Diverse Chlamydia-like agents associated with epitheliocystis infection in two cyprinid fish species, the common carp (Cyprinus carpio L.) and the gibel carp (Carassius auratus gibelio L.)

During a general annual fish health survey in natural waters and ponds, epitheliocystis infections were recorded in fingerlings of two cyprinid fish species, the cultured common carp and the wild gibel carp. Benign and heavy infections were equally observed without mortality. In addition to the general health inspection of fish, histopathological examinations of infected gills and molecular biological investigations of separated epitheliocysts were performed. Epitheliocysts were formed both in the interlamellar epithelial cells and in the lamella-free multilayered epithelium of the gill filaments. At the early stage of infection darkstaining inclusion bodies densely stuffed with some pathogenic agents were located at the centre of the cell, while in a progressive stage of the process inclusion bodies within the host cells were disseminated in the cytoplasm and stained pale. Molecular studies demonstrated three different agents related to Neochlamydia, Protochlamydia and Piscichlamydia based on sequence analysis of short regions of the 16S rRNA gene. Among them, Piscichlamydia is a primary fish pathogen, while Neochlamydia and Protochlamydia mostly infect free-living amoebae but have adapted thoroughly to fish

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

Method for cryopreservation of Paramoeba perurans

Paramoeba perurans causes amoebic gill disease (AGD), which is a major problem in aquaculture worldwide. The parasite can be cultured in vitro, but to this date, no method for long-term storage of the clones exists. In this study, we describe a method for cryopreservation of Paramoeba perurans. The method was successfully employed on four out the five clones we tested. The thawing success rate, that is the percentage of successfully thawed vials relative to the total number of vials that were thawed, differed for the clones and ranged from 25% to 100%. The age of the clones seemed to have a negative impact on the ability to survive cryopreservation.publishedVersio

Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae

Chlamydiae are obligate intracellular bacteria that share a unique but remarkably conserved biphasic developmental cycle that relies on a eukaryotic host cell for survival. Although the phylum was originally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. These so-called Chlamydia-like organisms (CLOs) are also referred to as ‘environmental chlamydiae', as many were initially isolated from environmental sources. However, these organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Their strict intracellular nature and the requirement for cell culture have been a confounding factor in characterizing the biology and pathogenicity of CLOs. Nevertheless, the genomes of seven CLO species have now been sequenced, providing new information on their potential ability to adapt to a wide range of hosts. As new isolation and diagnostic methods advance, we are able to further explore the richness of this phylum with further research likely to help define the true pathogenic potential of the CLOs while also providing insight into the origins of the ‘traditional' chlamydia

Paramoeba perurans and AGD in Norwegian aquaculture: effect of freshwater treatment against AGD on gill health in commercial production of Atlantic salmon (Salmo salar L.) and experimental testing of virulence of Paramoeba perurans

Gill diseases are a major and increasing challenge with respect to fish health and welfare during the marine stage in Atlantic salmon farming. The causative agents for gill diseases are complex, and several pathogens are often present at the same time. Amoebic gill disease (AGD), caused by Paramoeba perurans, is strongly associated with gill diseases. To control and prevent development of the disease and mortality, freshwater treatment has shown to be the most effective method. Treatments and handling of the fish causes stressful situations affecting the fish negatively. How these stressful situations within the production affects the gills health and the associated microbiota is less known. The main amin of this study was to map how stress in relation to commercial production (freshwater treatment) affects the gill health of Atlantic salmon. The gills were studied, and pathogens (prevalence and density) were mapped before and after treatment in addition to fish that died during treatment. Histopathological changes compatible with AGD lesions were observed, but there were no significant changes between the groups. The prevalence and density of some pathogens were significantly reduced after treatment, including P. perurans, Ichthyobodo spp, Cand. S. salmonis and Cand. P. salmonis. Other pathogens, including PRV1, P. theridion, SGPV, PMCV and Cand. B. cysticola, increased or had no change of prevalence and density in the gill tissue. The mortality prior to and during the treatment were high, but significantly reduced for the treated group. These results suggests that gill health prior to treatment, and presence of viruses will affect the outcome of the treatment and the mortality rate. A complementary challenge experiment was conducted to test the possible effect of microbiota on the virulence of clonal cultures of Paramoeba perurans during an experimental challenge. Characterization of microbiota present in the culture media of P. perurans showed a large variation of bacteria species, dominated by Vibrio splendidus variants. Although P. perurans cultured in pure culture of V. splendidus grew as well as the amoeba in the original culture media, the clones where not able to establish infection on the gills. The results indicates that the clone’s loss of ability to induce AGD in salmon could be related to the change in microbiota in the amoebas culture media or microbiota present on the salmon gill during infection with amoeba.Masteroppgave i fiskehelseFISK399MAMN-FIS

Experimental challenge of Atlantic salmon Salmo salar using clones of Paramoeba perurans, P. pemaquidensis and Tetramitus sp.

Salmon gill disease in Norway is in most cases associated with a range of different pathogens, stress and environmental factors. Paramoeba perurans and other amoebae have been isolated during such disease outbreaks. Other amoebae isolated from salmon with gill disease in Norway include P. pemaquidensis, Tetramitus sp. and Vannella sp. Here we tested the pathogenicity of the first 2 species in challenge experiments. We found that even when clonal cultures of P. pemaquidensis established an infection on the gills of salmon, it failed to cause gill disease, while Tetramitus sp. appeared to be unable to establish a lasting infection on the gills of healthy salmon. The result of the challenge with P. pemaquidensis confirms the results of similar studies performed in the USA and in Australia. Tetramitus sp. is probably a common amoeba in the marine environment, and its presence on the gills of farmed salmon may just be accidental. Based on this study, we conclude that P. perurans is the only known amoeba in marine salmon farming associated with amoebic gill disease in Norway.publishedVersio

Presence of DNA from Chlamydia-like organisms in the nasal cavities of grey seal pups (Halichoerus grypus) and three different substrates present in a breeding colony

BackgroundChlamydia-like organisms (CLO) have been found to be present in many environmental niches, including human sewage and agricultural run-off, as well as in a number of aquatic species worldwide. Therefore, monitoring their presence in sentinel wildlife species may be useful in assessing the wider health of marine food webs in response to habitat loss, pollution and disease. We used nasal swabs from live (n?=?42) and dead (n?=?50) pre-weaned grey seal pups and samples of differing natal substrates (n?=?8) from an off-shore island devoid of livestock and permanent human habitation to determine if CLO DNA is present in these mammals and to identify possible sources.ResultsWe recovered CLO DNA from 32/92 (34.7%) nasal swabs from both live (n?=?17) and dead (n?=?15) seal pups that clustered most closely with currently recognised species belonging to three chlamydial families: Parachlamydiaceae (n?=?22), Rhabdochlamydiaceae (n?=?6), and Simkaniaceae (n?=?3). All DNA positive sediment samples (n?=?7) clustered with the Rhabdochlamydiaceae. No difference was found in rates of recovery of CLO DNA in live versus dead pups suggesting the organisms are commensal but their potential as opportunistic secondary pathogens could not be determined.ConclusionThis is the first report of CLO DNA being found in marine mammals. This identification warrants further investigation in other seal populations around the coast of the UK and in other areas of the world to determine if this finding is unique or more common than shown by this data. Further investigation would also be warranted to determine if they are present as purely commensal organisms or whether they could also be opportunistic pathogens in seals, as well as to investigate possible sources of origin, including whether they originated as a result of anthropogenic impacts, including human waste and agricultural run-off

Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae.

Chlamydiae are obligate intracellular bacteria that share a unique but remarkably conserved biphasic developmental cycle that relies on a eukaryotic host cell for survival. Although the phylum was originally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. These so-called Chlamydia-like organisms (CLOs) are also referred to as 'environmental chlamydiae', as many were initially isolated from environmental sources. However, these organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Their strict intracellular nature and the requirement for cell culture have been a confounding factor in characterizing the biology and pathogenicity of CLOs. Nevertheless, the genomes of seven CLO species have now been sequenced, providing new information on their potential ability to adapt to a wide range of hosts. As new isolation and diagnostic methods advance, we are able to further explore the richness of this phylum with further research likely to help define the true pathogenic potential of the CLOs while also providing insight into the origins of the 'traditional' chlamydiae