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Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

In Encephalitozoon cuniculi like in other microsporidia, the primary transcript for SSU and LSU rRNAs includes only one internal transcribed spacer (ITS1) which separates SSU rRNA from the 5.8S region associated with LSU rRNA. The extraction of total RNA from E. cuniculi-infected MRC5 cells using a hot phenol/chloroform procedure enabled us to perform primer extension and S1 nuclease protection experiments in the aim of identifying rRNA maturation sites. Our data support a simple processing (four cleavage sites) with elimination of only nine nucleotides between SSU and LSU rRNA regions. Most of the presumed ITS1 sequence characterized by strain-dependent polymorphism therefore remains linked to SSU rRNA 3' end. A new secondary structure for the sixth domain of E. cuniculi LSU rRNA is proposed following the identification of its 3' terminus.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78 bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvalI digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.

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 (22:6omega3) 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 (18:1omega9) 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.

The microsporidian Encephalitozoon cuniculi can infect numerous mammals, including man. Three strains of E. cuniculi have been identified so far, the major marker being the number of a tetranucleotide repeats in the rDNA internal transcribed spacer. We investigated diversity at the chromosomal level through the electrophoretic karyotypes obtained from 15 E. cuniculi isolates from 5 different host species. All preparations provided patterns with 9-12 bands within a narrow molecular size range. Six karyotype forms were distinguished, involving subdivision of strain I into 3 types (A, B, C) and strain II into 2 types (D, E). The types A, B and C were mainly associated with isolates from rabbits of different geographical origins. The types D, E and F were characterized by a reduced chromosome size range, 2 of these appearing specific to a carnivorous host species (D in dog and F in blue fox). Hybridization experiments showed that all E. cuniculi isolates possess 11 chromosomes, with a size polymorphism entailing occasional electrophoretic comigration of heterologous chromosomes and differential migration of homologous ones. DNA rearrangements should occur during mitosis and the hypothesis of diploidy for the basic state of E. cuniculi seems likely.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

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.

With the use of Weber's modified trichrome and Uvitex 2B techniques, spores of microsporidia were detected in the stools of four travelers presenting clinically with chronic diarrhea. The general health of these patients was not impaired, and human immunodeficiency virus screening was negative. Immune evaluation, including the study of lymphocytic subpopulations, assay of serum immunoglobulins, and an intradermal multitest, showed normal results. Molecular identification of microsporidian species was based on the PCR amplification of a small-subunit rRNA sequence followed by HinfI endonuclease restriction. Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of E. intestinalis in immunocompetent individuals remains to be demonstrated.

Belonging to a large group of parasitic amitochondrial protozoans (Microspora), Encephalitozoon cuniculi infects humans and other mammals. Because of its medical importance and small genome size (2.9 Mbp), we are systematically sequencing its smallest (217 kbp) chromosome. The shotgun cloning strategy now has produced the sequence of randomly dispersed contigs representing more than 180 kbp of this chromosome. The present report describes analysis of the 4.3 kbp contig, which includes the complete coding regions of dihydrofolate reductase (DHFR), thymidylate synthase (TS), and serine hydroxymethyl transferase (SHMT) genes and the partial coding region of an aminopeptidase (AP) gene. In contrast to the other reported protozoan genes, DHFR and TS are encoded by two different open reading frames (ORFs). The SHMT gene is the first one identified in a protozoan and corresponds to the cytosolic form of the enzyme. No introns were detected, and the intergenic noncoding regions do not exceed 50 bp. The mean GC content is close to 60%, and there is a G or C third-base codon bias. Transcription and translation initiation signals also are analyzed, and a model for the mRNA-ssu rRNA interactions is proposed.

Pulsed field gel electrophoresis (PFGE) was used to separate chromosome-sized DNA from two species of microsporidia of fishes. The molecular karyotype of Glugea atherinae exhibits 16 DNA bands from 420 to 2,700 kb, and that of Spraguea lophii 12 bands from 230 to 980 kb. Until now they represent respectively the largest and the smallest genomes visualized for microsporidia: 19.5 Mb for G. atherinae and 6.2 Mb for S. lophii (the smallest nuclear genome in eukaryotic organism). We have analysed separately five strains of G. atherinae (individual cysts), with this technique. The electrophoretic spectra are the same for these strains, except for the absence of the 2,380-kb band in one case. Therefore, the karyotype seems to be rather well conserved for this species.

The glycemia of 4th instar larvae of Aedes aegypti (Diptera Culicidae) was determined over a 30 min to 36 hrs period following their immersion in water containing 0.00-0.01-0.02-0.1-1.0 mg per liter of a 12,000 I. U. per mg preparation of the delta-endotoxin of Bacillus thuringiensis israelensis. Seven major carbohydrate fractions were separated in the haemolymph, among which glycogen, trehalose and glucose were identified; the most concentrated of the 4 remaining unknown fractions, exhibits a similar chromatographic behavior but different chemical properties than glucuronic acid. The glycogen fraction shows two large amplitude peaks at 1 hr 30 and 6 hrs with 0.1 mg/l, whereas with the 2 lower doses (0.01 and 0.02 mg/l) a first drop from 30 min to 1 hr is followed by a peaking at 12 hrs. The haemolymph trehalose concentrations of intoxicated larvae increase relatively to controls 2 hrs after incubation with the lower dose; by contrast, they decrease with the 2 higher doses. The glucosemia increases in all cases, but more markedly with the higher dose as soon as 1 hr following the treatment. A positive correlation was found over the whole treatment duration between the glucosemia nd the trehalosemia of control larvae. The regression slope of this relationship decreases with increasing toxin doses and turns to negative values with 0.1 and 1.0 mg/l. These observations suggest the hypothesis that a uncontrolled increase of trehalase activity develops according to the increasing stages of intoxication. This clearly differenciates the effects of the B. T. i toxin from those of parasites, such as Fungi, Sporozoans or Nematodes in Mosquitoes.

A microsporidan parasite, Ormieresia carcini gen. n., sp. n., was found in the crab, Carcinus mediterraneus Czerniavsky. Its development and fine structure are the subject are the subject of the present study. The life cycle begins with a schizont surrounded by a unit membrane and containing a diplokaryon. The entire process of sporogony takes place in the host musculature. The sporogonic stages are enclosed in the pansporoblastic membrane. In each pansporoblast, sporogony gives rise to 8 sporoblasts; the octonucleate sporogonial plasmodium is lacking. In the course of schizogonic and sporogonic divisions, each kinetic center consists of 2 plaques, one located within and the other outside the nuclear envelope. The dividing sporonts and sporoblasts sevrets "metabolic" substances (granules, tubules) which are depostied in the pansporoblast. The uninucleate spore is long and cylindrical, measuring 19.1 X 2.4 micronm. A rectilinear manubrium traverses the spore. Its posterior end attenuates abruptly into a polar filament with 4 or 5 coils; its anterior end is attached to the polar cap, which is compressed by a double polar ring. The anterior part of the manubrium is surrounded by a polaroplast consisting of a "spongy" (vesicular) and a lamellar zone.

A cytochemical study of Microsporidia in Carcinus mediterraneus points out the following data: acid or very acid intrasporoblastic mucopolysaccharids, are found in Thelohamia maenadis which react as sulfomucins in opposition to external asulfated mucosubstances rich in electronegative groups. In Nosema pulvis, the mucopolysaccharid compound is nearly equivalent of that of Nosema orthocladii. Both species are also characterized by the abundance of sulfured proteins in their spore walls and the lack of stock substances.