Phylogenetic analyses of Candidatus Branchiomonas cysticola refine the taxonomic classification of Betaproteobacteria associated with epitheliocystis in fish

Candidatus Branchiomonas cysticola is recognized as the most prevalent bacterial agent causing epitheliocystis in Atlantic salmon (Salmo salar). Based on its partial 16S rRNA sequence, the bacterium has previously been found to be a member of Burkholderiales in the class Betaproteobacteria. Multilocus Sequence Analysis (MLSA) of the bacterium and 60 type strains of Betaproteobacteria using newly identified housekeeping genes (dnaK, rpoC, and fusA) and ribosomal subunit sequences (16S and 23S), instead supported the bacterium’s affiliation to Nitrosomodales. Taxonomic rank normalization by Relative Evolutionary Divergence (RED) showed the phylogenetic distinction between Cand. B. cysticola and its closest related type strain to be at the family level. A novel bacterial family named Branchiomonaceae has thus been proposed to include a monophyletic clade of Betaproteobacteria exclusively associated with epitheliocystis in fish.publishedVersio

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

<p>Abstract</p> <p>Background</p> <p>Since <it>Francisella noatunensis </it>was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of <it>F. noatunensis </it>epizootics would be an important tool for disease management. However, the high genetic similarity within the <it>Francisella </it>spp. makes strain typing difficult, but such typing of the related human pathogen <it>Francisella tullarensis </it>has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of <it>F. noatunensis</it>.</p> <p>Results</p> <p>Seven polymorphic VNTR loci were identified in the preliminary genome sequence of <it>F. noatunensis </it>ssp. <it>noatunensis </it>GM2212 isolate. These VNTR-loci were sequenced in <it>F. noatunensis </it>isolates collected from Atlantic cod (<it>Gadus morhua</it>) from Norway (n = 21), Three-line grunt (<it>Parapristipoma trilineatum</it>) from Japan (n = 1), Tilapia (<it>Oreochromis </it>spp.) from Indonesia (n = 3) and Atlantic salmon (<it>Salmo salar</it>) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique.</p> <p>Conclusions</p> <p>VNTRs can be used to separate isolates belonging to both subspecies of <it>F. noatunensis</it>. Low allelic diversity in <it>F. noatunensis </it>isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis.</p

Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon

Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells

Molecular tools for the detection and identification of Ichthyobodo spp. (Kinetoplastida), important fish parasites

-Ichthyobodo spp. are ectoparasitic flagellates of fish that may cause disease (ichthyobodosis), a common problem affecting the aquaculture industry worldwide. Ichthyobodosis in farmed fish is often associated with a range of other infectious agents and diagnosis in for example gill disease may be difficult. Sensitive and effective methods for detection and identification of Ichthyobodo spp. are needed to aid diagnosis of ichthyobodosis and epizootiological studies on Ichthyobodo spp. We have designed a specific quantitative real-time PCR assay targeting SSU rDNA for the detection of Ichthyobodo spp. infections. Also, several novel primer sets are presented for use in identification of Ichthyobodo spp. through PCR and sequencing. These PCR methods have been optimized and tested on samples from wild caught and farmed fish from different geographical areas in Norway. The real-time PCR assay has been tested for sensitivity and efficiency, and we present data demonstrating its use for absolute quantification of Ichthyobodo salmonis in tissue samples through RT-qPCR and qPCR. We demonstrate the use of the described set of molecular tools for the detection and sequencing of Ichthyobodo spp. from farmed and wild fish, and also show that they may aid the discovery of new Ichthyobodo species. The detection of light Ichthyobodo spp. infections through microscopy is time consuming and less sensitive compared to PCR methods. Initial real-time PCR testing and subsequent sequencing of positive samples is a powerful method that will increase diagnostic precision, aid carrier detection and promote species discoveries in the Ichthyobodonidae. Our preliminary observations indicate a high Ichthyobodo spp. diversity

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains

Tenacibaculosis is a bacterial ulcerative disease affecting marine fish and represents a major threat to aquaculture worldwide. Its aetiological agents, bacteria belonging to the genus Tenacibaculum, have been present in Norway since at least the late 1980’s and lead to regular ulcerative outbreaks and high mortalities in production of farmed salmonids. Studies have shown the presence of several Tenacibaculum species in Norway and a lack of clonality in outbreak-related strains, thus preventing the development of an effective vaccine. Hence, a thorough examination of the bacterial diversity in farmed fish presenting ulcers and the geographical distribution of the pathogens should provide important insights needed to strengthen preventive actions. In this study, we investigated the diversity of Tenacibaculum strains isolated in 28 outbreaks that occurred in Norwegian fish farms in the period 2017–2020. We found that 95% of the 66 strains isolated and characterized, using an existing MultiLocus Sequence Typing system, have not previously been identified, confirming the high diversity of this genus of bacteria in Norway. Several of these Tenacibaculum species seem to be present within restricted areas (e.g., Tenacibaculum dicentrarchi in western Norway), but phylogenetic analysis reveals that several of the strains responsible of ulcerative outbreaks were isolated from different localities (e.g., ST- 172 isolated from northern to southern parts of Norway) and/or from different hosts. Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines.publishedVersio

A novel protist parasite, Salmoxcellia vastator n. gen., n. sp. (Xcelliidae, Perkinsozoa), infecting farmed salmonids in Norway

Background In Norway, x-cell parasites associated with disease in farmed salmonids have been known as a rare phenomenon for two decades. These parasites cause systemic infections in farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), but have so far not been characterized and described. Methods The x-cells from several cases of diseased fish were studied using light and electron microscopy, and by phylogenetic analysis based on small subunit ribosomal RNA (SSU rRNA) gene sequences. Results We describe here the x-cell parasite as a new species in a new genus, Salmoxcellia vastator n. gen., n. sp. Phylogenetic analyses placed Salmoxcellia n. gen. together with Gadixcellia among the xcelliids, a group of perkinsozoan alveolates. The new genus and species were found to have vacuolate plasmodial x-cells filled with lipid droplets, and an electron-dense alveolar pellicle. Electron-dense cytoplasmic inclusions, which are characteristic of the other xcelliid genera Xcellia and Gadixcellia, are lacking in Salmoxcellia n. gen. These x-cell plasmodia divide by plasmotomy and occur as aggregates in the host tissues, particularly in blood-rich tissues such as those of the kidney, red musculature, heart and liver. Host reaction and the refractive lipid droplets in the x-cells result in S. vastator n. gen., n. sp. aggregates appearing as white patches in the tissues. Conclusions We describe a new genus and species of xcelliid protist parasites from two very important farmed fish species and provide molecular methods for detection. The new parasite is associated with disease, but more importantly it has a spoiling effect on farmed salmonid fillets, rendering them unsuitable for sale. Consequently, this parasite represents a threat to the aquaculture industry.publishedVersio

Bacteria associated with early life stages of the great scallop, Pecten maximus: impact on larval survival

International audienceA bacteriological study was carried out at a scallop (Pecten maximus) hatchery near Bergen, western Norway following a severe increase in mortality rates during the larval stages of the scallops. No larvae survived to settling, except for those in groups treated prophylactically with chloramphenicol. In order to identify pathogenic strains of bacteria, we performed a challenge test on 10- to 16-day-old larvae using isolated bacterial strains from the hatchery. Infection with six of these strains produced mortalities that were not statistically different from that resulting from infection with the known pathogen Vibrio pectenicida. However, about 5% of the strains tested in the challenge experiment produced higher motility rates than found in the unchallenged control group, indicating a possible probiotic effect. On the basis of 16S rDNA analysis on these strains, the phylogenetic tree indicated two groups of apparent pathogens: (1) one strain, LT13, grouped together with Alteromonas/Pseudoalteromonas; (2) a cluster of strains grouped together with Vibrio splendidus (LT06, LT21, LT73, PMV18 and PMV19). Strain LT13 was isolated from cultures of the microalga Chaetoceros calcitrans used for feed, while the other strains were isolated from larval cultures. Transmission electron microscopy showed intracellular bacteria that resembled bacteria in the groups Chlamydiaceae and Rickettsiaceae

Identification of housekeeping genes of Candidatus Branchiomonas cysticola associated with epitheliocystis in Atlantic salmon (Salmo salar L.)

Candidatus Branchiomonas cysticola is an intracellular, gram-negative Betaproteobacteria causing epitheliocystis in Atlantic Salmon (Salmo salar L.). The bacterium has not been genetically characterized at the intraspecific level despite its high prevalence among salmon suffering from gill disease in Norwegian aquaculture. DNA from gill samples of Atlantic salmon PCR positive for Cand. B. cysticola and displaying pathological signs of gill disease, was, therefore, extracted and subject to next-generation sequencing (mNGS). Partial sequences of four housekeeping (HK) genes (aceE, lepA, rplB, rpoC) were ultimately identified from the sequenced material. Assays for real-time RT-PCR and fluorescence in-situ hybridization, targeting the newly acquired genes, were simultaneously applied with existing assays targeting the previously characterized 16S rRNA gene. Agreement in both expression and specificity between these putative HK genes and the 16S gene was observed in all instances, indicating that the partial sequences of these HK genes originate from Cand. B. cysticola. The knowledge generated from the present study constitutes a major prerequisite for the future design of novel genotyping schemes for this bacterium.publishedVersio

Wild and farmed salmon (Salmo salar) as reservoirs for infectious salmon anaemia virus, and the importance of horizontal- and vertical transmission

The infectious salmon anaemia virus (ISAV) is an important pathogen on farmed salmon in Europe. The virus occurs as low- and high virulent variants where the former seem to be a continuous source of new high virulent ISAV. The latter are controlled in Norway by stamping out infected populations while the former are spreading uncontrolled among farmed salmon. Evidence of vertical transmission has been presented, but there is still an ongoing discussion of the importance of circulation of ISAV via salmon brood fish. The only known wild reservoirs are in trout (Salmo trutta) and salmon (Salmo salar). This study provides the first ISAV sequences from wild salmonids in Norway and evaluates the importance of this reservoir with respect to outbreaks of ISA among farmed salmon. Phylogenetic analyses of the surface protein hemagglutinin-esterase gene from nearly all available ISAV from Norway, Faeroe Islands, Scotland, Chile and wild salmonids in Norway show that they group into four major clades. Including virulent variants in the analysis show that they belong in the same four clades supporting the hypothesis that there is a high frequency of transition from low to high virulent variants in farmed populations of salmon. There is little support for a hypothesis suggesting that the wild salmonids feed the virus into farmed populations. This study give support to earlier studies that have documented local horizontal transmission of high virulent ISAV, but the importance of transition from low- to high virulent variants has been underestimated. Evidence of vertical transmission and long distance spreading of ISAV via movement of embryos and smolt is presented. We recommend that the industry focus on removing the low virulent ISAV from the brood fish and that ISAV-free brood fish salmon are kept in closed containment systems (CCS).publishedVersio