Bohumil Sak

Academy of Sciences of the Czech Republic, Praha, Praha, Czech Republic

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Publications (76)184.58 Total impact

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    ABSTRACT: Encephalitozoon cuniculi is a model microsporidian species with a mononucleate nucleus and a genome that has been extensively studied. To date, analyses of genome diversity have revealed the existence of four genotypes in E. cuniculi (EcI, II, III and IV). Genome sequences are available for EcI, II and III, and are all very divergent, possibly diploid and genetically homogeneous. The mechanisms that cause low genetic diversity in E. cuniculi (for example, selfing, inbreeding or a combination of both), as well as the degree of genetic variation in their natural populations, have been hard to assess because genome data have been so far gathered from laboratory-propagated strains. In this study, we aim to tackle this issue by analyzing the complete genome sequence of a natural strain of E. cuniculi isolated in 2013 from a steppe lemming. The strain belongs to the EcIII genotype and has been designated EcIII-L. The EcIII-L genome sequence harbors genomic features intermediate to known genomes of II and III lab strains, and we provide primers that differentiate the three E. cuniculi genotypes using a single PCR. Surprisingly, the EcIII-L genome is also highly homogeneous, harbors signatures of heterozygosity and also one strain-specific single-nucleotide polymorphism (SNP) that introduces a stop codon in a key meiosis gene, Spo11. Functional analyses using a heterologous system demonstrate that this SNP leads to a deficient meiosis in a model fungus. This indicates that EcIII-L meiotic machinery may be presently broken. Overall, our findings reveal previously unsuspected genome diversity in E. cuniculi, some of which appears to affect genes of primary importance for the biology of this pathogen.Heredity advance online publication, 3 February 2016; doi:10.1038/hdy.2016.4.
    Full-text · Article · Mar 2016 · Heredity
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    ABSTRACT: Transplant recipients have been identified as a new risk group for microsporidia infection. We characterize for the first time the prevalence of microsporidia in intestinal and urinary tracts of renal transplant recipients. Molecular examination of eighty-six patients showed 25.5 % of them were infected, 86 % were confirmed to have pathogens in their urine and 45.5 % in stool. Among positive patients 32 % had microsporidia confirmed in both urine and stool. Genotyping revealed Encephalitozoon cuniculi (59 %) and Enterocytozoon bieneusi (23 %) monoinfections as well as co-infections with both species (18 %). Moreover, we found diarrhea and fever as symptoms significantly associated with microsporidia presence. Our results indicate that microsporidial infection should be considered in diagnostics of renal transplant patients, especially in the urinary tract, even if asymptomatic. Molecular identification of microsporidia species is relevant due to their different susceptibility for treatment.
    No preview · Article · Jan 2016 · Clinical Microbiology and Infection
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    Full-text · Article · Jan 2016 · Folia parasitologica
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    ABSTRACT: The morphological, biological, and molecular characteristics of Cryptosporidium muris strain TS03 are described, and the species name Cryptosporidium proliferans n. sp. is proposed. Cryptosporidium proliferans obtained from a naturally infected East African mole rat (Tachyoryctes splendens) in Kenya was propagated under laboratory conditions in rodents (SCID mice and southern multimammate mice, Mastomys coucha) and used in experiments to examine oocyst morphology and transmission. DNA from the propagated C. proliferans isolate, and C. proliferans DNA isolated from the feces of an African buffalo (Syncerus caffer) in Central African Republic, a donkey (Equus africanus) in Algeria, and a domestic horse (Equus caballus) in the Czech Republic were used for phylogenetic analyses. Oocysts of C. proliferans are morphologically distinguishable from C. parvum and C. muris HZ206, measuring 6.8-8.8 (mean = 7.7 μm) × 4.8-6.2 μm (mean = 5.3) with a length to width ratio of 1.48 (n = 100). Experimental studies using an isolate originated from T. splendens have shown that the course of C. proliferans infection in rodent hosts differs from that of C. muris and C. andersoni. The prepatent period of 18-21 days post infection (DPI) for C. proliferans in southern multimammate mice (Mastomys coucha) was similar to that of C. andersoni and longer than the 6-8 DPI prepatent period for C. muris RN66 and HZ206 in the same host. Histopatologicaly, stomach glands of southern multimammate mice infected with C. proliferans were markedly dilated and filled with necrotic material, mucus, and numerous Cryptosporidium developmental stages. Epithelial cells of infected glands were atrophic, exhibited cuboidal or squamous metaplasia, and significantly proliferated into the lumen of the stomach, forming papillary structures. The epithelial height and stomach weight were six-fold greater than in non-infected controls. Phylogenetic analyses based on small subunit rRNA, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70, actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences revealed that C. proliferans is genetically distinct from C. muris and other previously described Cryptosporidium species.
    Full-text · Article · Jan 2016 · PLoS ONE
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    ABSTRACT: 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.
    No preview · Article · Jan 2016 · International Journal for Parasitology: Parasites and Wildlife
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    ABSTRACT: This study describes the prevalence of Encephalitozoon cuniculi in raw cow's milk and evaluates the effect of different milk pasteurization treatments on E. cuniculi infectivity for severe combined immunodeficient (SCID) mice. Using a nested polymerase chain reaction approach, 1 of 50 milking cows was found to repeatedly shed E. cuniculi in its feces and milk. Under experimental conditions, E. cuniculi spores in milk remained infective for SCID mice following pasteurization treatments at 72°C for 15 s or 85°C for 5 s. Based on these findings, pasteurized cow's milk should be considered a potential source of E. cuniculi infection in humans.
    Full-text · Article · Dec 2015 · Foodborne Pathogens and Disease
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    ABSTRACT: The prevalence of Cryptosporidium and microsporidia in feral horses, which have minimal contact with livestock and humans, is not currently known. We report the findings of a study on Cryptosporidium and microsporidia in 34 Mustangs and 50 Chincoteague ponies in the USA. Fecal samples were screened for presence of Cryptosporidium spp. by analysis of the small-subunit rRNA (SSU) and 60-kDa glycoprotein (gp60) genes, and Enterocytozoon bieneusi and Encephalitozoon spp. by analysis of the ribosomal internal transcribed spacer region (ITS). Cryptosporidium spp. and E. bieneusi were detected in in 28/84 (33.3%) and 7/84 (8.3%) samples, respectively. Sequence analysis of SSU and ITS revealed the presence of C. parvum (n=20) and E. bieneusi genotype horse 1 (n=7), respectively. Subtyping of C. parvum isolates at the gp60 locus showed the presence of subtype IIaA17G2R1 in Mustangs and subtypes IIaA13G2R1 and IIaA15G2R1 in Chincoteague ponies. Enterocytozoon bieneusi genotype horse 1 was detected in Mustangs (n=2) and Chincoteague ponies (n=5). No Cryptosporidium or E. bieneusi positive animals had diarrhea. The finding that Mustangs and Chincoteague ponies are host to the zoonotic pathogen C. parvum suggests that their infrequent contact with humans and livestock is sufficient to maintain transmission; however, we should also consider the possibility that C. parvum is an established parasite of Mustangs and Chincoteague ponies that persists in these animals independently of contact with humans or livestock.
    Full-text · Article · Dec 2015 · Experimental Parasitology
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    ABSTRACT: 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.
    No preview · Article · Nov 2015 · Veterinary Parasitology
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    ABSTRACT: 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.
    Full-text · Article · Aug 2015 · Parasitology Research
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    ABSTRACT: 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.
    Full-text · Article · Feb 2015 · Parasitology Research
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    ABSTRACT: A total of 219 and 124 individual faecal 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 C. 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.
    Full-text · Article · Jan 2015 · Veterinary Parasitology
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    ABSTRACT: Background Infectious diseases represent the greatest threats to endangered species, and transmission from humans to wildlife under increased anthropogenic pressure has been always stated as a major risk of habituation. Aims To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, one hundred mountain gorillas (Gorilla beringei beringei) from seven groups habituated either for tourism or for research in Volcanoes National Park, Rwanda were screened for the presence of microsporidia, Cryptosporidium spp. and Giardia spp. using molecular diagnostics. Results The most frequently detected parasites were Enterocytozoon bieneusi found in 18 samples (including genotype EbpA, D, C, gorilla 2 and five novel genotypes gorilla 4–8) and Encephalitozoon cuniculi with genotype II being more prevalent (10 cases) compared to genotype I (1 case). Cryptosporidium muris (2 cases) and C. meleagridis (2 cases) were documented in great apes for the first time. Cryptosporidium sp. infections were identified only in research groups and occurrence of E. cuniculi in research groups was significantly higher in comparison to tourist groups. No difference in prevalence of E. bieneusi was observed between research and tourist groups. Conclusion Although our data showed the presence and diversity of important opportunistic protists in Volcanoes gorillas, the source and the routes of the circulation remain unknown. Repeated individual sampling, broad sampling of other hosts sharing the habitat with gorillas and quantification of studied protists would be necessary to acquire more complex data.
    Full-text · Article · Nov 2014 · PLoS ONE
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    Full-text · Dataset · Oct 2014
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    Full-text · Dataset · Oct 2014
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    ABSTRACT: Microsporidia are ubiquitous, spore-forming, intracellular parasites infecting invertebrates and vertebrates. Some of them are important opportunistic pathogens in humans, including three species of genus Encephalitozoon. Intraspecies genetic variation with a different range of hosts is known in Encephalitozoon cuniculi distinguishing four genotypes. Recently, E. cuniculi is often observed in pet animals, mainly E. cuniculi genotype I in pet rabbits. This study described a fatal encephalitozoonosis in a group of pet rodents Steppe lemmings (Lagurus lagurus). The animals were presented with progressive weight loss, aggression, cannibalism, purulent conjunctivitis and hind limb paresis. Death occurred within 48 h after the onset of clinical signs. The group comprised of 15 animals was affected and died within a period of three months. Post-mortal examination did not show any macroscopic changes. Microsporidial vacuoles with typical spores were found in brain and kidney tissues and E. cuniculi DNA in all tested organs. The internal transcribed spacer region (ITS) of rRNA gene showed 100% homology with E. cuniculi genotype III previously identified in dogs, tamarin colonies from zoos, swine, birds and humans. Pet lemmings could represent a new potential source of the infection for their breeders.
    Full-text · Article · Sep 2014 · Veterinary Parasitology
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    ABSTRACT: Cryptosporidiosis is considered to be a widespread world zoonosis. The occurrence of Cryptosporidium species was investigated in Roma children in a district of Eastern Slovakia and, at the same time, also in children of non-Roma parents. In total, 103 children (54 boys and 49 girls) between 0 and 14 years of age were involved in this study. Fifty-three were Roma children and 50 children represented a non-Roma control group. Fecal samples were examined: immunologically [enzyme-linked immunosorbent assay (ELISA) test to prove antigen in the feces] and by molecular analysis [nested polymerase chain reaction (PCR)]. After the sequencing of the PCR, the products were identified as species of Cryptosporidium muris. Based on the results, the relative risk (RR) of the Cryptosporidium infection occurrence was calculated and we came to the conclusion that the risk of Cryptosporidium infection was almost 12 times higher in the Roma children compared to the non-Roma children.
    Full-text · Article · Mar 2014 · European Journal of Clinical Microbiology
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    ABSTRACT: Piglets from 4 to 8 weeks of age originated from a Cryptosporidium-free research breed were orally inoculated with 1×10(6) infectious oocysts of Cryptosporidium scrofarum. The number of shed oocysts per gram of faeces served to describe the infection intensity and prepatent period. In addition, faecal samples collected daily and tissue samples of the small and large intestine collected at 30 days post-inoculation were examined for the C. scrofarum small subunit ribosomal RNA gene using PCR. The piglets inoculated at 4-weeks of age remained uninfected, whereas 5-week-old and older animals were fully susceptible with a prepatent period ranging from 4 to 8 days. Susceptible pigs shed oocysts intermittently, and shedding intensity, reaching a mean maximum of 6000 oocysts per gram, did not differ significantly among infected animals. This study demonstrates that pigs become susceptible to C. scrofarum infection as late as 5-weeks of age. The mechanisms of age related susceptibility remain unknown.
    Full-text · Article · Feb 2014 · Veterinary Parasitology
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    ABSTRACT: A urinary tract coinfection, caused by Encephalitozoon cuniculi genotype II and Enterocytozoon bieneusi genotype D, was identified in an HIV-seronegative renal transplant recipient kept under lifelong immunosuppression. To our knowledge, this is the first report describing concurrent infection with these two microsporidia species in organ transplant recipients.
    Full-text · Article · Feb 2014 · Journal of clinical microbiology
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    ABSTRACT: Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.
    Full-text · Article · Feb 2014 · Emerging Infectious Diseases
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    ABSTRACT: Abstract Ancient DNA (aDNA) of Encephalitozoon intestinalis (Microsporidia, Fungi) was detected in archaeological material originated from New Town of Prague (Czech Republic) using molecular methods. Microsporidial aDNA was found in 3 samples originating from 2 objects, namely in well/cesspit (samples are from layers from 18th century) and in a well from the 18th/19th century. Feasibility of molecular detection of microsporidia extends the possibilities of paleoparasitology and could contribute to a better understanding of parasites shared between human and animals.
    Full-text · Article · Jan 2014 · Journal of Parasitology