Widespread distribution of human-infective Enterocytozoon bieneusi genotypes in small rodents in northeast China and phylogeny and zoonotic implications revisited.

Enterocytozoon bieneusi features high genetic diversity among host species and environmental sources and over 500 genotypes in 11 phylogenetic groups have been defined. Here we investigated 291 small rodents in Heilongjiang province, northeast China, for the presence of E. bieneusi by PCR of the ribosomal internal transcribed spacer (ITS). Nine of 60 (15.0 %) gray squirrels from a park in Harbin, 120 of 201 (59.7 %) guinea pigs from a pet shop in Harbin, and two of 30 (6.7 %) peridomestic rats from a pasture in Qiqihar were positive for the parasite. Six known genotypes (EbpB, SCC-1, SCC-2, D, S7 and HLJ-CP1) and two novel genotypes (NESQ1 and NEGP1) were identified by sequence analysis of the ITS, with EbpB, SCC-1, SCC-2 and NESQ1 found in squirrels, D, S7 and NEGP1 in guinea pigs, and EbpB and HLJ-CP1 in rats. Widespread distribution of human-infective Group 10 genotype S7 and Group 1 genotype D in guinea pigs raised our concerns about the importance of pet animals as zoonotic reservoirs of microsporidiosis. Co-occurrence of Group 1 genotypes D and HLJ-CP1 in cancer patients and rodents in Heilongjiang indicated a possibility of zoonotic transmission. The host range of Group 1 genotype EbpB previously considered pig-adapted was extended. A potential variant of genotype S7, namely NESQ1, went into the existing Group 10 in phylogenetic analysis. The other new genotype, NEGP1, was clustered in an undefined clade we proposed as Group 15. With the emerging epidemiologic evidence, the host specificity of existing E. bieneusi genotypes is now being challenged.

MICROSPORIDIA-ASSOCIATED GRANULOMATOUS DISEASE IN TWO AUSTRALIAN COLLECTIONS OF CAPTIVE INLAND BEARDED DRAGONS (POGONA VITTICEPS).

Microsporidia are obligate, intracellular fungi. In reptiles, they are most commonly reported in squamates. We report the first detection of microsporidiosis in inland bearded dragons (Pogona vitticeps) from Australia, and for the first time, mixed infections of microsporidium and adenovirus in asymptomatic inland bearded dragons. In one collection there were five individuals, one of which was lethargic, inappetent, and had lost weight. Two large ovarian granulomas were palpated (42 × 23 mm and 26 × 19 mm) and were surgically removed. This animal died shortly after surgery. Histological evaluation of these granulomas revealed granulomatous inflammation within or adjacent to ovarian tissue, containing numerous aggregates of microorganisms consistent with microsporidia. The organisms were confirmed as Encephalitozoon pogonae by polymerase chain reaction (PCR) and sequencing. Agamid adenovirus-1 was also detected. These two infectious agents were also detected by PCR in all the other bearded dragons in this collection (n = 5), all of which were asymptomatic. A single dragon from a second collection presented for a routine wellness examination after the sudden death of another dragon in the collection. This dragon had similar intracelomic masses to the dragon from the first collection. These were removed surgically, but the dragon died 5 wk later following 3 wk of treatment with 25 mg/kg fenbendazole PO q7 days. Necropsy samples were collected and the microsporidian Encephalitozoon pogonae was detected in oral-cloacal swabs, blood, and multiple tissues by PCR and sequencing. Agamid adenovirus-1 was not detected in this dragon.

Tetra disseminated microsporidiosis: a novel disease in ornamental fish caused by Fusasporis stethaprioni n. gen. n. sp.

A novel microsporidial disease was documented in two ornamental fish species, black tetra Gymnocorymbus ternetzi Boulenger 1895 and cardinal tetra Paracheirodon axelrodi Schultz 1956. The non-xenoma-forming microsporidium occurred diffusely in most internal organs and the gill, thus referring to the condition as tetra disseminated microsporidiosis (TDM). The occurrence of TDM in black tetra was associated with chronic mortality in a domestic farmed population, while the case in cardinal tetra occurred in moribund fish while in quarantine at a public aquarium. Histology showed that coelomic visceral organs were frequently necrotic and severely disrupted by extensive infiltrates of macrophages. Infected macrophages were presumed responsible for the dissemination of spores throughout the body. Ultrastructural characteristics of the parasite developmental cycle included uninucleate meronts directly in the host cell cytoplasm. Sporonts were bi-nucleated as a result of karyokinesis and a parasite-produced sporophorous vesicle (SPV) became apparent at this stage. Cytokinesis resulted in two spores forming within each SPV. Spores were uniform in size, measuring about 3.9 ± 0.33 long by 2.0 ± 0.2 µm wide. Ultrastructure demonstrated two spore types, one with 9-12 polar filament coils and a double-layered exospore and a second type with 4-7 polar filament coils and a homogenously electron-dense exospore, with differences perhaps related to parasite transmission mechanisms. The 16S rDNA sequences showed closest identity to the genus Glugea (≈ 92%), though the developmental cycle, specifically being a non-xenoma-forming species and having two spores forming within a SPV, did not fit within the genus. Based on combined phylogenetic and ultrastructural characteristics, a new genus (Fusasporis) is proposed, with F. stethaprioni n. gen. n. sp. as the type species.

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

Comparison of infectious agents detected from hatchery and wild juvenile Coho salmon in British Columbia, 2008-2018.

Infectious diseases are potential contributors to decline in Coho salmon (Oncorhynchus kisutch) populations. Although pathogens are theoretically considered to pose higher risk in high-density rearing environments like hatcheries, there is no direct evidence that hatchery-origin Coho salmon increase the transmission of infectious agents to sympatric wild populations. This study was undertaken to compare prevalence, burden, and diversity of infectious agents between hatchery-reared and wild juvenile Coho salmon in British Columbia (BC), Canada. In total, 2,655 juvenile Coho salmon were collected between 2008 and 2018 from four regions of freshwater and saltwater in BC. High-throughput microfluidics qPCR was employed for simultaneous detection of 36 infectious agents from mixed-tissue samples (gill, brain, heart, liver, and kidney). Thirty-one agents were detected at least once, including ten with prevalence >5%. Candidatus Brachiomonas cysticola, Paraneuclospora theridion, and Parvicapsula pseudobranchiocola were the most prevalent agents. Diversity and burden of infectious agents were substantially higher in marine environment than in freshwater. In Mainland BC, infectious burden and diversity were significantly lower in hatchery smolts than in wild counterparts, whereas in other regions, there were no significant differences. Observed differences in freshwater were predominantly driven by three parasites, Loma salmonae, Myxobolus arcticus, and Parvicapsula kabatai. In saltwater, there were no consistent differences in agent prevalence between hatchery and wild fish shared among the west and east coasts of Vancouver Island. Although some agents showed differential infectious patterns between regions, annual variations likely contributed to this signal. Our findings do not support the hypothesis that hatchery smolts carry higher burdens of infectious agents than conspecific wild fish, reducing the potential risk of transfer to wild smolts at this life stage. Moreover, we provide a baseline of infectious agents in juvenile Coho salmon that will be used in future research and modeling potential correlations between infectious profiles and marine survival.

Ultrastructure and phylogenetic characterization of the microsporidian parasite Heterosporis lessepsianus n. sp. (Microsporidia: Glugeidae) infecting the lizardfish Saurida lessepsianus (Pisces: Synodontidae) inhabiting the Red Sea.

Heterosporosis is an increasingly important microsporidian disease distributed worldwide, impacting wild and farmed-raised fish in both fresh and marine water environments. Twenty three out of 130 (17.69%) of the lizardfish Saurida lessepsianus were found to be naturally infected with microsporidian parasites. The rate of parasitic infection was increased during winter reaching 29.23% (19/65) and fall to 6.15% (4/65) in summer. The infection was recorded as numerous macroscopic whitish cysts reached 3.8-6.5 mm in diameter embedded in the abdominal cavity, skeletal muscles and mesenteric tissues of the infected fish inducing an enormous hypertrophy of infected tissues. Light microscopic examination revealed that parasitic foci were encapsulated by a host-derived fibrous membrane containing different developmental stages of the parasite. Spores were oval to pyriform in shape. Transmission electron microscopic study showed the presence of smooth membranes of the sarcoplasmic reticulum forming a thick, amorphous coat surrounding the various developmental stages of the examined parasite (meronts, sporont, sporoblasts, and spores). Mature spores were electron dense and uninucleate. The anchoring disk was found in a central position at the anterior end of the spore and a large vacuole was located at the posterior end. There was a definite number (7-8) of the polar filament turns. Molecular analysis based on the 16 small subunit (SSU) rDNA gene was performed to determine the phylogenetic position of the present parasite species. A 615 bp region of the 16SSU rDNA gene of the studied parasite was sequenced and deposited in GenBank under the accession number MF769371. Multiple sequence alignment demonstrated a high degree of similarity (>82%) with other twenty microsporidian species isolated from different aquatic hosts. The most closely related sequence was provided by the GenBank entry JF745533 for Heterosporis saurida isolated from the marine fish Saurida undosquamis with the highest percentage of identity (98%) and lowest divergence value (0.9). The ultrastructural characteristics and phylogenetic analysis support the recognition of a new species, herein named Heterosporis lessepsianus sp. n.

Occurrence of zoonotic Enterocytozoon bieneusi in cats in Brazil / Ocorrência de Enterocytozoon bieneusi zoonótico em gatos no Brasil

Abstract Enterocytozoon bieneusi is an opportunistic intestinal pathogen that infects humans and a wide variety of animals worldwide. Our aim in this study was to investigate the occurrence of E. bieneusi in a domestic cat population in Campo Grande, Mato Grosso do Sul, Brazil. Sixty fecal samples from diarrheic cats were subjected to polymerase chain reaction (PCR) and the amplicons were sequenced for identification. E. bieneusi was detected in two samples (3.3%), both identified as genotype D. This genotype has already been reported in animals and humans and is considered a zoonotic genotype. Our findings represent the first report of E. bieneusi in domestic cats in Brazil, reinforcing the importance of identifying this agent as a source of infection in animals and humans.

Resumo Enterocytozoon bieneusi é um patógeno intestinal oportunista que infecta humanos e uma variedade de animais em todo o mundo. O objetivo no presente estudo foi investigar a ocorrência de E. bieneusi em uma população de gatos domésticos em Campo Grande, Mato Grosso do Sul, Brasil. Sessenta amostras fecais de gatos diarréicos foram submetidas a reação em cadeia da polimerase (PCR) e os produtos de amplificação foram sequenciados para identificação molecular. E. bieneusi foi detectado em duas amostras (3,3%), ambos identificados como genótipo D. Esse genótipo tem sido relatado em animais e humanos e é considerado um genótipo zoonótico. Nossos resultados representam a primeira descrição de E. bieneusi em gatos domésticos no Brasil, reforçando a importância desse agente como fonte de infecção para animais e humanos.

Population screening and transmission experiments indicate paramyxid-microsporidian co-infection in Echinogammarus marinus represents a non-hyperparasitic relationship between specific parasite strains.

Phylogenetically distant parasites often infect the same host. Indeed, co-infections can occur at levels greater than expected by chance and are sometimes hyperparasitic. The amphipod Echinogammarus marinus presents high levels of co-infection by two intracellular and vertically transmitted parasites, a paramyxid (Paramarteilia sp. Em) and a microsporidian strain (Dictyocoela duebenum Em). This co-infection may be hyperparasitic and result from an exploitative 'hitchhiking' or a symbiotic relationship between the parasites. However, the best-studied amphipod species are often collected from contaminated environments and may be immune-compromised. Immune-challenged animals frequently present co-infections and contaminant-exposed amphipods present significantly higher levels of microsporidian infection. This suggests the co-infections in E. marinus may result from contaminant-associated compromised immunity. Inconsistent with hyperparasitism, we find that artificial infections transmit Paramarteilia without microsporidian. Our population surveys reveal the co-infection relationship is geographically widespread but find only chance co-infection between the Paramarteilia and another species of microsporidian, Dictyocoela berillonum. Furthermore, we identify a haplotype of the Paramarteilia that presents no co-infection, even in populations with otherwise high co-infection levels. Overall, our results do not support the compromised-immunity hypothesis but rather that the co-infection of E. marinus, although non-hyperparasitic, results from a relationship between specific Paramarteilia and Dictyocoela duebenum strains.

A case study of Desmozoon lepeophtherii infection in farmed Atlantic salmon associated with gill disease, peritonitis, intestinal infection, stunted growth, and increased mortality.

BACKGROUND: In September 2008, a disease outbreak characterized by acute, severe gill pathology and peritonitis, involving the gastrointestinal tract, was observed in an Atlantic salmon (Salmo salar L.) farm in north-western Norway. During subsequent sampling in November 2008 and January 2009, chronic proliferative gill inflammation and peritonitis was observed. Cumulative mortalities of 5.6-12.8% and severe growth retardation were observed. Routine diagnostic analysis revealed no diseases known to salmon at the time, but microsporidian infection of tissues was observed. METHODS: To characterize the disease outbreak, a combination of histopathology, in situ hybridization (ISH), chitin, calcofluor-white (CFW) staining, and real-time PCR were used to describe the disease progression with visualization of the D. lepeophtherii stages in situ. RESULTS: The presence of the microsporidian Desmozoon lepeophtherii was confirmed with real-time PCR, DNA sequencing and ISH, and the parasite was detected in association with acute lesions in the gills and peritoneum. ISH using a probe specific to small subunit 16S rRNA gene provided an effective tool for demonstrating the distribution of D. lepeophtherii in the tissue. Infection in the peritoneum seemed localized in and around pre-existing vaccine granulomas, and in the gastrointestinal walls. In the heart, kidney and spleen, the infection was most often associated with mononuclear leucocytes and macrophages, including melanomacrophages. Desmozoon lepeophtherii exospores were found in the nuclei of the gastrointestinal epithelium for the first time, suggesting a role of the gastrointestinal tract in the spread of spores to the environment. CONCLUSIONS: This study describes the progression of D. lepeophtherii disease outbreak in an Atlantic salmon farm without any other known diseases present. Using different methods to examine the disease outbreak, new insight into the pathology of D. lepeophtherii was obtained. The parasite was localized in situ in association with severe tissue damage and inflammation in the gills, peritoneal cavity and in the gastrointestinal (GI) tract that links the parasite directly to the observed pathology.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

The honeybee disease nosemosis type C is a serious problem since its causative agent, microsporidium Nosema ceranae, is widespread among adult honey bees. Some of the feasible alternative treatments that are used to control this disease are plant extracts. The aim of the present work was to evaluate the effects of essential oils of Chilean plant species, such as Cryptocarya alba, which is used against N. ceranae, and to identify and quantify the majority active compounds in the EO as well as their potential use for the control of nosemosis. Essential oils were obtained using the stripping steam technique with Clevenger equipment and were subsequently analyzed by Gas chromatography-mass spectrometry. Mortality was recorded daily over at least 8days as worker honeybees were exposed to a range of doses of EO dispersed in a sucrose solution. C. alba oil appears to be nontoxic to A. mellifera adults at the tested concentration (the same concentration inhibits the growth of N. ceranae), showing that this oil can be used for the treatment of nosemosis. EO effectiveness was demonstrated against N. ceranae by calculating the percentage of decrease in infected bees from untreated infected groups vs infected groups treated with EO or the reference drug fumagillin. It was determined that a dose of 4µg EO/bee was most effective in controlling N. ceranae development. We determined innocuous doses of C. alba essential oil for honeybees. We demonstrated the antifungal activity of C. alba EO at 4µg/bee against N. ceranae and compared it to its major monoterpenes, such as ß-phellandrene (20µg/bee), eucalyptol (20µg/bee) and α-terpineol (20µg/bee). The major compounds of C. alba EO, α-terpineol, eucalyptol and ß-phellandrene, had significant effects against Apis mellifera infection by N. ceranae, but the antifungal effect of the complete essential oil on N. ceranae was larger than the effect of α-terpineol, eucalyptol or ß- phellandrene separately, showing that C. alba oil may be a candidate for the treatment or prevention of nosemosis.

Microsporidiosis in Four Tortoises ( Testudo hermanni boettgeri).

Microsporidia are obligate intracellular, spore-forming fungi. A wide range of vertebrate and invertebrate hosts can be infected; however, cases of infected turtles or tortoises have not yet been described. This is the first description of 4 cases in tortoises ( Testudo spp), which showed general signs of illness as well as intestinal and respiratory signs until death occurred or they were euthanized. Granulomatous and necrotizing inflammation was visible in the lung and liver. Fungal organisms were present in heart blood, lung, liver, and intestine. Because of their morphology, staining properties (eg, positive in periodic acid-Schiff and silver reaction), and electron microscopic appearance, they were classified as microsporidia.

First record of Tetramicra brevifilum in lumpfish (Cyclopterus lumpus, L.).

A microsporidian species with 98.3-98.4% nucleotide identity to Tetramicra brevifilum (Journal of Fish Diseases, 3, 1980, 495) was diagnosed in lumpfish (Cyclopterus lumpus, L.) broodstock held at a breeding and rearing facility in western Ireland. The fish were wild-caught from the west coast of Ireland, and the first case was diagnosed one year after capture. Clinical signs included severe bloating, lethargy, exophthalmos, anorexia, white patches on the cornea and externally visible parasitic cysts on skin and fins. Necropsy revealed severe ascites, white nodules and vacuoles in all the internal organs and partial liquefaction of the skeletal muscle. On histological examination, microsporidian xenomas were observed in all internal organs, the skin, skeletal muscle, gills and the eyes. The microsporidian species was identified by molecular analysis and transmission electron microscopy. This is the first record of T. brevifilum infecting lumpfish, and the disease is considered to be of potential significance to the rising aquaculture industry of this species.

Clonal Evolution of Enterocytozoon bieneusi Populations in Swine and Genetic Differentiation in Subpopulations between Isolates from Swine and Humans.

Enterocytozoon bieneusi is a widespread parasite with high genetic diversity among hosts. Its natural reservoir remains elusive and data on population structure are available only in isolates from primates. Here we describe a population genetic study of 101 E. bieneusi isolates from pigs using sequence analysis of the ribosomal internal transcribed spacer (ITS) and four mini- and microsatellite markers. The presence of strong linkage disequilibrium (LD) and limited genetic recombination indicated a clonal structure for the population. Bayesian inference of phylogeny, structural analysis, and principal coordinates analysis separated the overall population into three subpopulations (SP3 to SP5) with genetic segregation of the isolates at some geographic level. Comparative analysis showed the differentiation of SP3 to SP5 from the two known E. bieneusi subpopulations (SP1 and SP2) from primates. The placement of a human E. bieneusi isolate in pig subpopulation SP4 supported the zoonotic potential of some E. bieneusi isolates. Network analysis showed directed evolution of SP5 to SP3/SP4 and SP1 to SP2. The high LD and low number of inferred recombination events are consistent with the possibility of host adaptation in SP2, SP3, and SP4. In contrast, the reduced LD and high genetic diversity in SP1 and SP5 might be results of broad host range and adaptation to new host environment. The data provide evidence of the potential occurrence of host adaptation in some of E. bieneusi isolates that belong to the zoonotic ITS Group 1.

Single and multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of farmed and wild crustacean hosts.

Almost half of all known microsporidian taxa infect aquatic animals. Of these, many cause disease in arthropods. Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolated Hepatospora spp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship between Hepatospora isolated from E. sinensis and C. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyse Hepatospora isolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminate Hepatospora isolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description of H. eriocheir in Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential for Hepatospora to exist as a cline across wide geographies and habitats.

Identification of Giardia duodenalis and Enterocytozoon bieneusi in an epizoological investigation of a laboratory colony of prairie dogs, Cynomys ludovicianus.

Since 2005, black-tailed prairie dogs (Cynomys ludovicianus) have been collected for use as research animals from field sites in Kansas, Colorado, and Texas. In January of 2012, Giardia trophozoites were identified by histology, thin-section electron microscopy, and immunofluorescent staining in the lumen of the small intestine and colon of a prairie dog euthanized because of extreme weight loss. With giardiasis suspected as the cause of weight loss, a survey of Giardia duodenalis in the laboratory colony of prairie dogs was initiated. Direct immunofluorescent testing of feces revealed active shedding of Giardia cysts in 40% (n=60) of animals held in the vivarium. All tested fecal samples (n=29) from animals in another holding facility where the index case originated were PCR positive for G. duodenalis with assemblages A and B identified from sequencing triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and ß-giardin (bg) genes. Both assemblages are considered zoonotic, thus the parasites in prairie dogs are potential human pathogens and indicate prairie dogs as a possible wildlife reservoir or the victims of pathogen spill-over. Molecular testing for other protozoan gastrointestinal parasites revealed no Cryptosporidium infections but identified a host-adapted Enterocytozoon bieneusi genotype group.

Observations on the occurrence of Spraguea lophii in Mediterranean lophiids.

In the Mediterranean Sea, anglerfish Lophius budegassa and Lophius piscatorius support an important fishery, and landings have increased in recent years. These species are infected by the microsporean Spraguea lophii, a parasite that infects their nervous system giving rise to multiple cysts. Due to the high value of these fish and conspicuous nature of the infection, we determined the apparent prevalence of this parasite in both Lophius spp. Specimens were obtained from commercial catches along the NW Mediterranean Sea during 2013. The parasite was observed in the nerves from the peripheral nervous system of the individuals and was detected at the prevalence of 69.2% (202/292) and 100% (58/58) in L. budegassa and L. piscatorius, respectively. Non-significant differences were noted between sexes of L. budegassa (χ(2) = 0.683; p = 0.409), although a positive correlation was noted between host size and prevalence (χ(2) = 6.134; p = 0.013). During the sampling, two specimens of L. budegassa with atypical morphological characteristics, pigment anomalies and blindness and infected with S. lophii xenomas were described.

Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia.

Abstract Zebrafish are a powerful model organism to study disease. Like other animal models, Danio rerio colonies are at risk of pathogenic infection. Microsporidia, a group of intracellular fungus-like parasites, are one potential threat. Microsporidian spores germinate and spread causing pathological changes in the central nervous system, skeletal muscle, and other anatomic sites. Infection can impair breeding, cause other morbidities, and ultimately be lethal. Previously, detecting microsporidia in zebrafish has required sacrificing animals for histopathologic analysis or microscopic examination of fresh tissues. Here, we show that fish with microsporidial infection often have autofluorescent nodules, and we demonstrate infectious spread from nodule-bearing fish to healthy D. rerio. Histologic analyses revealed that fluorescent nodules are granulomatous lesions composed of spores, degenerating muscle, and inflammatory cells. Additional histologic staining verified that microsporidia were present, specifically, Pseudoloma neurophilia. Polymerase chain reaction (PCR)-based testing confirmed the presence of P. neurophilia. Further PCR testing excluded infection by another common zebrafish microsporidial parasite, Pleistophora hyphessobryconis. Collectively, these studies show that P. neurophilia can induce skeletal muscle granulomas in D. rerio, a previously unknown finding. Moreover, since granulomas autofluoresce, microscopic screening for P. neurophilia infection is feasible in live fish, avoiding the need to sacrifice fish for surveillance for this pathogen.

In vitro cultivation model for Heterosporis saurida (Microsporidia) isolated from lizardfish, Saurida undosquamis (Richardson).

Heterosporis saurida is a microsporidian that infects lizardfish, Saurida undosquamis (Richardson, 1848), in the Arabian Sea. Spores were isolated from infected lizardfish and used to infect derived fish cell lines: common carp brain (CCB), epithelioma papulosum cyprinid (EPC), fathead minnow epithelial (FHM), rainbow trout gonad (RTG), bluegill fry (BF-2) and chinook salmon embryo (CHSE). Non-fish cell lines were also tested that include: insect (SF-9), rabbit (RK-13) and African green monkey (Vero E6). No growth of H. saurida was observed in any fish cell line, SF-9 or Vero E6 cell lines. H. saurida spores grew only in RK-13 cell line and were detected by immunofluorescence. Developmental stages of H. saurida were seen in RK-13 cells by light and transmission electron microscopy, and species identification was confirmed by sequencing. This study demonstrated that H. saurida was able to proliferate in the mammalian RK-13 cell line, which thus represents an in vitro model for conducting molecular genetics and cell-pathogen interaction studies of Heterosporis.

A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae.

The presence of a new microsporidium is believed to be responsible for an emaciative syndrome observed in farmed gilthead sea bream (Sparus aurata) from different facilities along the Spanish coast. Infected fish were approximately half the average weight and significant mortality was attributed to the condition in some facilities. Clinical signs included anorexia, cachexia and pale internal organs. The microsporidium was found mainly in the intestinal mucosa and occasionally in the submucosa. Morphological, histopathological, ultrastructural and molecular phylogenetic studies were conducted to characterise this organism. This microsporidium undergoes intranuclear development in rodlet cells and enterocytes, and cytoplasmic development mainly in enterocytes and macrophages. The nucleus-infecting plasmodium contains several diplokarya and displays polysporous development which occurs without an interfacial envelope. In the host cell cytoplasm, the parasite develops within a membrane-bound matrix. In both infection locations, the polar tube precursors appear as disks, first with lucent centres, then as fully dense disks as they fuse to form the polar filament, all before division of the plasmodium into sporoblasts. Up to 16 intranuclear spores result from the sporogonic development of a single plasmodium, whereas more than 40 spores result from several asynchronous reproductive cycles in the cytoplasmic infection. Fixed spores are ellipsoidal and diplokaryotic, with five to six coils of an isofilar polar filament in a single row. ssrDNA-based molecular phylogenetic inference places this parasite as a sister clade to crustacean-infecting species of the Enterocytozoonidae and closer to Enterocytozoon bieneusi than to other fish-infecting microsporidians presenting intranuclear development, i.e. Nucleospora, Paranucleospora and Desmozoon. Our studies result in the erection of a new species, Enterospora nucleophila, within the family Enterocytozoonidae, and the description of this family is amended accordingly to accommodate the features of known species assigned to it. Severe histopathological damage occurs in intense infections and this microsporidian is considered a serious emerging threat in sea bream production.

Morphological and phylogenetic description of a new xenoma-inducing microsporidian, Microsporidium aurata nov. sp., parasite of the gilthead seabream Sparus aurata from the Red Sea.

A new species of Microsporidia found in the marine teleost Sparus aurata collected from Hurghada coasts along the Red Sea, Egypt was described based on light and ultrastructural studies. Twenty three (30.6%) out of 75 of the examined fish were parasitized with a microsporidian parasite. Numerous macroscopic whitish cysts embedded in the peritoneal cavity were observed to infect many organs of the body including muscles, connective tissues, and the intestinal epithelium. The infection was developed as tumor-like masses of often up to 5 mm in diameter inducing an enormous hypertrophy to the infected organs. Fresh spores appeared mostly ovoid to pyriform in shape reaching a size of 1.7 ± 0.5 (1.5-2.5) µm × 1.3 ± 0.4 (1-2) µm; they possessed a large vacuole at the posterior end. These spores were located within a sporophorous vesicle which was bound by a thick amorphous wall. The ultrastructural features support the placement of the present species within the genus Microsporidium. The developmental stages were enclosed within a xenoma structure that was bounded by a double-layered cyst wall. The life cycle of the microsporidian pathogen described herein included four stages: proliferation (merogony), sporogony, sporoblast, spores, and liberation. Mature spores appeared electron dense, uninucleate, and were ellipsoidal in shape. At the anterior end of the spore, the anchoring disk was found in a central position. There was a definite number (5-11) of turns of the polar tube. A 538-bp region of the SSU rDNA gene of the studied species was sequenced (GenBank accession number: KF0220444). Multiple sequence alignment calculated a high degree of similarity (>92%) with six microsporidian species. The most closely related sequence was provided by the GenBank entry AF151529 for Microsporidium prosopium isolated from Hyperoplus lanceolatus differing in 67 nucleotide positions in its SSU rDNA with the highest percentage of identity (97.2%) and the lowest divergence value (0.20). Variations in the morphology of the spores and developmental stages between the two species revealed that the two species are different. The site of infection in the host and description of the onset of parasite development are strong criteria for the placement of the microsporidian parasite of the fish S. aurata within the genus Microsporidium as a new species, and we propose to name it Microsporidium aurata nov. sp.