Detection and diagnosis of Panulirus argus virus 1 in captive spiny lobsters using qPCR in conjunction with histopathology and transmission electron microscopy.

Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus described in the Caribbean spiny lobster, Panulirus argus. PaV1 infection in decapod species that commonly co-occur with P. argus, including the spotted spiny lobster Panulirus guttatus, has not been previously described. In 2016, 14 Caribbean and 5 spotted spiny lobsters were collected near Summerland Key, Florida, to supplement the resident population of the Audubon Aquarium of the Americas in New Orleans, Louisiana. After 5 months in quarantine, Caribbean and spotted spiny lobsters began to exhibit clinical signs of lethargy and dying in the molt. Initial histologic evaluation revealed intranuclear inclusion bodies in circulating hemocytes in the spongy connective tissue of the epidermis, suggesting a viral infection. Samples of hepatopancreas and hemolymph from deceased Caribbean and spotted spiny lobsters tested negative for white spot syndrome virus and positive for PaV1 using real-time quantitative polymerase chain reaction (qPCR). Intranuclear, eosinophilic to amphophilic, Cowdry type A inclusion bodies observed primarily within fixed phagocytes and circulating hemocytes in the hepatopancreas of freshly euthanized Caribbean spiny lobsters were consistent with PaV1 infection. Transmission electron microscopy revealed that hemocytes associated with hepatopancreatic tubules contained viral inclusions with location, size, and morphology consistent with previously described PaV1 infection. These findings highlight the significance of using molecular diagnostics in conjunction with histopathology and electron microscopy in the investigation and diagnosis of PaV1 in spiny lobsters. Further study is required to investigate the relationship of PaV1-associated mortality events and microscopic lesions in the spotted spiny lobster.

Microsporidia Ameson earli sp. n. and A. michaelis (Sprague 1965) infecting blue crabs Callinectes sapidus from shedding facilities in Louisiana.

During a survey for pathogens and commensals of blue crabs in commercial soft shell shedding facilities in Louisiana, we discovered an occurrence of microsporidiosis in two of forty examined crabs. Judging from spore shape and size, tissue tropism and external signs of muscle pathology, the causative agent of infections was identified as Ameson michaelis, a muscle-infecting species that has been repeatedly detected in populations of Callinectes sapidus in Louisiana since 1965. However, retrospective ultrastructural examination revealed that in one of Ameson-infected crabs, infection was caused by a parasite with ultrastructural characters not completely compliant with the ones of A. michaelis. The major difference was the absence of microtubule-like appendages attached to the exospore, typical of A. michaelis and other Ameson spp. SSUrDNA-inferred pairwise evolutionary distances between the novel species and other Ameson spp. ranged from 0.006 to 0.051; it was 0.039 in the case of A. michaelis. Hence, we describe here a new species in the genus Ameson, and name it after Prof. Earl Weidner, our colleague and friend, an outstanding microsporidiologist and the author of pioneer papers on the ultrastructure and physiology of A. michaelis.

Evidence from the resurrected family Polyrhabdinidae Kamm, 1922 (Apicomplexa: Gregarinomorpha) supports the epimerite, an attachment organelle, as a major eugregarine innovation.

BACKGROUND: Gregarines are a major group of apicomplexan parasites of invertebrates. The gregarine classification is largely incomplete because it relies primarily on light microscopy, while electron microscopy and molecular data in the group are fragmentary and often do not overlap. A key characteristic in gregarine taxonomy is the structure and function of their attachment organelles (AOs). AOs have been commonly classified as "mucrons" or "epimerites" based on their association with other cellular traits such as septation. An alternative proposal focused on the AOs structure, functional role, and developmental fate has recently restricted the terms "mucron" to archigregarines and "epimerite" to eugregarines. METHODS: Light microscopy and scanning and transmission electron microscopy, molecular phylogenetic analyses of ribosomal RNA genes. RESULTS: We obtained the first data on fine morphology of aseptate eugregarines Polyrhabdina pygospionis and Polyrhabdina cf. spionis, the type species. We demonstrate that their AOs differ from the mucron in archigregarines and represent an epimerite structurally resembling that in other eugregarines examined using electron microscopy. We then used the concatenated ribosomal operon DNA sequences (SSU, 5.8S, and LSU rDNA) of P. pygospionis to explore the phylogeny of eugregarines with a resolution superior to SSU rDNA alone. The obtained phylogenies show that the Polyrhabdina clade represents an independent, deep-branching family in the Ancoroidea clade within eugregarines. Combined, these results lend strong support to the hypothesis that the epimerite is a synapomorphic innovation of eugregarines. Based on these findings, we resurrect the family Polyrhabdinidae Kamm, 1922 and erect and diagnose the family Trollidiidae fam. n. within the superfamily Ancoroidea Simdyanov et al., 2017. Additionally, we re-describe the characteristics of P. pygospionis, emend the diagnoses of the genus Polyrhabdina, the family Polyrhabdinidae, and the superfamily Ancoroidea.

Streptococcus dysgalactiae: A Pathogen of Feral Populations of Silver Carp from a Fish Kill Event.

In August 2018, a series of large fish kills involving only Silver Carp Hypophthalmichthys molitrix occurred on the Mississippi River in northern Louisiana. Clinical signs observed in moribund animals included erratic swimming behavior, such as spiraling and spinning at the surface. A moribund specimen was captured by dip net near the surface at Lake Providence Landing in East Carroll Parish, northern Louisiana, and was submitted for analysis. An aseptic necropsy was performed, and diagnostic procedures, including bacteriology, parasitology, histopathology, virology, and electron microscopy, revealed that a gram-positive coccus was the primary pathogen. Pure cultures of the organism were obtained from the brain, and it was the predominant colony type isolated from the spleen, kidney, and liver. Bacterial sepsis caused by the gram-positive coccus and involving multiple organ systems was diagnosed histologically. Bacterial colonization and necrotic lesions were seen in the spleen, liver, kidney, heart, eye, and brain. Numerous cocci were observed dividing intracellularly in phagocytic cells of the kidney and brain by transmission electron microscopy. The organism was identified as Streptococcus dysgalactiae ssp. dysgalactiae by conventional biochemical methods and subsequently by the API 20 Strep system. The identity of the pathogen was later confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of the 16S ribosomal RNA gene. Multilocus sequence analysis clustered this isolate along with two other S. dysgalactiae isolates from fish in a divergent phyletic group that was separate from other S. dysgalactiae ssp. dysgalactiae isolates from terrestrial animals, implying a possible novel clade that is pathogenic for fish.

Two new species of Unikaryon (Microsporidia) hyperparasitic in microphallid metacercariae (Digenea) from Florida intertidal crabs.

The genus Unikaryon (Microsporidia) holds exclusively hyperparasites of Platyhelminthes. Four species of Unikaryon are presently known from trematodes infecting mollusks and fish, and one from a cestode infecting a fish. Here we report two species of Unikaryon from microphallid trematode metacercariae parasitizing the brachyuran crabs, Panopeus herbstii and Pachygrapsus transversus, collected from intertidal habitats in Florida. The first microsporidium, which we assign here to a new species, Unikaryon panopei sp. n., was isolated from Microphallus sp. encysted in Panopeus herbstii from Tampa Bay. The specific designation for the second Unikaryon sp. (Unikaryon sp. 2), which occurred in metacercaria of Diacetabulum sp. found in P. transversus from the Florida Keys, is pending due to the lack of SSrDNA sequence data. Light and electron microscopy demonstrates that both species display characteristics of the genus Unikaryon including the arrangement of spores in sets of two, large posterior vacuole, and eccentric position of the polar filament. Spores of Unikaryon panopei sp. n., unlike those of Unikaryon sp. 2, assemble in large membrane-bound masses containing hundreds of organisms, and display a larger number of polar filament coils - 7-8, compared to 4-5 in Unikaryon sp. 2 The SSUrDNA-inferred phylogenetic analysis places Unikaryon panopei in one clade with Unikaryon legeri, the only other molecularly characterized member of the genus, with 94% of SSUrDNA similarity. These findings increase the number of species parasitizing trematodes and broaden the host range of Unikaryon spp.

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

Microsporidia Can Acquire Lamin-like Intermediate Filaments and Cell Adhesion Catenin-cadherin Complexes from the Host (?).

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with ß and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A. michaelis and A. algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin-GFP to see whether developing A. algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin-cadherin complexes from the host.

Development of Anncaliia algerae (Microsporidia) in Drosophila melanogaster.

Representatives of the genus Anncaliia are known as natural parasites of dipteran and coleopteran insects, amphipod crustaceans, but also humans, primarily with immunodeficiency. Anncaliia algerae-caused fatal myositis is considered as an emergent infectious disease in humans. A. (=Nosema, Brachiola) algerae, the best studied species of the genus, demonstrates the broadest among microsporidia range of natural and experimental hosts, but it has never been propagated in Drosophila. We present ultrastructural analysis of development of A. algerae in visceral muscles and adipocytes of Drosophila melanogaster 2 wk after per oral experimental infection. We observed typical to Anncaliia spp. features of ultrastructure and cell pathology including spore morphology, characteristic extensions of the plasma membrane, and presence of "ridges" and appendages of tubular material at proliferative stages. Anncaliia algerae development in D. melanogaster was particularly similar to one of A. algerae and A.(Brachiola) vesicularum in humans with acute myositis. Given D. melanogaster is currently the most established genetic model, with a fully sequenced genome and easily available transgenic forms and genomic markers, a novel host-parasite system might provide new genetic tools to investigate host-pathogen interactions of A. algerae, as well to test antimicrosporidia drugs.

Encephalitozoon cuniculi and Vittaforma corneae (Phylum Microsporidia) inhibit staurosporine-induced apoptosis in human THP-1 macrophages in vitro.

Obligately intracellular microsporidia regulate their host cell life cycles, including apoptosis, but this has not been evaluated in phagocytic host cells such as macrophages that can facilitate infection but also can be activated to kill microsporidia. We examined two biologically dissimilar human-infecting microsporidia species, Encephalitozoon cuniculi and Vittaforma corneae, for their effects on staurosporine-induced apoptosis in the human macrophage-differentiated cell line, THP1. Apoptosis was measured after exposure of THP-1 cells to live and dead mature organisms via direct fluorometric measurement of Caspase 3, colorimetric and fluorometric TUNEL assays, and mRNA gene expression profiles using Apoptosis RT2 Profiler PCR Array. Both species of microsporidia modulated the intrinsic apoptosis pathway. In particular, live E. cuniculi spores inhibited staurosporine-induced apoptosis as well as suppressed pro-apoptosis genes and upregulated anti-apoptosis genes more broadly than V. corneae. Exposure to dead spores induced an opposite effect. Vittaforma corneae, however, also induced inflammasome activation via Caspases 1 and 4. Of the 84 apoptosis-related genes assayed, 42 (i.e. 23 pro-apoptosis, nine anti-apoptosis, and 10 regulatory) genes were more affected including those encoding members of the Bcl2 family, caspases and their regulators, and members of the tumour necrosis factor (TNF)/TNF receptor R superfamily.

A new microsporidium, Apotaspora heleios n. g., n. sp., from the Riverine grass shrimp Palaemonetes paludosus (Decapoda: Caridea: Palaemonidae).

We report a new microsporidium from a key species of the estuarine communities of the Gulf States, the Riverine grass shrimp, Palaemonetes paludosus. A milky-white shrimp was found in the Mobile Bay Delta, a large, oligohaline-freshwater wetland in Alabama, USA. Light microscopy of smears and thick sections of the abdominal tissues demonstrated infection with microsporidian spores enclosed in sporophorous vesicles (SVs) in sets of eight. Broadly oval spores measured 2.9 ±â€¯0.06 × 1.7 ±â€¯0.03 µm (2.5-3.3 × 1.6-1.9 µm, n = 11). SVs with a persistent membrane ranged from 4.4 to 5.6 µm in diameter. Subcuticular epithelium and underlying musculature were packed with sporonts, sporoblasts, and spores. Electron microscopy demonstrated diplokaryotic meronts that gave rise to sporont mother cells with a large single nucleus. The meront plasma membrane turned into a SV envelope, and the sporont wall segregated internally. The sporont nucleus underwent meiosis followed by two mitotic divisions accompanied by internal budding to produce four sporonts, each dividing in two uninucleate sporoblasts. Eight-spore SVs were filled with fibrillary-tubular secretions. Spores possessed 90-110-nm thick envelopes (exospore, 40-60 nm + endospore, 30-50 nm), a triangle-shaped nucleus, isofilar polar filament of 10-13 coils arranged in two-three rows, bipartite polaroplast, and a mushroom-shaped polar disk. The SSU rDNA sequence of the novel species was deposited in GenBank under Accession number MG 708238. SSU rDNA-based phylogenetic analysis indicated that the Riverine grass shrimp microsporidium was a new species and placed it in one branch with two species of Potaspora, xenoma-forming microsporidia from freshwater perciform fishes. Because morphological and developmental characters of the novel species did not fit the diagnosis of the genus Potaspora, and, based on SSU rDNA-inferred phylogenetic analyses, different host specificity, pathogenesis, and ecological considerations, we erect here the new genus Apotaspora for the Riverine grass shrimp microsporidium and name the new species Apotaspora heleios. Grouping together fish and crustacean parasites on SSU rDNA phylogenetic trees suggests that polyxenous life cycles might be a common feature of extinct and/or extant members of the studied lineage of the Microsporidia.

Molecular and Morphological Characterization of Anncaliia azovica sp. n. (Microsporidia) Infecting Niphargogammarus intermedius (Crustacea, Amphipoda) from the Azov Sea.

Five out of one hundred adults of Niphargogammarus intermedius caught at the Azov sea shore were found to be infected with microsporidia. The infection was found in the subcuticular fat body and myocytes. Parasites developed in direct contact with host cells, displayed a disporoblastic sporogony and diplokaryotic arrangement of nuclei at all stages. Spores were oval, 4.6-5.8 × 2.6-3.0 µm. Exospore appendages, vesicular-tubular secretions, and the anisofilar polar filament indicated a similarity to Anncaliia species. Sporont surfaces displayed ridges of amorphous material. Meronts and sporonts formed protoplastic extensions, similar to A. vesicularum and A. meligheti. Mature spores possessed a bipartite polaroplast. The polar tube was arranged in one row of 13-18 coils including 0-3 distal coils of lesser diameter. Partial sequencing of SSU, ITS, and LSU regions of rRNA gene (GenBank accessions: KY288064-KY288065) confirmed this new species to be congeneric with A. algerae (#AF069063) and A. meligheti (#AY894423). The SSU gene of this novel microsporidium shared 99.4% sequence similarity to A. algerae and 98.9% to A. meligheti. Genes for HSP70 and RPB1 amplified with primers designed for A. algerae orthologs displayed 99.7% and 97.4% similarity, respectively, between A. algerae and the novel microsporidium. A new species, Anncaliia azovica, is described based on morphological and molecular characterization.

A Microsporidian Infection in Phoronids (Phylum Phoronida): Microsporidium phoronidi n. sp. from a Phoronis embryolabi.

Microsporidia-like spores (2.0-3.0 × 1.3-1.5 µm) were discovered upon examination of histological sections taken from Phoronis embryolabi Temereva, Chichvarkhin 2017 found inhabiting burrows of shrimps Nihonotrypeae japonica (Decapoda, Callianassidae) from the Sea of Japan, Russia. Ultrastructural examination of spores revealed one nucleus and a uniform polar filament of 7-11 coils. Representatives of the phylum Phoronida have never been recorded as hosts of microsporidia. Parasites developed in vasoperitoneal tissue and caused formation of multinucleate syncytia. Basing on unique host and fine morphology, we assign the novel finding to Microsporidium phoronidi n. sp. and place provisionally in the collective genus Microsporidium.

The microsporidium Nosema disstriae (Thomson 1959): Fine structure and phylogenetic position within the N. bombycis clade.

A microsporidium Nosema disstriae (Thomson) is a parasite of the forest tent caterpillar Malacasoma disstria (Lepidoptera: Lasiocampidae), a notable defoliator of deciduous trees in North America. The goal of this paper was to demonstrate the ultrastructure of N. disstriae and to determine the position of this microsporidium within the N. bombycis clade (NBC) using comparative morphology and multiple molecular phylogenetic markers: RPB1, LSU-, ITS- and SSU-rDNA. As a part of this goal, the revision of the described members of the NBC has been performed. The ultrastructure of proliferating stages and spores of N. disstriae were similar to previously described Nosema spp. parasitizing lepidopteran species. Meronts produced tubular-like structures on their surfaces and exhibited a tight association with host mitochondria. All stages were diplokaryotic and developed without interfacial envelopes. Disporoblastic sporogony produced typical Nosema-type spores with 9-12 polar filament coils. A vesicle with immature spores was once recognized on sections, concordant with the previous record of octosporous sporogony in the N. disstriae life cycle. Rarely, spores with thinner envelopes and large posterior vacuoles were seen in the midgut. Tracheae were most heavily infected. Midgut, surrounding muscles, haemocytes and fat body also contained microsporidia. SSUrRNA-inferred phylogenies were consistent with previously published articles and did not resolve the relation within the NBC clade. The RPB1-inferred trees and concatenated RPB1 and LSU-ITS-SSUrDNA-based trees demonstrated clustering of N. disstriae with N. antheraeae as early divergent species within the NBC.

Nannizziopsis guarroi infection in 2 Inland Bearded Dragons (Pogona vitticeps): clinical, cytologic, histologic, and ultrastructural aspects.

Chrysosporium-related infections have been increasingly reported in reptiles over the last 2 decades. In this report, we describe clinical, cytologic, histopathologic, and ultrastructural aspects of Chrysosporium-related infection in 2 Inland Bearded Dragons (Pogona vitticeps). Case 1 was presented for an enlarging raised lesion over the left eye and multiple additional masses over the dorsum. Case 2 was submitted to necropsy by the referring veterinarian for suspected yellow fungus disease. Impression smears of the nodules in case 1 revealed granulomatous to pyogranulomatous inflammation and many septate, variably long, 4-10 µm wide, often undulated hyphae, and very rare conidia. Postmortem impression smears of the superficial lesions of case 2 contained large numbers of solitary conidia and arthroconidia and low numbers of hyphae with similar morphology to case 1. Histopathology of the 2 cases revealed severe, multifocal, chronic, ulcerative, nodular pyogranulomatous dermatitis, with myriad intralesional septate hyphae, and arthroconidia. Fungal culture and molecular sequencing in both cases indicated infection with Nannizziopsis guarroi.

Establishing a New Species Encephalitozoon pogonae for the Microsporidian Parasite of Inland Bearded Dragon Pogona vitticeps Ahl 1927 (Reptilia, Squamata, Agamidae).

The microsporidium parasitizing Inland Bearded Dragons Pogona vitticeps, and developing primarily in macrophages within foci of granulomatous inflammation of different organs, is described as a new species Encephalitozoon pogonae. Establishing the new species was based on sequencing the ITS-SSUrDNA region of the ribosomal gene and consequent SSUrDNA-inferred phylogenetic analyses, as well as on comparison of pathogenesis, host specificity, and ultrastructure among Encephalitozoon species and isolates. The new species is closely related to E. lacertae and E. cuniculi. Analysis of the literature suggests that this microsporidium has been reported previously as an unidentified microsporidian species or isolate of E. cuniculi and may represent a common infection in bearded dragons. All stages of E. pogonae develop in parasitophorous vacuoles. Uninucleate spores on methanol-fixed smears measured 2.1 × 1.1 µm, range 1.7-2.6 × 0.9-1.7 µm; on ultrathin sections spores measured 0.8-1.1 × 1.8-2.2 µm. Ultrastructural study revealed 3-6 polar filament coils, a mushroom-shaped polar disk, and a polar sac embracing half of the volume occupied by the lamellar polaroplast. In activated spores, polar filament everted eccentrically. The overall morphology and intracellular development of E. pogonae were similar to other Encepahalitozoon spp. We also review the existing data on microsporidia infecting reptiles.

Morphology and phylogeny of Agmasoma penaei (Microsporidia) from the type host, Litopenaeus setiferus, and the type locality, Louisiana, USA.

Since June 2012, samples of wild caught white shrimp, Litopenaeus setiferus, from the Gulf of Mexico, Plaquemines and Jefferson Parishes (Louisiana, USA) with clinical signs of microsporidiosis have been delivered to the Louisiana Aquatic Diagnostic Laboratory for identification. Infection was limited predominantly to female gonads and was caused by a microsporidium producing roundish pansporoblasts with eight spores (3.6×2.1 µm) and an anisofilar (2-3+4-6) polar filament. These features allowed identification of the microsporidium as Agmasoma penaei Sprague, 1950. Agmasoma penaei is known as a microsporidium with world-wide distribution, causing devastating epizootic disease among wild and cultured shrimps. This paper provides molecular and morphological characterisation of A. penaei from the type host and type locality. Comparison of the novel ssrDNA sequence of A. penaei from Louisiana, USA with that of A. penaei from Thailand revealed 95% similarity, which suggests these geographical isolates are two different species. The A. penaei sequences did not show significant homology to any other examined taxon. Phylogenetic reconstructions using the ssrDNA and alpha- and beta-tubulin sequences supported its affiliation with the Clade IV Terresporidia sensu Vossbrink 2005, and its association with parasites of fresh and salt water crustaceans of the genera Artemia, Daphnia and Cyclops.

Emerging microsporidian infections in Russian HIV-infected patients.

Microsporidia were identified in stool specimens by histochemistry and PCR of 30 (18.9%) of 159 HIV-infected patients presenting to the S. P. Botkin Memorial Clinical Hospital of Infectious Diseases, St. Petersburg, Russia. The higher prevalence of Encephalitozoon intestinalis, in 21 (12.8%) patients, than of Enterocytozoon bieneusi, in 2 patients (1.2%), was unexpected. Encephalitozoon cuniculi was detected in three patients: one with strain I and two with strain II. Encephalitozoon hellem was detected in one patient, and two patients were identified as being infected by Microsporidium species. One patient was infected with both E. intestinalis and E. cuniculi. In two patients, the microsporidian species were not identifiable. No statistically significant differences in gender, age, and stage of AIDS were observed between the microsporidian-positive and -negative HIV-infected patients. HIV-infected patients diagnosed with microsporidian infection, however, were significantly more likely to exhibit ≤ 100 CD4(+) T cells/µl blood (20/30 patients [67%]; odds ratio [OR], 3.150; 95% confidence interval [CI(95)], 1.280 to 7.750; P = 0.0116) and weight loss of >10% of the baseline (19/30 patients [63%]; odds ratio, 2.995; CI(95), 1.100 to 8.158; P = 0.0352) than HIV-infected patients not diagnosed with microsporidian infection. In summary, this is the first report describing the diagnosis of microsporidian infection of HIV-infected patients in Russia and the first detection of E. cuniculi strain II in a human.

New microsporidia parasitizing bark lice (Insecta: Psocoptera).

Two species of bark lice, Xanthocaecilius sommermanae Mockford and Polypsocus corruptus Hagen, collected in a canopy Malaise trap placed in Great Smoky Mountains National Park as part of a survey of the park's fauna, were found to be infected with microsporidia. Diagnosis was originally based on light microscopy, and was confirmed by PCR amplification and electron microscopy. This is the first record of microsporidia infection in the insect order Psocoptera. Four morphological spore types corresponded to four original SSUrDNA sequences (Genbank accession no. FJ865221-24), suggesting infection with four microsporidia species. Two of those species were examined by electron microscopy. We describe here one new genus and two new species based on morphological and sequence data: Antonospora psocopterae sp. n. with elongated diplokaryotic spores, 4.4+/-0.05 x 1.9+/-0.03 microm and Mockfordia xanthocaeciliae gen. n. sp. n. with ovocylindrical monokaryotic spores, 2.5+/-0.10 x 1.4+/-0.02 microm. A. psocopterae displayed high sequence (95%) and structural similarity with Antonospora scoticae, fell within a well supported dichotomy with A. scoticae inside the Antonospora-Paranosema clade in phylogenetic analyses by NJ, PS and ML. M. xanthocaeciliae did not exhibit much sequence or structural similarity with any of known microsporidia species, except Encephalitozoon spp. M. xanthocaeciliae fell within one clade with Encephalitozoon spp. in phylogenies and shared with encephalitozoons structural resemblance and about 80% of SSUrDNA sequence identity. The other two species were not described and provisionally were placed to the collective genus Microsporidium as Microsporidium sp. 1 and Microsporidium sp. 4 from bark lice because of insufficient morphological data. The finding that samples fixed and stored for months in propylene glycol ("antifreeze") are good enough for DNA sequence analysis and can be used for morphological analyses (if no better fixation alternatives are available), is promising for future surveys for microsporidia.

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA).

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.

Morphology and taxonomy of the microsporidium Liebermannia covasacrae n. sp. from the grasshopper Covasacris pallidinota (Orthoptera, Acrididae).

During a survey for grasshopper pathogens in Argentina in 2005-2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6+/-0.28 x 1.4+/-0.12 microm (range 2.2-3.4 x 1.1-1.7 microm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3-5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.