Microsporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi is probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8 E. cuniculi isolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54-59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54-58 kDa as well as 28-40 kDa by Western blot immunodetection using murine antisera raised against E. cuniculi, Encephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the different E. cuniculi isolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses. Fok I digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5'-GTTT-3' was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3 E. cuniculi strains which may become important in the epidemiology of human E. cuniculi infections. Furthermore, as additional E. cuniculi isolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.

Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK-13, MDCK, HT-29, Caco-2, Vero, and I047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane-bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction-based methods with restriction endonuclease digestion, double-stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 l of stool to detect one microsporidian after viewing 50 fields at a final magnification of ؋1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEMnegative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.

Microsporidiosis with concurrent megabacteriosis in budgerigar (Melopsittacus undulatus) chicks contributed to significant economic floss in a commercial pet bird aviary in Mississippi. Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied. Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No. 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3). The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species. The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon hellem, an organism that has only been identified in humans. Encephalitozoon hellem causes keratoconjunctivitis and respiratory infections in humans with acquired immunodeficiency syndrome. This report presents the first confirmed case of microsporidiosis in budgerigars. The finding of E. hellem in pet birds may be important in elucidating the epidemiology of human infections with this organism.