Occurrence of microsporidians Glugea hertwigi and Pleistophora ladogensis, in smelt Osmerus eperlanus from two German rivers, North Sea coast.

Monthly samples of smelt Osmerus eperlanus (Linnaeus, 1758) were collected from July 1985 to May 1986, in the river Elbe (Germany), and examined for infections with microsporidians. Two microsporidians were found: Glugea hertwigi Weissenberg, 1911, infecting the digestive tract and Pleistophora ladogenis Voronin, 1978, infecting the skeletal musculature. G. hertwigi infection led to the formation of xenomas, whereas P. ladogensis was characterized by diffuse infections, with the production of macroscopic visible thread-like or oval-shaped infection foci. Development of G. hertwigi in the host cells showed characteristics typical of the genus Glugea. The ultrastructural development of P. ladogensis showed features typical of the genus Pleistophora, without evidence of the production of 2 types of spores. Host reaction consisted of inflammatory tissue surrounding some of the infection foci as well as phagocytosis of spores. G. hertwigi was only found in juvenile smelt (<10 cm in length), whereas P. ladogensis infected smelts from 6 to 26 cm in length. Prevalence increased with fish length to a maximum value of 9.6%. Seasonal fluctuations in prevalence of infection were also found, with the lowest value in the winter months (2.5% in January 1986) and the highest in summer (11.8% in July 1985). The differences in prevalence of infection with fish length and date of sampling were significant. Additionally, samples of smelt caught in April 1986 from the rivers Eider and Ems revealed infections with P. ladogensis in the first river system only.

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.

Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis).

A captive garter snake (Thamnophis sirtalis) showed marked circumferential thickening of the body wall due to a chronic fibrous to necrotizing inflammatory reaction extending from the serosa to the skin. The lesions were associated with the presence of intra- and extracellular microsporidian organisms. With ultrastructural and molecular analyses the microsporidia were identified as Heterosporis anguillarum. This organism causes a morphologically similar disease in the Japanese eel (Anguilla japonica), but this is the first time it has been identified in an infected reptile.

Ultrastructure and molecular phylogenetics of a new isolate of Pleistophora pagri sp. nov. (Microsporidia, Pleistophoridae) from Pagrus pagrus in Egypt.

The spore morphology and molecular systematic of a new microsporidian which was isolated from the common sea bream Pagrus pagrus (F: Sparidae Linnaeus, 1758) from the Red Sea, Egypt have been studied. Fifty-six out of 300 (18.7%) of this fish were infected with microsporidian parasites. The infection was appeared as whitish, ellipsoid, round, or elongated nodules embedded in the epithelial lining of the peritoneum and also in the intestinal epithelium. Light microscopic study revealed that nodules were encapsulated by a fibrous layer encircling numerous mature spores measuring 1.7 ± 0.6 (1.5-2.7 µm) × 1.5 ± 0.3 µm (1.2-1.8 µm) in size. Ultrastructure of spores was characteristic for the genus Pleistophora: dimorphic, uninucleate spores (each spore possesses three to five polar filament coils) and a posterior vacuole. Also, the early recognizable stages of the parasite within nodules include uninucleated, binucleated, and multinucleated meronts followed by detachment of the plasmalemma of the sporont producing sporoblasts which mature to spores that consist of a spore coat and spore contents. Also, we analyzed the small subunit ribosomal gene (SSUrDNA) using PCR and sequencing specimens from the marine populations of P. pagrus fish from the Red Sea. From blast searches, sequence analysis, and phylogenetic analysis, we did not find corresponding GenBank entries to our species. Comparison of the nucleotide sequences showed that the sequence of our microsporidium was most similar to five Pleistophora species with degrees of identity (>91.5%). It was most similar (97.8% identity) to that of Pleistophora hyphessobryconis (account no. GU126672) differing in 19 nucleotide positions and with lower divergence value, Pleistophora ovariae (96.2% identity, account no. AJ252955), Pleistophora hippoglossoideos (91.9% identity, account no. AJ252953), Pleistophora mulleri (91.9% identity, account no. EF119339), and Pleistophora typicalis (91.9% identity, account no. AJ252956). So, they likely represent new species named Pleistophora pagri sp. n. with accession number JF797622 and a GC content of 53%.

Ultrastructure, development, and molecular phylogeny of Pleistophora hyphessobryconis, a broad host microsporidian parasite of Puntius tetrazona.

A potentially fatal microsporidial infection targeting the skeletal muscles of the tiger barb Puntius tetrazona was described. Ultrastructural and molecular analyses of infected tissues confirmed that the causative parasite was Pleistophora hyphessobryconis. Compared to P. hyphessobryconis observed in other hosts, those infecting tiger barb demonstrated differences in ultrastructure that may be related to host adaptation. Phylogenetic analysis revealed that classifications based on different methods of analysis (molecular, morphologic, or developmental) do not always coincide, and suggesting that the genetic relationships between Pleistophora and Ovipleistophora may need to be redefined. Transparent mutants of tiger barb can be artificially infected by P. hyphessobryconis, and the dynamic process and spatial distribution of P. hyphessobryconis infection can be observed in real time. These transparent fish mutants are a valuable model to study microsporidial infection in vivo.

Morphological and ultrastructural description of Pleistophora dammami sp. n. infecting the intestinal wall of Saurida undosquamis from the Arabian Gulf, Saudi Arabia.

Pleistophora dammami sp. n. is described from Saurida undosquamis from the Arabian Gulf in Saudi Arabia. Infection appeared as whitish cysts in the intestinal wall. Cysts ranged in size from 1 to 4 mm. The prevalence of the infection across both fish sexes was 17.5% (24/420). Two kinds of spores were recognized, microspores and macrospores, and each were ovoid in shape. The microspores measured ~2.5 × 2.0 µm in size, while the macrospores measured ~6.0 × 3.0 µm. Ultrastructurally, the parasite did not form xenoma but it formed cysts surrounded by thick cyst wall. All stages of development as meronts, sporonts, sporoblast and spores occurred in the cytoplasm of the host cells within sporophorous vesicles. The stages of development occurred asynchronously and thus all stages were randomly distributed within the cysts. Meronts were elliptical and multinucleated, with unpaired nuclei which constantly divided giving rise to new sporonts. During the transition to sporonts, the border of the meronts increased in thickness to form dense discontinuous cell coat. Later, the sporont divided into sporoblast cells which gradually differentiated the typical organelles of the spores. In mature spores, the polar filament was arranged in 20-24 coils in two rows either side of the posterior vacuole. All ultrastructural and morphological criteria indicate that the described species belongs to the genus Pleistophora.

Morphological and molecular biological characterization of Pleistophora aegyptiaca sp. nov. infecting the Red Sea fish Saurida tumbil.

One hundred three out of 225 (45.8%) of the Red Sea fish Saurida tumbil were infected with microsporidian parasites. The infection was recorded as tumor-like masses (whitish macroscopic cysts) or xenomas often up to 2 cm in diameter and embedded in the peritoneal cavity. Generally, the infection was increased during winter 63.8% (86 out of 135) and fall to 18.9% (17 out of 90) in summer. Light microscopic study revealed that xenomas were encapsulated by a fibrous layer encircling numerous sporophorous vesicles filled with mature spores measuring 1.7 ± 0.6 (1.5-2.7 µm) × 1.5 ± 0.3 µm (1.2-1.8 µm) in size. Ultrastructural microscopic study showed the presence of smooth membranes of the sarcoplasmic reticulum forming a thick, amorphous coat surrounding various developmental stages of the parasite. The various recognizable stages of the parasite were uninuclear, binucleated, and multinucleated meronts followed by detachment of the plasmalemma of the sporont from the sporophorous vesicle producing sporoblasts. Mature spores consist of a spore coat and spore contents. The spore contents consist of the uninucleated sporoplasm and a posterior vacuole located at the posterior end. The polar tube consists of a straight shaft and a coiled region (26-32 coils) arranged in many rows along the inside periphery of the spore. The polaroplast consisted of an anterior region of closely and loosely packed membranes. Molecular analysis based on the small subunit rDNA gene was performed to determine the phylogenetic position of the present species. The percentage identity between this species and a range of other microsporidia predominantly from aquatic hosts demonstrated a high degree of similarity (>92%) with eight Pleistophora species. Comparison of the nucleotide sequences and divergence showed that the sequence of the present microsporidium was most similar to that of Pleistophora anguillarum (99.8% identity) differing in 13 nucleotide positions. So, the present species was recorded and phylogenetically positioned as a new species of Pleistophora.

Detection and species identification of microsporidial infections using SYBR Green real-time PCR.

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)(-1), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

Ultrastructure, development, and host-parasite relationship of a new species of the genus Pleistophora--a microsporidian parasite of the marine fish Epinephelus chlorostignei.

The life cycle of a new microsporidian of the genus Pleistophora is described. This parasite infects the epithelial cells of the gut and the peritoneal cavity of the Red Sea fish, Epinephelus chlorostignei. All stages develop within a special structure, the sporophorocyst, which is covered by a thick dense wall. This wall grows along with the growth of the parasites inside. Meronts are uni- to binucleate, which divide and constantly give rise to sporonts. During transition to sporonts, the cell border of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually, a uniform thick sporont wall is formed; then, the sporont cells detach themselves from the wall (future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores, and consists of two layers. Mature spores measure about 2.0 x 1.8 microm. They possess a polar filament with 20-28 coils, a posterior vacuole, and a polaroplast made up of an outer part of dense and closely spaced lamellae encircling an inner part of widely spaced lamellae. All morphological and ultrastructural features indicate that the described microsporidian parasite belongs to the genus Pleistophora.