid pathogens. Furthermore, it was sought to establish if the sea bass isolate possessed at least 2 ITS regions, 1 of which cont...

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...

No abstract

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

No abstract

No abstract