Mass mortality event of round sardinella Sardinella aurita Valenciennes associated with Glugea Thélohan, 1891 microsporidian infection off the southern Italian coast.

Intracellular parasites of the genus Glugea Thélohan, 1891 (Microsporidia) comprise about 34 putative species capable of causing high morbidity and mortality in freshwater and marine teleost fishes. In this study, we report on the first mass mortality event associated with Glugea sp. infecting free-ranging round sardinella Sardinella aurita in the southern Tyrrhenian Sea (Italy). Here, we describe the ultrastructure of mature spores of this microsporidian and characterize it molecularly, as well as report its phylogenetic position. Most of the affected fish showed an irregular swelling of its abdomen. At necropsy, a variable number of xenomas, spherical to ellipsoidal in shape, were found in the peritoneal cavity strongly attached to the viscera of all fish. Histological analysis revealed varying severity of chronic inflammation along with occasional necrosis in visceral organs associated with multiple xenoma proliferation. These pathological findings were considered the main cause of this mass mortality event. Morphologically, the present material was closely related to G. sardinellesis and G. thunni. The phylogenetically closest taxa to the newly SSU rDNA sequence were G. thunni and an erroneusly identified  G. plecoglossi, which were very closely related to each other, also suggesting that all these sequences might belong to the same species.

Pseudokabatana alburnus n. gen. n. sp., (Microsporidia) from the liver of topmouth culter Culter alburnus (Actinopterygii, Cyprinidae) from China.

We describe the type species of a novel genus of microsporidian parasite, Pseudokabatana alburnus n. gen. n. sp., infecting the liver of topmouth culter, Culter alburnus Basilewsky, 1855, from Lake Poyang off Xingzi county, Jiangxi Province, China. The parasite elicits formation of spherical xenomas of up to 1.2 mm in diameter containing all observed life stages from early merogonal plasmodia to mature spores contained within the cytoplasm of host hepatocytes. Merogonal plasmodia existed in direct contact with the host cytoplasm and contained up to 20 visible nuclei. Plasmotomy of the multinucleate plasmodium led to formation of uninucleate cells in which the nucleus underwent further division to form bi-nucleate presporonts, sporonts (defined by cells with a thickened endospore) and eventually sporoblasts (containing pre-cursors of the spore extrusion apparatus). Mature spores were pyriform and monokaryotic, measuring 2.3 ± 0.19 µm long and 1.3 ± 0.10 µm wide. Spores possessed a bipartite polaroplast and 5-6 coils of a polar filament, in a single rank. The obtained partial SSU rRNA gene sequence, 1383 bp in length, did not match any of microsporidia available in GenBank. SSU rDNA-based phylogenetic analysis indicated a new taxon branching with Kabatana rondoni, a parasite infecting the skeletal muscle of Gymnorhamphichthys rondoni from the Amazon River. Due to different host and tissue tropism, the novel taxon did not fit the diagnostic criteria for the genus Kabatana. Further, based on SSU rDNA-inferred phylogenetic analyses, different ultrastructural features of developmental stages, and ecological considerations, a new genus Pseudokabatana and type species Pseudokabatana alburnus n. sp. was erected for the parasite in topmouth culter.

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua.

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno-positive results for vimentin, α-smooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua-associated xenoma-like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.

Integrative Approach for the Reliable Detection and Specific Identification of the Microsporidium Loma morhua in Atlantic Cod (Gadus morhua).

Microsporidia are fungal parasites that infect diverse invertebrate and vertebrate hosts. Finfish aquaculture supports epizootics due to high host density and the high biotic potential of these parasites. Reliable methods for parasite detection and identification are a necessary precursor to empirical assessment of strategies to mitigate the effects of these pathogens during aquaculture. We developed an integrative approach to detect and identify Loma morhua infecting Atlantic cod. We show that the spleen is more reliable than the commonly presumed gills as best organ for parasite detection in spite of substantial morphological plasticity in xenoma complexes. We developed rDNA primers with 100% sensitivity in detecting L. morhua and with utility in distinguishing some congeneric Loma species. ITS sequencing is necessary to distinguish L. morhua from other congeneric microsporidia due to intraspecific nucleotide variation. 64% of L. morhua ITS variants from Atlantic cod have a 9-nucleotide motif that distinguishes it from Loma spp. infecting non-Gadus hosts. The remaining 36% of ITS variants from Atlantic cod are distinguished from currently represented Loma spp., particularly those infecting Gadus hosts, based on a 14-nucleotide motif. This research approach is amenable to developing templates in support of reliable detection and identification of other microsporidian parasites in fishes.

Microsporidians xenomas of anglerfish from NE Atlantic waters.

The presence of emergent visible parasites at commercial valuable fish species is increasingly causing problems at fisheries and seafood industries. Microsporidians have been previously reported to appear forming apparent xenomas complexes in anglerfish species, but no effort has been carried out to simultaneously integrate epidemiological data, phenotypic, genotypic and fine structural characterizations in the same parasite sample. In this work, specimens of Lophius budegassa and Lophius piscatorius from NE Atlantic waters were sampled and examined to provide information about specific site of infection and demographic data of two groups of different sizes of xenomas present at both fish species. Histological descriptions and scanning and transmission electron microscopy were carried out on fresh spores of Lophius budegassa for ultrastructural studies. In both types of xenomas, it was observed simultaneously the microsporidian genus Spraguea in the form of two different types of spores. Molecular analyses of both xenomas from the two fish species, based on the small subunit ribosomal DNA gene, were also performed to genetically support the morphological diagnostic provided.

Immunohistochemical localization of inflammatory cells and cell cycle proteins in the gills of Loma salmonae infected rainbow trout (Oncorhynchus mykiss).

Microsporidial gill diseases particularly those caused by Loma salmonae incur significant economic losses to the salmonid aquaculture industry. The gill responses to infection include the formation of xenomas and the acute hyperplastic inflammatory responses once the xenomas rupture releasing infective spores. The aim of this work was to characterize the inflammatory responses of the gill to both the presence of the xenomas as well as the hyperplasia associated with L. salmonae infection in the rainbow trout gill following an experimental infection using immunohistochemistry. Hyperplastic lesions demonstrated numerous cells expressing PCNA as well as an apparent increased expression of caspase-3 and number of apoptotic cells (TUNEL positive cells). There was an expression of TNFα in individual cells within the gill and increased expression of a myeloid cell line antigen indicating the presence of granulocyte infiltration of both the hyperplastic lesions as well as the xenomas. Similar immune-reactivity was seen in gill EGCs. Hyperplastic gill lesions showed a marked infiltration of CD8+ cells and expression of MHC class I antigens. These findings suggest that L. salmonae xenomas may be subject to infiltration by the host immune cells as well as the mounting or a marked cellular cytotoxic immunoreaction in the resultant hyperplasia following xenoma rupture and spore release.

Areospora rohanae n.gen. n.sp. (Microsporidia; Areosporiidae n. fam.) elicits multi-nucleate giant-cell formation in southern king crab (Lithodes santolla).

This paper utilises histological, ultrastructure and molecular phylogenetic data to describe a novel genus and species (Areospora rohanae n.gen., n.sp.) within the phylum Microsporidia. Phylogenetic and morphological distinction from other known lineages within the phylum also provide strong support for erection of a new family (Areosporiidae n. fam) to contain the parasite. Recognised via lesions observed by workers in king crab processing facilities in southern Chile, the parasite elicits giant cell formation in infected crabs. Merogony within haemocytes and fixed phagocytes proceeds apparent fusion of infected cells to produce multinucleate syncitia in which further development of the parasite occurs. Subsequent recruitment of adjacent cells within the haemal spaces of the hepatopancreas, the podocytes of the gill, and particularly in the subcuticular connective tissues, characterises the pathogenesis of A. rohanae. In late stages of infection, significant remodelling of the subcuticular tissues corresponds to the clinical lesions observed within processing plants. Sporogony of A. rohanae also occurs within the syncitial cytoplasm and culminates in production of bizarre spores, ornamented with distinctive tubular bristles. Spores occur in sets of 8 within a sporophorous vesicle. The description of A. rohanae offers considerable insight into the pathogenesis of giant-cell forming Microsporidia, signifies a new lineage of giant-cell forming Microsporidia in marine hosts, and may reflect emergence of a commercially-significant pathogen in the southern ocean Lithodes santolla fishery.

A severe microsporidian disease in cultured Atlantic Bluefin Tuna (Thunnus thynnus).

One of the most promising aquaculture species is the Atlantic bluefin tuna (Thunnus thynnus) with high market value; disease control is crucial to prevent and reduce mortality and monetary losses. Microsporidia (Fungi) are a potential source of damage to bluefin tuna aquaculture. A new microsporidian species is described from farmed bluefin tunas from the Spanish Mediterranean. This new pathogen is described in a juvenile associated with a highly severe pathology of the visceral cavity. Whitish xenomas from this microsporidian species were mostly located at the caecal mass and ranged from 0.2 to 7.5 mm. Light and transmission electron microscopy of the spores revealed mature spores with an average size of 2.2 × 3.9 µm in size and a polar filament with 13-14 coils arranged in one single layer. Phylogenetic analysis clustered this species with the Glugea spp. clade. The morphological characteristics and molecular comparison confirm that this is a novel microsporidian species, Glugea thunni. The direct life-cycle and the severe pathologies observed makes this parasite a hard risk for bluefin tuna cultures.

Characterizing the Xenoma of Vairimorpha necatrix Provides Insights Into the Most Efficient Mode of Microsporidian Proliferation.

Microsporidia are a group of obligated intracellular parasites that can infect nearly all vertebrates and invertebrates, including humans and economic animals. Microsporidian Vairimorpha necatrix is a natural pathogen of multiple insects and can massively proliferate by making tumor-like xenoma in host tissue. However, little is known about the subcellular structures of this xenoma and the proliferation features of the pathogens inside. Here, we characterized the V. necatrix xenoma produced in muscle cells of silkworm midgut. In result, the whitish xenoma was initially observed on the 12th day post infection on the outer surface of the midgut and later became larger and numerous. The observation by scanning electronic microscopy showed that the xenoma is mostly elliptical and spindle with dense pathogen-containing protrusions and spores on the surface, which were likely shedding off the xenoma through exocytosis and could be an infection source of other tissues. Demonstrated with transmission electron microscopy and fluorescent staining, the xenoma was enveloped by a monolayer membrane, and full of vesicle structures, mitochondria, and endoplasmic reticulum around parasites in development, suggesting that high level of energy and nutrients were produced to support the massive proliferation of the parasites. Multiple hypertrophic nuclei were found in one single xenoma, indicating that the cyst was probably formed by fusion of multiple muscle cells. Observed by fluorescence in situ hybridization, pathogens in the xenoma were in merongony, sporogony, and octosporogony, and mature stages. And mature spores were pushed to the center while vegetative pathogens were in the surface layer of the xenoma. The V. necatrix meront usually contained two to three nuclei, and sporont contained two nuclei and was wrapped by a thick membrane with high electron density. The V. necatrix sporogony produces two types of spores, the ordinary dikaryotic spore and unicellular octospores, the latter of which were smaller in size and packed in a sporophorous vesicle. In summary, V. necatrix xenoma is a specialized cyst likely formed by fusion of multiple muscle cells and provides high concentration of energy and nutrients with increased number of mitochondria and endoplasmic reticulum for the massive proliferation of pathogens inside.