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The morphological, biological, and molecular characteristics of Cryptosporidium avian genotype V are described, and the species name Cryptosporidium avium is proposed to reflect its specificity for birds under natural and experimental conditions. Oocysts of C. avium measured 5.30-6.90 μm (mean = 6.26 μm) × 4.30-5.50 μm (mean = 4.86 μm) with a length to width ratio of 1.29 (1.14-1.47). Oocysts of C. avium obtained from four naturally infected red-crowned parakeets (Cyanoramphus novaezealandiae) were infectious for 6-month-old budgerigars (Melopsittacus undulatus) and hens (Gallus gallus f. domestica). The prepatent periods in both susceptible bird species was 11 days postinfection (DPI). The infection intensity of C. avium in budgerigars and hens was low, with a maximum intensity of 5000 oocysts per gram of feces. Oocysts of C. avium were microscopically detected at only 12-16 DPI in hens and 12 DPI in budgerigars, while PCR analyses revealed the presence of specific DNA in fecal samples from 11 to 30 DPI (the conclusion of the experiment). Cryptosporidium avium was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus). Naturally or experimentally infected birds showed no clinical signs of cryptosporidiosis, and no pathology was detected. Developmental stages of C. avium were detected in the ileum and cecum using scanning electron microscopy. Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. avium is genetically distinct from previously described Cryptosporidium species.

Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoeniceus; order Passeriformes) were examined for the presence of Cryptosporidium by PCR amplification and sequence analysis of the 18S ribosomal RNA (18S rRNA), actin, and 70-kDa heat shock protein (HSP70) genes. Twelve birds (17.1 %) were positive for the Cryptosporidium 18S rRNA gene: six birds were positive at the proventriculus site only and six birds were positive at the proventriculus and intestinal sites. Sequence analysis of the 18S rRNA, actin and HSP70 genes showed the presence of the gastric species Cryptosporidium galli in a single proventriculus sample and a closely related genotype, which we have named Cryptosporidium avian genotype VI, in all other positive samples. These findings contribute to our understanding of Cryptosporidium diversification in passerines, the largest avian order.

Within the microsporidian genus Encephalitozoon , three species, Encephalitozoon cuniculi , Encephalitozoon hellem and Encephalitozoon intestinalis have been described. Several orders of the Class Aves (Passeriformes, Psittaciformes, Apodiformes, Ciconiiformis, Gruiformes, Columbiformes, Suliformes, Podicipediformes, Anseriformes, Struthioniformes, Falconiformes) and of the Class Mammalia (Rodentia, Lagomorpha, Primates, Artyodactyla, Soricomorpha, Chiroptera, Carnivora) can become infected. Especially E. cuniculi has a very broad host range while E. hellem is mainly distributed amongst birds. E. intestinalis has so far been detected only sporadically in wild animals. Although genotyping allows the identification of strains with a certain host preference, recent studies have demonstrated that they have no strict host specificity. Accordingly, humans can become infected with any of the four strains of E. cuniculi as well as with E. hellem or E. intestinalis, the latter being the most common. Especially, but not exclusively, immunocompromised people are at risk. Environmental contamination with as well as direct transmission of Encephalitozoon is therefore highly relevant for public health. Moreover, endangered species might be threatened by the spread of pathogens into their habitats. In captivity, clinically overt and often fatal disease seems to occur frequently. In conclusion, Encephalitozoon appears to be common in wild warm-blooded animals and these hosts may present important reservoirs for environmental contamination and maintenance of the pathogens. Similar to domestic animals, asymptomatic infections seem to occur frequently but in captive wild animals severe disease has also been reported. Detailed investigations into the epidemiology and clinical relevance of these microsporidia will permit a full appraisal of their role as pathogens.

Diversity of Enterocytozoon bieneusi genotypes in wild small rodent populations still remains incomplete and only few molecular studies have been conducted among these hosts. Therefore, the aim of this study was to determine whether small rodents, i.e., Apodemus agrarius, Apodemus flavicollis, Mus musculus and Myodes glareolus act as hosts of E. bieneusi and can play an important role in spore spreading in the environment of south-western Poland. Molecular analyses were conducted to determine pathogen genotypes. A total of 191 fecal and 251 spleen samples collected from 311 rodent individuals were examined for the occurrence of E. bieneusi by PCR amplifying ITS gene. The overall prevalence of E. bieneusi in rodent samples was 38.9%. The nucleotide sequences of ITS region of E. bieneusi revealed the presence a total of 12 genotypes with two being already known, i.e., D and gorilla 1 genotypes. The remaining ten are novel genotypes (WR1-WR10) which segregated into three groups in a neighbor joining phylogeny. This study reports for the first time E. bieneusi occurrence in wild living rodents in Poland and shows extensive genetic diversity within E. bieneusi isolates of rodent origin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bats from the families Rhinolophidae (n = 90) and Vespertilionidae (n = 191) in the USA and Czech Republic were screened for the presence of Cryptosporidium by microscopic and molecular analysis of faecal samples collected from rectum of dissected animals and from the ground beneath roosting sites. Cryptosporidium oocysts were not detected in any of the 281 faecal specimens examined using the aniline-carbol-methyl violet staining method. Nested PCR amplification, sequencing and phylogenetic analysis of the small ribosomal subunit rRNA and actin genes were used to identify isolates and infer evolutionary relationships. Cryptosporidium parvum was identified in a western small-footed bat (Myotis ciliolabrum) from the USA and a common pipistrelle bats (Pipistrellus pipistrellus) from the Czech Republic. Two novel genotypes were identified and named Cryptosporidium bat genotype III and IV. Bat genotype III was found in two big brown bats (Eptesicus fuscus) from the USA. Bat genotype IV was detected in two common pipistrelle bats from the Czech Republic.

Faecal samples were collected from 352 horses on 23 farms operating under six different management systems in the Czech Republic and Poland during 2011 and 2012. Farms were selected without previous knowledge of parasitological status. All faecal samples were screened for Cryptosporidium spp. presence using microscopy, following aniline-carbol-methyl violet staining and PCR analysis of the small-subunit (SSU) rRNA and the 60-kDa glycoprotein (gp60) genes. Cryptosporidium muris-positive samples were additionally genotyped at four minisatellite markers: MS1 (encoding a hypothetical protein), MS2 (encoding a 90-kDa heat shock protein), MS3 (encoding a hypothetical protein) and MS16 (encoding a leucine-rich repeat family protein). Cryptosporidium spp. was detected by PCR in 12/352 (3.4%) samples from 4 out of 13 farms. None of the samples tested by microscopy was positive. There was no relationship between Cryptosporidium prevalence and age, sex, diarrhoea or management system; however, Cryptosporidium was found only on farms where horses were kept on pasture during the day and in a stable overnight. Sequence analyses of SSU and gp60 genes revealed the presence of C. muris RN66 (n = 9), Cryptosporidium parvum IIaA15G2R1 (n = 1), Cryptosporidium tyzzeri IXbA22R9 (n = 1), and Cryptosporidium horse genotype VIaA15G4 (n = 1). The C. muris subtypes were identified as MS1-M1, MS2-M4, novel MS2-M7 and MS16-M1 by multilocus sequence of three minisatellite loci. The MS3 locus was not amplified from any isolate. This is the first report of C. tyzzeri and C. muris subtypes from horses.

A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screened for the presence of Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi DNA by genus-specific nested PCR. Isolates were genotyped by sequence analysis of SSU rRNA, GP60, TRAP-C1, COWP, and HSP70 loci in Cryptosporidium, and the ITS region in microsporidia. Cryptosporidium spp. was detected on 3/18 horse farms and 1/15 farms where donkeys were kept. Overall, five (2.3%) horse and two (1.6%) donkey specimens were PCR positive for Cryptosporidium. Genotyping at SSU and GP60 loci revealed that three isolates from horses and donkeys were C. parvum subtype family IIaA16G1R1, one isolate from a horse was, C. muris RN66, and one isolate from a donkey was C. muris TS03. An isolate from a horse shared 99.4% and 99.3% similarity with Cryptosporidium hominis and C. cuniculus, respectively, at the SSU locus. This isolate shared 100% identity with C. hominis at the TRAP-C1, COWP, and HSP70 loci, and it was from the novel gp60 subtype family IkA15G1. Microsporidia were found on 6/18 horse and 2/15 donkey farms. E. bieneusi was identified in 6.8% (15/219) and 1.6% (2/124), and Encephalitozoon cuniculi was identified in 1.8% (4/219) and 1.6% (2/124), of horses and donkeys, respectively. Three genotypes of E. cuniculi (I, II and III) were detected in horses, and E. cuniculi genotype II was detected in donkeys. Four genotypes of E. bieneusi (horse1, horse 2, CZ3, D) were described in horses. An additional five horses and two donkeys were positive for E. bieneusi, but the isolated were not genotyped. Neither Cryptosporidium nor microsporidia prevalence were affected by sex, age, type of breeding, or whether the host was a horse or a donkey., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ancient DNA (aDNA) of Encephalitozoon intestinalis (Microsporidia, Fungi) was detected in archaeological material originated from New Town of Prague (Czech Republic) with the use of molecular methods. Microsporidial aDNA was found in 3 samples originating from 2 objects, in a well/cesspit (samples from layers from the 18th century) and in a well from the 18th/19th century. The ability to use molecular methods to detect microsporidia extends the range of paleoparasitological inquiry, and could contribute to a better understanding of parasites shared between human and animals.

The morphological, biological, and molecular characteristics of Cryptosporidium hedgehog genotype are described, and the species name Cryptosporidium erinacei n. sp. is proposed to reflect its specificity for hedgehogs under natural and experimental conditions. Oocysts of C. erinacei are morphologically indistinguishable from Cryptosporidium parvum, measuring 4.5-5.8 μm (mean=4.9 μm) × 4.0-4.8 μm (mean=4.4 μm) with a length to width ratio of 1.13 (1.02-1.35) (n=100). Oocysts of C. erinacei obtained from a naturally infected European hedgehog (Erinaceus europaeus) were infectious for naïve 8-week-old four-toed hedgehogs (Atelerix albiventris); the prepatent period was 4-5 days post infection (DPI) and the patent period was longer than 20 days. C. erinacei was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus), Mongolian gerbils (Meriones unguiculatus), or golden hamsters (Mesocricetus auratus). Phylogenetic analyses based on small subunit rRNA, 60 kDa glycoprotein, actin, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, and heat shock protein 70 gene sequences revealed that C. erinacei is genetically distinct from previously described Cryptosporidium species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81-5.96 μm (mean=5.16)×4.23-5.29 μm (mean=4.83) with a length to width ratio of 1.07±0.06 (n=400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4-6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250-4000 oocysts per gram of feces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB/c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cryptosporidiosis belongs to the important parasitic infections with zoonotic potential and the occurrence in European countries is rare. The first cases of cryptosporidiosis caused by Cryptosporidium hominis detected in the Slovak republic were described here. Collection of examined humans consisted of five family members. Faecal specimens were examined by formalin sedimentation, by the Sheather's sugar flotation and by immunochromatography and visualised by the Ziehl-Neelsen acid fast stain. A fragment of the Cryptosporidium small subunit ribosomal RNA gene was amplified by nested polymerase chain reaction and species was determined by restriction fragment length polymorphism analysis with the endonucleases SspI and VspI. C. hominis was found in faeces of two immunocompetent siblings (a 7-year-old boy and a 2-year-old girl). The symptoms occurred only in the boy as gastrointestinal disorders lasting 5 days, and manifested by abdominal pain, an elevated body temperature (37.2 °C), mild diarrhoea, accompanied by lassitude, depression and anorexia. Ultrasonic scan revealed enlarged spleen and mezenteric lymph nodes. Microscopic examination of the stool sample revealed numerous Cryptosporidium oocysts. The DNA typing identified C. hominis subtype IbA10G2. Cryptosporidium was also detected in the boy's sister without any complications and symptoms. Their father, mother and grandmother were parasitologically negative. The source of infection remained unknown. Human cases in present study reflect necessity of systematic attention on intestinal parasites diagnostic inclusive of cryptosporidia.

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.

To determine the occurrence of potentially human pathogenic microsporidia (Enterocytozoon bieneusi and Encephalitozoon spp.) in wild mice, we examined 289 East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) trapped at 74 localities in an area across the Czech-German border. Microsporidia were detected at 33 localities, in 34% of M. m. musculus and 33% of M. m. domesticus examined specimens. Single-species infection was detected in 23 mice for E. hellem, 42 mice for E. cuniculi and 25 mice for E. bieneusi. No Encephalitozoon intestinalis positive animals were identified. Moreover, co-infections were detected in 6 animals; E. bieneusi co-existed with E. cuniculi or E. hellem in 3 mice. The natural infection of E. hellem has never been recorded in mice before. No differences were found by a statistical analysis of microsporidia occurrence between the House Mouse subspecies. Although the gender-dependent infestation of microsporidia was statistically supported in M. m. musculus, no significant differences were observed when the occurrence of microsporidia was estimated for all males and females irrespective of the House Mouse subspecies. The results of this report document the low host specificity of detected microsporidia species and imply the importance of synanthropic rodents as a potential source of human microsporidial infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)