Epitheliocystis Agents Research Articles

Molecular interactions in an holobiont-pathogen model: Integromics in gilthead seabream infected with Sparicotyle chrysophrii

The gill ectoparasite Sparicotyle chrysophrii is the major parasitic pathogen in gilthead seabream (Sparus aurata) aquaculture with limited management and treatment strategies. In this work, we assessed the modulation of gilthead seabream gill microbiota during sparicotylosis by sequencing the complete 16S rRNA gene, and the host's molecular pathways occurring at local and systemic levels performing RNA sequencing of gill, spleen and liver. The results obtained and other available data from the same animals were integrated to unveil routes and interactions in this host-microbiota-parasite system. Sparicotylosis significantly modulated gill microbiota decreasing diversity in fish with high infection intensity (H). LEfSe analysis revealed nine microbial biomarkers, highlighting 2013Ark19i (Candidatus Ichthyocystis sparus), already present in control fish, but dramatically increasing in H fish. This bacterium, proposed as the causing agent of epitheliocystis, correlated with the bacterial cysts observed in histopathology, revealing a secondary bacterial infection in these animals. In the RNA sequencing study, 759, 337, and 603 differentially expressed transcripts (DET) were found in gills, spleen and liver, respectively, with 16 commonly regulated transcripts, including genes related to the parasite pathogenesis, such as haemoglobin subunits or hypoxia-inducible factors. Relevant regulated pathways in infected fish were immune system; response to stress, starvation and hypoxia; apoptosis; and haemostasis. Several DET and enriched pathways were shared with a previous plasma proteomics study, particularly in liver. The integrative analysis separated genes and pathways specifically affected by the primary (parasite) and secondary (bacterium) infections. These results show how sparicotylosis changes the gill microbiota, facilitating the growth of pathogenic bacteria and development of secondary infections aggravating the disease outcome. They also evidence host molecular pathways involved in sparicotylosis pathogenesis. Key genes and interactions identified using this integrative approach contribute to the understanding of the disease and can constitute targets for further research pointing to solutions for this infection.

Host associated genotypes in the family Branchiomonaceae with proposal of Ca. Branchiomonas mykissicola n. sp. from rainbow trout (Oncorhynchus mykiss)

Candidatus Branchiomonas cysticola is the primary etiological agent of epitheliocystis and the most prevalent bacterium associated with gill disease (GD) in farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Norway. Partial sequencing of seven of the bacterium's partial housekeeping (HK) genes (aceE, dnaK, lepA, gltA, rplB, rpoC, and typA) revealed few genetic differences, irrespective of geographic origin, reservoirs, and fish populations. Candidatus Branchiomonas cysticola sequenced from gill samples of wild and farmed salmon in Norway and United Kingdom likely represents a single clonal complex (CC). Highly divergent genotypes were, however, identified from gill samples of rainbow trout. Real-time reverse transcriptase PCR (real-time RT-PCR) and fluorescence in-situ hybridization (FISH) showed putative host-specific properties of the bacterial genotypes isolated from salmon and rainbow trout. Sequencing of the 16S and 23S rRNA ribosomal subunits from gills of these two hosts and from other fish species testing positive for Ca. B. cysticola revealed single nucleotide polymorphisms (SNPs). These results support the existence of host-associated bacteria and suggest a strong selection towards host-specific variants of Branchiomonas. A novel host-specific bacterial species from gill samples of rainbow trout, named Candidatus Branchiomonas mykissicola, has therefore been proposed.

Epitheliocystis in Greater Amberjack: Evidence of a Novel Causative Agent, Pathology, Immune Response and Epidemiological Findings.

Epitheliocystis is a fish gill disease caused by a broad range of intracellular bacteria infecting freshwater and marine fish worldwide. Here we report the occurrence and progression of epitheliocystis in greater amberjack reared in Crete (Greece). The disease appears to be caused mainly by a novel Betaproteobacteria belonging to the Candidatus Ichthyocystis genus with a second agent genetically similar to Ca. Parilichlamydia carangidicola coinfecting the gills in some cases. After a first detection of the disease in 2017, we investigated epitheliocystis in the following year’s cohort of greater amberjack juveniles (cohort 2018) transferred from inland tanks to the same cage farm in the open sea where the first outbreak was detected. This cohort was monitored for over a year together with stocks of gilthead seabream and meagre co-farmed in the same area. Our observations showed that epitheliocystis could be detected in greater amberjack gills as early as a month following the transfer to sea cages, with ionocytes at the base of the gill lamellae being initially infected. Cyst formation appears to trigger a proliferative response, leading to the fusion of lamellae, impairment of gill functions and subsequently to mortality. Lesions are characterized by infiltration of immune cells, indicating activation of the innate immune response. At later stages of the outbreak, cysts were no longer found in ionocytes but were observed in mucocytes at the trailing edge of the filament. Whole cysts appeared finally to be expelled from infected mucocytes directly into the water, which might constitute a novel means of dispersion of the infectious agents. Molecular screening indicates that meagre is not affected by this disease and confirms the presence of previously described epitheliocystis agents, Ca. Ichthyocystis sparus, Ca. Ichthyocystis hellenicum and Ca. Similichlamydia spp., in gilthead seabream. Prevalence data show that the bacteria persist in both gilthead seabream and greater amberjack cohorts after first infection.

Epitheliocystis caused by a novel Chlamydia emerging in Spinibarbus denticulatus in China.

Epitheliocystis is an emerging and global aquaculture disease caused by a diverse range of bacteria of the order Chlamydiales. Here we report a case of epitheliocystis caused by a novel Chlamydia bacterium, which resulted in 40% mortality in cultured cyprinids (Spinibarbus denticulatus). The affected fish exhibited lethargy, were observed swimming near the oxygen pump and subsequently died. Histopathology analysis revealed that lesions were concentrated mainly on the gills. The epithelial cells of the damaged gill lamellae showed hyperplasia, fusion and adhesion, and were characterized by inflammation and necrosis. Inclusion bodies were observed in some proliferating epithelial cells at the tips of the gill lamellae and were accompanied by different degrees of mucous cell proliferation. Transmission electron microscopy examination clearly showed the morphological characteristics of chlamydia-like bacteria in epithelial cells. In addition, 16S rRNA sequencing (752 bp) and molecular phylogenetic analyses revealed that epitheliocystis agents detected in S. denticulatus belonged to a novel family, Chlamydiaceae. This is the first report of epitheliocystis in cultured fish in China, and the findings in this study increase the range of hosts affected by epitheliocystis.

Epitheliocystis in rohu Labeo rohita (Hamilton, 1822) is caused by novel Chlamydiales

Epitheliocystis, an emerging aquaculture disease, has been reported in >90 fish species from 14 countries. In the present study, we report epitheliocystis from cultured rohu Labeo rohita, an Indian major carp, which is the major cultured species in the Indian subcontinent. The affected fish exhibited lethargy, swimming near the water surface followed by mortality. In wet mounts of gills, infestation with dactylogyrids, trichodinids and myxosporeans was observed. Subsequently, following euthanasia, the affected fish were dissected and subjected to routine bacteriological, virological and histopathological investigations. The bacteria could not be isolated from internal organs, namely kidney and liver. In addition, cytopathic effects were not observed in the Cyprinus carpio koi fin (CCKF) cell line following inoculation of filtered tissue homogenate from pooled kidney samples. In histology of gill sections, amorphous intracytoplasmic inclusions typical of epitheliocystis were observed in epithelial cells, and the presence of chlamydial DNA in gills was confirmed by amplification of 16S rRNA gene sequence of Chlamydiales. The sequencing of amplicons (774 bp) revealed that the epitheliocystis agents from rohu belong to family Candidatus Parilichlamydiaceae in the order Chlamydiales. Since the sequences of epitheliocystis agent from rohu showed <95% similarity with any member of the genera in above family, therefore, these agents appear to be novel, and it is proposed that these may be known as Candidatus Panilichalamydia rohitae. Using in-situ hybridisation, the 16S rRNA gene amplicons labelled with digoxigenin, linked to the epitheliocysts. The present study adds to the growing range of host species affected by epitheliocystis.

Epitheliocystis in fish: An emerging aquaculture disease with a global impact

Epitheliocystis is a skin and gill disease in fish caused by pathogenic intracellular bacteria. The disease has been reported in at least 90 species of marine and freshwater fish in both the southern and northern hemispheres. It affects a number of commercially important aquaculture species, including salmon, kingfish and bream. In infected fish, cysts typically develop in the gill epithelia, promoting the fusion of gill lamellae. Infections can lead to respiratory distress and death, particularly in cultured and juvenile fish with cases rarely reported in wild fish. Modern molecular techniques are challenging the conventional wisdoms regarding the epidemiology of epitheliocystis, showing now that a number of distinct bacterial pathogens from completely different phyla can cause this disease. Here, we review the state of knowledge, including updates on aetiology, host range, diagnosis and treatments. Traditionally, bacteria from the phylum Chlamydiae were the only known pathogenic agents of epitheliocystis, but aetiology is now recognized as being more complex, including a range of Proteobacteria. Notwithstanding recent advances in identifying the pathogens, the reservoirs and modes of transmission remain largely unknown. Recent genome sequencing of the growing number of epitheliocystis agents suggests that many bacteria causing this disease are unique to individual species of fish. Environmental conditions that approach or exceed animals' physiological tolerances (e.g. atypical temperature, salinity or pH levels) are thought to contribute to disease development and progression. Empirical data and evidence concerning epidemiology, aetiology and treatments are, however, in many cases limited, highlighting the need for more work to better characterize this disease across the different hosts and locales affected.

Candidatus Actinochlamydia pangasiae sp. nov. (Chlamydiales, Actinochlamydiaceae), a bacterium associated with epitheliocystis in Pangasianodon hypophthalmus.

Chlamydial infections are recognised as causative agent of epitheliocystis, reported from over 90 fish species. In the present study, the farmed striped catfish Pangasianodon hypophthalmus (14-15cm, 70-90g) with a history of cumulative mortality of about 23% during June and July 2015, were brought to the laboratory. The histopathological examination of gills from the affected fish revealed presence of granular basophilic intracellular inclusions, mostly at the base of the interlamellar region and in gill filaments. A concurrent infection with Trichodina spp., Ichthyobodo spp. and Dactylogyrus spp. was observed in the gills. The presence of chlamydial DNA in the gills of affected fish was confirmed by amplification and sequencing of 16S rRNA gene. BLAST-n analysis of these amplicons revealed maximum similarity (96%) with Candidatus Actinochlamydia clariae. On the basis of phylogenetic analysis, it was inferred that the epitheliocystis agents from striped catfish were novel and belonged to the taxon Ca. Actinochlamydia. It is proposed that epitheliocystis agents from striped catfish will be named as Ca. Actinochlamydia pangasiae. The 16S rRNA gene amplicons from novel chlamydiae were labelled and linked to inclusions by insitu hybridisation. This is the first report of epitheliocystis from India in a new fish host P. hypophthalmus.

Epitheliocystis Distribution and Characterization in Brown Trout (Salmo trutta) from the Headwaters of Two Major European Rivers, the Rhine and Rhone

We present a first description of the distribution and characterization of epitheliocystis infections in brown trout (Salmo trutta) from the upper catchments of two major European rivers, the Rhine and the Rhone. Overall, epitheliocystis was widely distributed, with 70% of the Rhine and 67% of the Rhone sites harboring epitheliocystis positive brown trout. The epitheliocystis agents Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola could be identified in both catchments, although their relative proportions differed from site to site. Additionally, in two rivers in the Rhine catchment, a new species of Candidatus Similichlamydia was identified. Based on the histology, infection intensity, and severity of pathological changes were significantly more pronounced in mixed chlamydial infections, whereas single infections showed only low numbers of cysts and mild pathology. Infections could be found over a wide range of temperatures, which showed no correlation to infection prevalence or intensity.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria.

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

The emergence of epitheliocystis in the upper Rhone region: evidence for Chlamydiae in wild and farmed salmonid populations.

We present the first study comparing epitheliocystis in a wild and farmed salmonid in Europe. Sampling three tributaries to the Lake Geneva, including one from headwaters to river mouth, revealed an unequal distribution of epitheliocystis in brown trout (Salmo trutta). When evaluated histologically and comparing sites grouped as wild versus farm, the probability of finding infected trout is higher on farms. In contrast, the infection intensities, as estimated by the number of cysts per gill arch, were higher on average and showed maximum values in the wild trout. Sequence analysis showed the most common epitheliocystis agents were Candidatus Piscichlamydia salmonis, all clustering into a single clade, whereas Candidatus Clavichlamydia salmonicola sequences cluster in two closely related subspecies, of which one was mostly found in farmed fish and the other exclusively in wild brown trout, indicating that farms are unlikely to be the source of infections in wild trout. A detailed morphological analysis of cysts using transmission electron microscopy revealed unique features illustrating the wide divergence existing between Ca. P. salmonis and Ca. C. salmonicola within the phylum Chlamydiae.

“Candidatus Similichlamydia laticola”, a Novel Chlamydia-like Agent of epitheliocystis in Seven Consecutive Cohorts of Farmed Australian Barramundi, Lates calcarifer (Bloch)

Six consecutively hatched cohorts and one cohort of pre-hatch eggs of farmed barramundi (Lates calcarifer) from south Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To identify and characterise the bacteria, 59 gill samples and three pre-hatch egg samples were processed for histology, in situ hybridisation and 16S rRNA amplification, sequencing and comprehensive phylogenetic analysis. Cases of epitheliocystis were observed microscopically and characterised by membrane-enclosed basophilic cysts filled with a granular material that caused hypertrophy of the epithelial cells. In situ hybridisation with a Chlamydiales-specific probe lead to specific labelling of the epitheliocystis inclusions within the gill epithelium. Two distinct but closely related 16S rRNA chlamydial sequences were amplified from gill DNA across the seven cohorts, including from pre-hatch eggs. These genotype sequences were found to be novel, sharing 97.1 - 97.5% similarity to the next closest 16S rRNA sequence, Ca. Similichlamydia latridicola, from Australian striped trumpeter. Comprehensive phylogenetic analysis of these genotype sequences against representative members of the Chlamydiales order and against other epitheliocystis agents revealed these Chlamydia-like organisms to be novel and taxonomically placed them within the recently proposed genus Ca. Similichlamydia. Following Fredricks and Relman’s molecular postulates and based on these observations, we propose the epitheliocystis agents of barramundi to be known as “Candidatus Similichlamydia laticola” (sp. nov.).

Candidatus Syngnamydia Venezia, a Novel Member of the Phylum Chlamydiae from the Broad Nosed Pipefish, Syngnathus typhle

Chlamydia are obligate intracellular bacteria and important pathogens of humans and animals. Chlamydia-related bacteria are also major fish pathogens, infecting epithelial cells of the gills and skin to cause the disease epitheliocystis. Given the wide distribution, ancient origins and spectacular diversity of bony fishes, this group offers a rich resource for the identification and isolation of novel Chlamydia. The broad-nosed pipefish (Syngnathus typhle) is a widely distributed and genetically diverse temperate fish species, susceptible to epitheliocystis across much of its range. We describe here a new bacterial species, Candidatus Syngnamydia venezia; epitheliocystis agent of S. typhle and close relative to other chlamydial pathogens which are known to infect diverse hosts ranging from invertebrates to humans.

A Novel Betaproteobacterial Agent of Gill Epitheliocystis in Seawater Farmed Atlantic Salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although ‘Candidatus Piscichlamydia salmonis’, previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as ‘Candidatus Branchiomonas cysticola’, was the cyst-forming agent in these samples. While histology and ultrastructure of ‘Ca. B. cysticola’ cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.

Characterization of ‘Candidatus Clavochlamydia salmonicola’: an intracellular bacterium infecting salmonid fish

The phylum Chlamydiae contains obligate intracellular bacteria, several of which cause disease in their hosts. Morphological studies have suggested that this group of bacteria may be pathogens of fish, causing cysts in epithelial tissue - epitheliocystis. Recently, the first genetic evidence of a chlamydial aetiology of this disease in seawater reared Atlantic salmon from Norway and Ireland was presented, and the agent was given the name 'Candidatus Piscichlamydia salmonis'. In this article we present molecular evidence for the existence of a novel Chlamydiae that also may cause epitheliocystis in Norwegian salmonids. This novel Chlamydiae has been found in salmonid fish from freshwater, and based on its partial 16S rRNA gene, it may constitute a third genus in the family Chlamydiaceae, or a closely related sister family. By using whole-mount RNA-RNA hybridization we demonstrate how infected cells are distributed in a patchy manner on a gill arch. The morphology of the novel Chlamydiae includes the characteristic head-and-tail cells that have been described earlier from salmonid fish suffering from epitheliocystis. We propose the name 'Candidatus Clavochlamydia salmonicola' for this agent of epitheliocystis in freshwater salmonids.

Epitheliocystis in fish

Epitheliocystis is a condition affecting the gills and skin of fish, which has been reported from more than 50 freshwater and marine species. It is caused by intracellular Gram-negative bacteria. Mortalities have been associated with epitheliocystis infections in cultured fish. This review provides an update of our current understanding of this condition, including characterization of the pathogen using immunohistochemical and molecular studies. In most fish species the epitheliocystis agent was negative to an antibody specific for chlamydial genus-specific lipopolysaccharide antigen. Recently, four epitheliocystis agents from four different fish species have been characterized using molecular analysis. While they all belong to the order Chlamydiales, in a lineage separate from the Chlamydiaceae, they are distinct organisms and similarity analysis showed that they had highest similarity values with other chlamydia-like bacteria isolated from various sources, including humans or pig. This confirms the high diversity and host specificity of the pathogen. Further molecular analysis should result in an increased understanding of this condition. To date the pathogen has not been cultured, making experimental studies difficult. High stocking densities, presence of nutrients, season, temperature and fish age have been identified as potential risk factors for the manifestation of this condition.

Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon ( Phycodurus eques ), Silver Perch ( Bidyanus bidyanus ), and Barramundi ( Lates calcarifer )

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.

Virus-like particles associated with heart and skeletal muscle inflammation (HSMI)

The first cases of heart and skeletal muscle inflammation (HSMI), in Atlantic salmon Salmo salar were registered in 1999 in the Hitra/Frøya area of Norway. The disease has since spread south to Rogaland, i.e. the southernmost county with salmon farming in Norway. The disease outbreaks usually start 5 to 9 mo after release into seawater but may occur as early as 2 wk after sea release. The present study focuses on possible pathogens associated with HSMI. It was not possible to find any parasites or bacteria that could explain HSMI, and none of the well-known viruses (infectious salmon anaemia virus, Norwegian salmonid alphavirus, infectious pancreatic necrosis virus, Atlantic salmonid paramyxovirus) were consistently present. Use of transmission electron microscopy showed the presence of epitheliocystis agent in 3 of 4 farms included in this study, and several virus-like particles. Type I and Type II virus particles, previously described for salmon suffering from haemorrhagic smolt syndrome (HSS), and erythrocytic inclusion body syndrome (EIBS) virus were consistently present in salmon suffering from HSMI in all 4 farms included in this study. The 2 HSS viruses (Type I and Type II) were also cultured in Atlantic salmon kidney (ASK) cells from salmon suffering from HSMI. However, a causal relationship between the observed virus particles and HSMI remains to be demonstrated.

Epitheliocystis disease in cultured pacu (Piaractus mesopotamicus) in Brazil.

Epitheliocystis disease was diagnosed in a cultured pacu (Piaractus mesopotamicus Holmberg, 1893) stock on the basis of gross pathological (greyish-white, pinpoint- or pinhead-sized focal areas, connected strongly to the gill lamellae) and histopathological examinations (hypertrophic cells 10-40 microns in size, having a well-defined wall and a central inclusion or characteristic granular content stained with Giemsa). The disease did not occur among fishes (Cyprinus carpio L., Ctenopharyngodon idella Cuv. and Val., Sarotherodon mosambicus Peters, Hoplias malabaricus Bloch) kept together with the affected pacus. About 30-40% of the pacu stock succumbed. Since branchial lesions and heavy mortality were observed in this pacu population, the aetiological role of the epitheliocystis agent could be suspected. Based upon ultrastructural examinations this agent is considered to be a chlamydia or a chlamydia-like organism.

A Morphological Study of the Epitheliocystis Agent in Farmed Atlantic Salmon

Abstract During the last few years there have been several reports of epitheliocystis on farmed Atlantic salmon Salmo salar along the Norwegian coast, and heavy mortalities have been associated with the disease in some cases. Epitheliocystis has previously been described from juvenile steelhead Oncorhynchus mykiss, juvenile lake trout Salvelinus namaycush, and several different nonsalmonid species. These studies of epitheliocystis suggest that the agent belongs to the order Rickettsiales or Chlamydiales. The epitheliocystis agent found in Atlantic salmon was an intracellular, gram-negative bacteria with three distinctive developmental stages: reticulate bodies (RB), intermediate bodies (IB), and elementary bodies (EB). All three stages were present within membrane-bound vacuoles in gill epithelial cells. The bodies were surrounded by two trilaminar membranes and contained ribosomes and nucleic acids. The nucleic acids in the IB and EB were condensed to a nucleoid, while the nucleic acids in the RB were fo...

Epitheliocystis Infection in Cultured White Sturgeon (Acipenser transmontanus): Antigenic and Ultrastructural Similarities of the Causative Agent to the Chlamydiae

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250-300 g) white sturgeon (Acipenser transmontanus) from a private culture facility with a 4-8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4-0.8 x 0.5-1.4 microns but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2-0.4 x 0.3-0.6 microns although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3-0.4 microns oval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chalmydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.