Occurrence of microsporidia as emerging pathogens in Slovak Roma children and their impact on public health.

INTRODUCTION AND OBJECTIVE: Microsporidia are identified as ubiquitous organisms of almost every animal group and are now recognized as emerging opportunistic pathogens of human. The risk factors include immunodeficiency, lack of sanitation, and exposure to contaminated water and infected animals. In Slovakia, the places with an increased risk of infection due to the presence of risk factors and routes of transmission are represented by Roma settlements. Therefore, the aim of this work was to study the occurrence of Encephalitozoon spp. and E. bieneusi in children living in Roma settlements. MATERIALS AND METHODS: Stool samples were examined of 72 clinically healthy children coming from a group of the non-integrated Roma minority for the presence of microsporidia Encephalitozoon spp. and E. bieneusi. Microsporidian spores were detected by standard Rylux D, staining and by PCR and DNA sequencing. RESULTS: Of the total number of 72 stool smears examined, 22 were positive, which represented 30.6%. By the Real Time PCR, E. bieneusi was detected in 3 samples (4.2 %) and E. cuniculi in 19 samples (26.4 %). By comparing the sequences with sequences in the GenBank, E. cuniculi genotype I (Accession No. AJ005581.1) and E. bieneusi genotype A (Accession No. AF101197.1). CONCLUSIONS: Microsporidia, as newly emerging pathogens of humans and animals, are characterised by the production of spores which are environmentally resistant. Diseases caused by them have a cosmopolitan occurrence. Although E. bieneusi and E. cuniculi belong to the most frequently diagnosed species of microsporidia in humans, in Slovakia, this is the first confirmed evidence of E. bieneusi genotype A, as well as E. cuniculi genotype I in humans by the molecular method.

Molecular identification and genotyping of Microsporidia in selected hosts.

The work is described by microscopic analysis, the serological analysis (IFAT) and the molecular analysis of isolates from clinical samples (blood, faeces and urine) from ten domestic rabbits (Oryctolagus cuniculus), breed Malický, four New Zealand domestic rabbits, 11 sows of breed Slo0076akian Improved White and 15 clinically healthy laboratory BALB/c mice. The aim of the study was to validate the suitability of species-unspecific primer pairs 530F and 580R for genotype determination of the Microsporidia strain and species-specific primer pairs ECUNF and ECUNR, SINTF and SINTR and EBIER1 and EBIEF1 for the determination of E ncephalitozoon cuniculi, Encephalitozoon intestinalis and Enterocytozoon bieneusi species for diagnostic purposes. Sequences of animals were compared with those from the GenBank database. In rabbits, two murine genotypes II and four canine genotypes III were identified. Genotype II was identified in mice. The Encephalitozoon intestinalis identified in the sample from swine showed no genetic heterogeneity.

Application of specific primers in the diagnosis of Encephalitozoon spp.

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.

Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia.

Encephalitozoon cuniculi is one of the mamalian microsporidian pathogens that can affect a number of different species of animals as well as humans. The presence of specific serum antibodies to Encephalitozoon cuniculi was studied in a group of animals and humans from Eastern Slovakia by the indirect immunofluorescence of antibodies (IFA). 456 people, 571 rabbits, 457 mice, 193 dogs, 72 cats, and 59 sheep were examined. Specific anti-E. cuniculi antibodies were found in 26 out of 456 human sera examined (5.7%). The highest occurrence of antimicrosporidial antibodies was found in the group of immunodeficiency patients - 37.5%. In the group of animals, the highest positivity was observed in rabbits - 41.7%, and in dogs - 37.8. The relative high prevalence, especially in rabbits and dogs as potential sources of microsporidial infection for humans, indicates the importance of performing the screening examinations in animals with aim of reducing or halting the spread of this disease.