Identification and Phylogenetic Relationships of Microsporidia by Riboprinting

ABSTRACT. The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish ( Chloroscombrus chrysurus ) is related although not identical to Microgemma oviodea , a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei , are not related to the fish isolates.     

Comparative Study of Microsporidian Spores by Flow Cytometric Analysis

ABSTRACT. Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii ) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.     

Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

Redescription of Microsporidium takedai (Awakura, 1974) as Kabatana takedai (Awakura, 1974) comb. n

Ultrastructural study of the microsporidian Microsporidium takedai from the muscles of masu salmon Oncorhynchus masou proved that this species can be assigned to the genus Kabatana Lom, Dyková and Tonguthai, 2000. The parasites develop within disintegrated sarcoplasm without any delimiting boundary or cyst. Cylindrical multinucleate meronts proliferate by serial constrictions into uninucleate stages which repeat the process. Eventually, the uninucleate stages transform into uninucleate sporonts, which divide once to produce sporoblasts, thus functioning as sporoblast mother cells. Spores, with a subterminally located anchoring disc and 3 to 4 turns of the polar tube coil, average 3.3 by 1.9 microm in size. The exospore is divided into small fields; the endospore frequently makes small invaginations into the spore inside. Phylogenetic analysis using SSU rDNA sequence consistently placed Kabatana takedai in a group consisting of Microgemma sp., Spraguea lophii and Glugea americanus. The K. takedai could easily be separated from the other species in the same group by 2 inserts in the SSU rDNA sequence.     

The ultrastructure of spores (Protozoa: Microsporida) from Lophius americanus, the angler fish

Spinal and cranial ganglia of American angler fish, Lophius americanus, are often infected with microsporidia. This protozoon elicits the formation of large, spore-filled, hypertrophied host cells, cysts. Previous reports of microsporidia in European lophiids identify the parasite as Spraguea lophii, a genus which has recently been shown to be dimorphic. The spores from L. americanus are monomorphic (2.8 X 1.5 micron) and uninucleate. Each spore contains a polar tube that forms six to nine coils. Spraguea lophii differs from the microsporidium described in L. americanus in several ways. Spraguea lophii has two spore types: a large spore (4.0 X 1.25 micron) containing a diplokaryon and three to four polar tube coils and a smaller uninucleate spore (3.5 X 1.5 micron) with five to six polar tube coils. Because of these major differences, the microsporidium from L. americanus is removed from the genus Spraguea and returned to its original genus, Glugea, as a new species, G. americanus n. sp. Other ultrastructural characteristics of G. americanus are included: the posterior vacuole encloses two distinct membranous structures; one is tubular and resembles a "glomerular tuft" and the second is lamellar and composed of concentric membrane whorls, additionally, the straight or manubroid portion of the polar tube proceeds beyond the posterior vacuole before it turns anteriorly and begins to coil.     

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.     

Experimental infection of American winter flounder (Pseudopleuronectes americanus) with Glugea stephani (Microsporida)

Young-of-the-year Pseudopleuronectes americanus were captured in Sandy Hook Bay, New Jersey and maintained in aquaria at 16–19° C for 115 days in 1982 and 127 days in 1983. Half of them were experimentally exposed to Glugea stephani and the others were sham exposed. During the course of these experiments fish from both groups died as a result of G. stephani infection, demonstrating Glwgea -induced mortality in this species. Infections were found in both control and experimentally exposed fish. Mortality of Glwgea -infected fish in the exposed group was 63% while that in the controls was 28.5%.     

The occurrence of Glugea stephani (Hagenmuller, 1899) in American winter flounder, Pseudopleuronects americanus (Walbaum) from the New York-New Jersey lower bay complex

American winter flounder, Pseudopleuronectes americanus , Collected in the New York-New Jersey bay complex, contained cysts in the intestinal tract which were subsequently identified as the microsporidan Glugea stephani . These fish represent 13 collections taken at approximately monthly intervals over a one and a half year period. The winter flounder varied in size from 8 to 33 cm and consequently represented a fish population of diverse age. During this study, a total of 1840 apparently healthy fish were dissected and examined for the presence of Glugea cysts , with an overall parasite infection rate of 6.63%. There was no correlation between size of the fish and percentage of infection. This percentage represents the mean carrying rate of this parasite in the fish population, since these are the fish that have survived the initial infection. This report extends the reported host range to the New York-New Jersey area. As a further aid in the characterization of G. stephani , we have included ultrastructural details of the spore morphology.     

Infection of plaice Pleuronectes platessa L. with Glugea (Nosema) stephani (Hagenmüller 1899) (Protozoa: Microsporidia) in a fish farm and under experimental conditions

One tank of plaice in the W.F.A. Fish Cultivation Unit at Hunterston, known to habour an infection with the microsporidian Glugea stephani , was sampled over a 2 year period and showed a mean level of infection of 49.4 % with an apparent trend towards a decrease. Experimental oral transmission was achieved, without the use of intermediate or carrier hosts, and the dependence of G. stephani on high temperatures was demonstrated. Most fish were able to tolerate the infection, but some mortalities in young fish were attributed to the parasite, particularly in the 2 months following the experimental infection. The parasite in the fish farm is believed to have been introduced with plaice stocks imported from more southern waters.     

Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

微孢子虫一新种的描述及分子鉴定

随着集约化、高密度养殖业的发展, 传播性寄生虫病给水产业造成了严重的损失, 微孢子虫是养殖鱼类的主要病原生物之一, 迄今, 已报道的鱼类寄生微孢子虫多达100 余种。       

Parasitology of the English Sole, Parophrys vetulus Girard in Oregon, U.S.A.

A total of 877 juvenile English sole ( Parophrys vetulus Girard) from the Yaquina Bay estuary and742 juvenileandadultsole from the Pacific Ocean off Oregon were examined forparasites. Fifteen species of parasites were found in juvenile English sole on the estuarine nursery ground. Differences in the prevalence and intensity of parasite infection between size classes of juvenile sole and between sole occupying the upper and lower estuary were determined. An additional 14 parasite species were found in offshore English sole, bringing the total observed in all fish to 29 species. Parasites acquired only in the estuary included the microsporidan Glugea stephani , the acanthocephalan Echinorhynchus lageniformis , and the nematode Philometra americana . Those acquired only in offshore areas included the trematodes Otodistomum veliporum and Zoogonus dextrocirrus , the leech Oceanobdella sp. and three species of copepods. An attempt to use parasite data to indicate the presence of distinct English sole stocks along the Oregon coast was inconclusive.     

The microsporidian spore invasion tube. IV. Discharge activation begins with pH-triggered Ca2+ influx

The microsporidian spore extrusion apparatus activates with a calcium influx from Spraguea lophii spore wall/plasma membrane; this influx requires preconditioning with an extrasporular shift in medium pH to the alkaline in the presence of the polyanions mucin or polyglutamate. Undischarged S. lophii spores display calcium bound to the wall/plasma membrane with a characteristic calcium-chlorotetracycline fluorescence; this fluorescence declines significantly during spore discharge. S. lophii spores do not discharge when spore wall/plasma membrane calcium is removed with EGTA. Extrasporular mucin or polyglutamate and a pH shift to the alkaline appear to be necessary preconditions for the triggering of the influx of spore wall/plasma membrane-bound 45Ca2+. Ionophore A-23187 also effectively activates spore discharge without other extrasporular polyanions. Micromolar concentrations of the calcium antagonists lanthanum or verapamil prevent spore discharge, and micromolar concentrations of calmodulin inhibitors chlorpromazine and trifluroperazine prevent spore discharge. Calmodulin, visualized with a calmodulin antibody and a peroxidase conjugate, is localized particularly on the plasma membrane and the polaroplast membranes of the extrusion apparatus.     

Shoaling behaviour of sticklebacks infected with the microsporidian parasite, <Emphasis Type="Italic">Glugea anomala</Emphasis>

We compared the shoaling behaviour of three-spined sticklebacks, Gasterosteus aculeatus, infected with the microsporidian, Glugea anomala, to that of non-infected conspecifics. Infected fish lost significantly more weight than non-infected fish during a period of food deprivation, suggesting a metabolic cost to parasitism. In binary shoal choice tests, non-infected test fish showed an association preference for a shoal of non-infected over a shoal of infected conspecifics; infected test fish displayed no preference. Infected fish, however, showed a higher overall tendency to shoal than non-parasitised fish. Furthermore, infected fish occupied front positions within a mixed school. We consider the behavioural differences between infected and uninfected fish in the context of their potential benefits to the fish hosts and the parasites.     

Effect, distribution, and prevalence of Glugea stephani (Microspora) in winter flounder (Pleuronectes americanus) living near two pulp and paper mills in Newfoundland

A study was conducted to determine the effects, geographical distribution, and prevalence of a microsporan parasite, Glugea stephani, in winter flounder (Pleuronectes americanus) in Newfoundland. Fish were captured by SCUBA divers in several coastal areas, including 2 embayments where pulp and paper mill effluent was discharged, as well as a number of pristine sites. Fish health was assessed by comparing histological profiles, condition factors (K), organosomatic indices and blood values between infected and uninfected samples. Multifocal xenomas of G. stephani were observed in several organs of fish taken near contaminated sites, whereas infected samples captured at a pristine site harbored the cysts only in the wall of the digestive tract. Proliferative inflammation, granuloma formation, and focal necrosis were associated with the infection primarily in the liver and kidney. Condition factors and blood values were lower and ovarian development inhibited or delayed in infected flounder. The multifocal infection occurred only in flounder in 2 embayments in western Newfoundland where pulp and paper mill effluent was discharged. Prevalence varied seasonally, with a peak in autumn and a low in spring. It is likely that the multifocal infection was associated with immunodepression after exposure to the contaminant.     

Seasonal prevalence of the microsporidan, Glugea stephani (Hagenmuller), in winter flounder, Pseudopleuronectes americanus (Walbaum), from the New York-New Jersey Lower Bay Complex

During a 32 month period 26 monthly collections of winter flounder were conducted from various locations in the New York-New Jersey Lower Bay Complex. A total of 3125 flounder were captured and examined for the presence of the microsporidan, Glugea stephani. Of the total number of fish collected, 260 (8.32%) were infected with the protozoan. At least one G. stephani infected flounder was captured and identified each month, which indicates that the infection is present on a year-round basis. The monthly infection prevalence ranged from 0.63 to 25%. Increased parasitism corresponded with elevated water temperatures. Fish size was not a statistically significant factor for infection.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

The immunomodulating effect of the microsporidan Glugea stephani on the humoral response and immunoglobulin levels in winter flounder, Pseudopleuronectes americanus

A quantitative study of serum immunoglobulin levels and haemagglutination titres has shown that an injection of the microsporidan Glugea stephani caused a decrease in the humoral response of the winter flounder. These examples demonstrate the effect of temperature on the decrease in total IgM levels and haemagglutination titres in winter flounder simultaneously injected with either G. stephani or horse red blood cells.
Transfer of serum from fish injected with spores 3 weeks earlier may also implicate soluble serum factors, such as prostaglandins or leukotrienes, causing the observed decrease in serum IgM levels.     

Validation of a quantitative PCR diagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas

Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 x 10(1) to 7.91 x 10(6) copies microg(-1) host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 x 10(1) to 8.62 x 102 copies microg(-1) host DNA.     

Intracellular Development of the Microsporidan Glugea anomala Moniez in Hypertrophying Migratory Cells of the Fish Gasterosteus aculeatus L., an Example of the Formation of "Xenoma" Tumors

SYNOPSIS. The conclusion drawn in 1921 that the large nuclei in the cytoplasmic cortex of Glugea cysts are not vegetative nuclei of the microsporidan but nuclei of the hypertrophied host cell was based on the discovery of early developmental stages in the mesenchyme of stickleback larvae experimentally fed Glugea spores. This observation had been made on serial sections from experiments done in 1912. The intracellular development of the microsporidan could be followed up in this material only thru the 1st stages of schizogony. Renewed infection experiments, done still in 1921 on a much broader basis, have fully confirmed the previous findings, as briefly stated in 1922. On this material, the intracellular development of G. anomala has been followed up in recent years from uninucleate host cells 7 μ in diameter, interpreted as wandering cells in the mesenchyme, until they became macroscopic multinucleate cysts, in which schizogony and sporogony of the microsporidan produced innumerable vegetative stages and spores of Glugea. The details of the developmental processes are described in the present paper.
The multinucleate host cell and the intracellular parasites together form one of the symbiotic complexes for which the term "xenom" or "xenoma" has been used by me since 1949. By a sequence of amitotic nuclear divisions, the uninucleate host cell in the Glugea xenomas of Gasterosteus becomes plurinucleate in contrast to the usual structure of other xenomas of fish.
Already in 1921, I thought that the host cell in the Glugea xenomas may have phagocytic properties. The observation of accumulation of granules from pigment cells in some of the Glugea xenomas has now verified this supposition.