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In our experiment, 3 species-specific primer pairs cultivated in cell lines were used: Encephalitozoon cuniculi-specific primer pairs (ECUNF and ECUNR), Encephalitozoon hellem-specific primer pairs (EHELF and EHELR), and Encephalitozoon intestinalis-specific primer pairs (SINTF and SINTR). The PCR products were estimated to be 550 bp in E. cuniculi, 547 bp in E. hellem and 545 bp in E. intestinalis respectively, which can prove the precision and reliability of this method in the species identification of the genus Encephalitozoon. All 3 primer pairs were species-specific and none of them amplified gene sequences from other Encephalitozoon spp.
The development of an immune response of immunocompetent BALB/c and C57BL/6 mice with different levels of resistance to microsporidial infection and immunodeficient SCID mice was analysed in relation to the route of infection with Encephalitozoon intestinalis. Cell-mediated immune responses were characterised by measurement of IFN-γ and quantification of CD4+, CD8+ and NK cells. Intraperitoneal (i.p.) infection induced higher levels of IFN-γ in ex vivo cultures of peritoneal exudate cells or splenocytes than peroral (p.o.) infection. Numbers of CD4+ and CD8+ T lymphocytes in the spleen increased markedly at later time points (14 and 21 DPI) after i.p. infection of BALB/c mice, whereas in C57BL/6 mice the highest increase was after p.o. infection. Considerable IFN-γ production in ex vivo cultures of Peyer's patch lymphocytes of both mouse strains was observed after p.o. infection. Intraperitoneal infection of SCID mice induced marked influx of inflammatory cells into the peritoneum with NK cells. The results contribute to the development of an animal model of microsporidiosis caused by E. intestinalis.
Encephalitozoon intestinalis is one of the most common microsporidial species found in humans worldwide but it has rarely been identified in animals. The presence of this pathogen has been detected in a few species of domestic, captive and wild mammals as well as in three species of birds. The aim of the present study was to examine fecal samples obtained from mammals housed in the Poznan Zoological Garden, Poland, for the presence of potentially human-infectious microsporidia. A total of 339 fresh fecal samples collected from 75 species of mammals belonging to 27 families and 8 orders were examined for the presence of microsporidian spores. Microsporidian spores were identified in 3 out of 339 (0.9%) examined fecal samples. All samples identified as positive by chromotrope 2R and calcofluor white M2R were also positive by the FISH assay. Using multiplex FISH in all 3 fecal samples, only spores of E. intestinalis were identified in 2 out of 14 Ring-tailed lemurs (Lemur catta) and in one out of 17 Red ruffed lemurs (Varecia variegata rubra). To our knowledge this is the first diagnosis of E. intestinalis in Ring-tailed and Red ruffed lemurs. It should be mentioned that both lemur species are listed by the IUCN Red List of Threatened Species. Although the lemurs were asymptomatically infected, the possibility of widespread infection or death of these animals remains in the event of an elevated stress or a decrease in their immunological functions.
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