The effect of salinity on enterocytozoon hepatopenaei infection in Penaeus vannamei under experimental conditions.

BACKGROUND: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. RESULTS: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at three different salinities (2 ppt, 15 ppt, and 30 ppt) while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt. CONCLUSION: The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt.

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.

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.

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.

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.

Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis.

Nosema ceranae is an intracellular microsporidian parasite that infects epithelial cells of the honey bee (Apis mellifera) midgut. Previous studies have shown that Nosema may alter cell renewal and apoptosis in honey bees. We found that the amount of apoptotic cells progressively declines from the anterior towards posterior regions of the midgut in Nosema-infected sensitive bees. There was no such pattern in the infected Nosema tolerant honey bees and controls. These data provide additional evidence that N. ceranae appears to alter apoptosis in its host cells for its own advantage.

Three Cases of Neurologic Syndrome Caused by Donor-Derived Microsporidiosis.

In April 2014, a kidney transplant recipient in the United States experienced headache, diplopia, and confusion, followed by neurologic decline and death. An investigation to evaluate the possibility of donor-derived infection determined that 3 patients had received 4 organs (kidney, liver, heart/kidney) from the same donor. The liver recipient experienced tremor and gait instability; the heart/kidney and contralateral kidney recipients were hospitalized with encephalitis. None experienced gastrointestinal symptoms. Encephalitozoon cuniculi was detected by tissue PCR in the central nervous system of the deceased kidney recipient and in renal allograft tissue from both kidney recipients. Urine PCR was positive for E. cuniculi in the 2 surviving recipients. Donor serum was positive for E. cuniculi antibodies. E. cuniculi was transmitted to 3 recipients from 1 donor. This rare presentation of disseminated disease resulted in diagnostic delays. Clinicians should consider donor-derived microsporidial infection in organ recipients with unexplained encephalitis, even when gastrointestinal manifestations are absent.

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.

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.

Ultrastructure and molecular phylogeny of Pleistophora hyphessobryconis (Microsporidia) infecting hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a Brazilian aquaculture facility.

A microsporidian infecting the skeletal muscle of hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a commercial aquaculture facility in Brazil is described. Affected fish exhibited massive infections in the skeletal muscle that were characterized by large opaque foci throughout the affected fillets. Histologically, skeletal muscle was replaced by inflammatory cells and masses of microsporidial developmental stages. Generally pyriform spores had a wrinkled bi-layer spore wall and measured 4·0 × 6·0 µm. Multinucleate meronts surrounded by a simple plasma membrane were observed. The polar filament had an external membrane and a central electron dense mass. The development of sporoblasts within a sporophorous vesicle appeared synchronized. Ultrastructural observations and molecular analysis of 16S rDNA sequences revealed that the microsporidian was Pleistophora hyphessobryconis. This study is the first report of a P. hyphessobryconis infection in a non-ornamental fish.

A new microsporidium, Potaspora macrobrachium n.sp. infecting the musculature of pond-reared oriental river prawn Macrobrachium nipponense (Decapoda: Palaemonidae).

This paper described a novel microsporidian infection in the pond-reared oriental river prawn Macrobrachium nipponense. A conspicuous symptom of the infection was progressive white opacity associated with the musculature. Although neither bacteria nor viruses were detected in routine diagnostic tests, apparently degenerated microsporidian cells or spores were frequently observed in wet smears of the musculature from diseased prawns. Histological observations also revealed characteristics typical of microsporidian infection throughout the host. Transmission electron microscopy revealed multiple life stages of a microsporidian parasite within the cytoplasm of host muscle cells. In addition, partial small subunit ribosomal RNA (SSU rRNA) gene was obtained by a nested PCR using microsporidian specific primers. A consensus sequence was then deposited in GenBank (accession no. KU307278) and subjected to a general BLASTn search that yielded hits only for microsporidian sequence records. Phylogenetic analysis showed that the isolate was most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 87%, was with Potaspora spp. Based on histological, ultrastructure and molecular phylogenetic data, we erected a new species, Potaspora macrobrachium for the novel microsporidium. The description of microsporidium in this important commercial host was fundamental for future consideration of factors affecting stock health and sustainability.

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h(-1)) from 10 °C to the fish's critical thermal maximum (CT(Max)). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U(crit)) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT(Max) and U(crit) tests (i.e. a reduction of 30-40%), it appears that the Atlantic cod can largely compensate for these Loma-induced cardiorespiratory limitations. For example, (i) CT(Max) (21.0 ± 0.3 °C) and U(crit) (~1.75 BL s(-1)) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

Enterocytozoon bieneusi genotypes in children in Northeast China and assessment of risk of zoonotic transmission.

The prevalence (7.5%, 19/255) and genotypes of Enterocytozoon bieneusi in children of various age categories and clinical presentations were determined herein. The co-occurrence of the known genotypes (CS-4, EbpC, and Henan-IV) in children and pigs in the same study area, the phylogenetic characterization of novel genotypes (NEC1 to NEC5), and the assessment of potential risk factors associated with zoonotic transmission robustly suggested that pigs could be a significant source of human E. bieneusi infections in northeast China.

Caenorhabditis elegans as a model for intracellular pathogen infection.

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.

A new species of Glugea Thélohan, 1891 in the red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae) from China.

A new microsporidian species is described from farmed red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae). Large numbers of spherical whitish xenomas were observed throughout the visceral organs of the host. Histological examination showed that the microsporidia caused several xenomas that were embedded in the intestinal muscularis externa or submucosa. Light and transmission electron microscopy examination of the spores also revealed morphological features typical of species of Glugea Thélohan, 1891. This microsporidian parasite has two different types of mature spores: microspores and macrospores. The spores are elongate-ovoid, with a large posterior vacuole. The polaroplast is bi-partite, with anterior and posterior parts comprising densely packed lamellae and loose membranes, respectively, and occupies approximately the anterior half of the spore. The polar filament is anisofilar, with 12-13 coils in a single layer almost touching the posterior spore wall. Comparison of the small subunit rDNA sequences revealed 92.7-98.1% identity with the sequences available from other Glugea spp. from piscine hosts. Phylogenetic analysis demonstrated that the microsporidian species studied clustered within the Glugea clade with strong support. Based on the differences in the morphological characteristics and molecular data, the microsporidian infecting P. major is considered to represent a species new to science, Glugea pagri n. sp.

Opportunistic microsporidiosis unveiled by fine-needle aspiration cytology of cervical lymph node with literature review.

Microsporidia are highly specialized obligate intracellular organisms closely related to fungi, traditionally linked to diarrheal diseases in acquired immunodeficiency syndrome patients. Over the past two decades, an increasing incidence of extraintestinal infections affecting various organ systems, especially in immunocompromised individuals, has been observed. The report presents a unique case of lymph node microsporidiosis in a 38-year-old male, positive for human immunodeficiency virus, with coinfections of hepatitis B and C. Fine-needle aspiration cytology (FNAC) from cervical lymph node yielded pus-like, necrotic material with periodic acid-Schiff stained smear uncovering small round to oval spores on microscopy suspicious for microsporidia. Based on polymerase chain reaction and sequencing done with aspiration material, the causative agent was identified as Vittaforma corneae. This rare encounter highlights the significance of recognizing unique morphological characteristics of infectious organisms and employing appropriate ancillary techniques for precise identification. The case underscores the crucial role of FNAC in diagnosing opportunistic infections involving the lymph nodes and the growing significance of molecular tests for specific pathogen confirmation.

Pathogenesis of Experimental Infection of Nile Tilapia (Oreochromis niloticus) with Nucleospora Braziliensis Pathology and Proteomic of Microsporidia.

The recent discovery of disease caused by Nucleospora braziliensis in Nile tilapia (Oreochromis niloticus) is important as it has highlighted the high prevalence of infection and associated mortality in cultured fish. Thus, this study conducted an experimental infection of this microsporidium to evaluate pathological alterations and conduct proteomic analysis. For pathological observation, samples of brain, eyes, gall bladder, gut, heart, kidney, liver, muscle, skin, spleen, and stomach tissue, were collected, and liquid chromatography-mass spectrometry (LC-MS/MS) was performed for proteomic analysis. The most prevalent lesions were brownish color of the liver, gill filament fusion, gut ischemia, hemorrhage of the lips and fins, hepatomegaly, spleen atrophy, splenomegaly, and stomach congestion. The most common microscopic lesions were degeneration, hemorrhage, and inflammation in the brain, gills, gut, kidney, liver, muscle, spleen, and stomach. The digested peptides were identified by LC-MS/MS and the intersection of each group showed that in the spleen there were 121 exclusive proteins in the infected sample and 252 in the control, while in the kidney, 129 proteins were identified in the infected specimen compared to 83 in the control. In conclusion, this study demonstrates the proteome profile of O. niloticus kidney and spleen tissue in response to infection with N. braziliensis.

Microsporidial stromal keratitis: treatment outcomes, clinical manifestations, confocal microscopy and histopathology findings: a retrospective observational study.

OBJECTIVE: Microsporidial stromal keratitis (MSK) is an uncommon disease. Only several case series have been reported. We aimed to describe the clinical manifestations, histopathology and treatment outcomes of MSK. METHODS AND ANALYSIS: Retrospective data of MSK diagnosed between January 2009 and December 2020 at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand were retrieved. The diagnosis was made based on corneal scraping, corneal biopsy and corneal button histopathology findings. Detailed clinical characteristics, histopathological findings and treatment outcomes were reviewed and analysed. RESULTS: 21 patients with MSK with a mean age of 63.8 years (SD 12.2) had an indolent disease onset with a median of 9 months (IQR 2.2-12.0). Five patients (23.8%) experienced ocular traumas. Herpes stromal keratitis was the most common preliminary diagnosis (33.3%), followed by non-specific ulcers and fungal keratitis. The most common corneal finding was multifocal grey-white lesions with anterior to mid-stromal infiltration and fluffy borders (66.7%). Pathogens were identified by modified trichrome staining of corneal scrapings in 11 of 14 cases (78.6%). Histopathological examination showed positive Ziehl-Neelsen staining in 17 of 19 cases (89.5%). All patients received surgical treatment, with 18 receiving therapeutic penetrating keratoplasty (TPK), 2 undergoing deep anterior lamellar keratoplasty and 1 undergoing femtosecond laser-assisted anterior lamellar keratoplasty. The overall cure rate was 76.2% after the first surgery and 95.2% after the second surgery. CONCLUSION: MSK can be easily underdiagnosed. Clues to diagnosis included a history of chronic refractory stromal infiltration and typical corneal findings of deep stromal infiltration, without epithelial defects. TPK is the preferred treatment for MSK.