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Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC: V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)-1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.

Seven isolates of Encephalitozoon hellem from human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating that E. hellem has a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships among E. hellem strains.