Bohumil Sak

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

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Publications (67)172.05 Total impact

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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.
    Parasitology Research 02/2015; 114(4). DOI:10.1007/s00436-015-4353-y · 2.33 Impact Factor
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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.
    Veterinary Parasitology 01/2015; 208(3-4). DOI:10.1016/j.vetpar.2015.01.007 · 2.55 Impact Factor
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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.
    PLoS ONE 11/2014; 9(11):e109751. DOI:10.1371/journal.pone.0109751 · 3.53 Impact Factor
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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.
    Veterinary Parasitology 09/2014; DOI:10.1016/j.vetpar.2014.07.008 · 2.55 Impact Factor
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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.
    European Journal of Clinical Microbiology 03/2014; DOI:10.1007/s10096-014-2082-2 · 2.54 Impact Factor
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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.
    Veterinary Parasitology 02/2014; 202(3-4). DOI:10.1016/j.vetpar.2014.02.012 · 2.55 Impact Factor
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    ABSTRACT: A urinary tract co-infection, caused by Encephalitozoon cuniculi genotype II and Enterocytozoon bieneusi genotype D, was identified in HIV-seronegative renal transplant recipient kept under life-long immunosuppression. To our knowledge, this is the first report describing concurrent infection with these two microsporidia species in organ transplant recipients.
    Journal of clinical microbiology 02/2014; 52(5). DOI:10.1128/JCM.03328-13 · 4.23 Impact Factor
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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.
    Emerging Infectious Diseases 02/2014; 20(2):217-24. DOI:10.3201/eid2002.121797 · 7.33 Impact Factor
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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.
    Journal of Parasitology 01/2014; 100(3). DOI:10.1645/13-232.1 · 1.26 Impact Factor
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    ABSTRACT: 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 4.5-5.8μm (mean=4.9μm)×4.0-4.8μm (mean=4.4μm) 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, 60kDa 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.
    Veterinary Parasitology 01/2014; 201(1-2). DOI:10.1016/j.vetpar.2014.01.014 · 2.55 Impact Factor
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    ABSTRACT: 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.
    Annals of agricultural and environmental medicine: AAEM 12/2013; 20(4):695-8. · 3.06 Impact Factor
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    ABSTRACT: From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1-6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.
    Parasitology Research 11/2013; DOI:10.1007/s00436-013-3707-6 · 2.33 Impact Factor
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    ABSTRACT: The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses, was identified as the cause of the diarrheal disease cryptosporidiosis in an immunocompetent man in the Czech Republic. This is the first report of human illness caused by the Cryptosporidium hedgehog genotype.
    Journal of clinical microbiology 10/2013; 52(1). DOI:10.1128/JCM.02456-13 · 4.23 Impact Factor
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    ABSTRACT: SUMMARY This study focuses on mapping the life cycle of Cryptosporidium muris in two laboratory rodents; BALB/c mice and the southern multimammate rat Mastomys coucha, differing in their prepatent and patent periods. Both rodents were simultaneously experimentally inoculated with viable oocysts of C. muris (strain TS03). Animals were dissected and screened for the presence of the parasite using a combined morphological approach and nested PCR (SSU rRNA) at different times after inoculation. The occurrence of first developmental stages of C. muris in stomach was detected at 2·5 days post-infection (dpi). The presence of Type II merogony, appearing 36 h later than Type I merogony, was confirmed in both rodents. Oocysts exhibiting different size and thickness of their wall were observed from 5 dpi onwards in stomachs of both host models. The early phase of parasitization in BALB/c mice progressed rapidly, with a prepatent period of 7·5-10 days; whereas in M. coucha, the developmental stages of C. muris were first observed 12 h later in comparison with BALB/c mice and prepatent period was longer (18-21 days). Similarly, the patent periods of BALB/c mice and M. coucha differed considerably, i.e. 10-15 days vs chronic infection throughout the life of the host, respectively.
    Parasitology 10/2013; 141(02):1-17. DOI:10.1017/S0031182013001637 · 2.35 Impact Factor
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    ABSTRACT: Infectious diseases pose one of the greatest threats to endangered species, and a risk of gastrointestinal parasite transmission from humans to wildlife has always been considered as a major concern of tourism. Increased anthropogenic impact on primate populations may result in general changes in communities of their parasites, and also in a direct exchange of parasites between humans and primates. To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, we conducted a long-term monitoring of microsporidia, Cryptosporidium and Giardia infections in western lowland gorillas at different stages of the habituation process, humans, and other wildlife in Dzanga-Sangha Protected Areas in the Central African Republic. We detected Encephalitozoon cuniculi genotypes I and II (7.5%), Enterocytozoon bieneusi genotype D and three novel genotypes (gorilla 1-3) (4.0%), Giardia intestinalis subgroup A II (2.0%) and Cryptosporidium bovis (0.5%) in gorillas, whereas in humans we found only G. intestinalis subgroup A II (2.1%). In other wild and domestic animals we recorded E. cuniculi genotypes I and II (2.1%), G. intestinalis assemblage E (0.5%) and C. muris TS03 (0.5%). Due to the non-specificity of E. cuniculi genotypes we conclude that detection of the exact source of E. cuniculi infection is problematic. As Giardia intestinalis was recorded primarily in gorilla groups with closer human contact, we suggest that human-gorilla transmission has occurred. We call attention to a potentially negative impact of habituation on selected pathogens which might occur as a result of the more frequent presence of humans in the vicinity of both gorillas under habituation and habituated gorillas, rather than as a consequence of the close contact with humans, which might be a more traditional assumption. We encourage to observe the sections concerning hygiene from the IUCN best practice guidelines for all sites where increased human-gorilla contact occurs.
    PLoS ONE 08/2013; 8(8):e71840. DOI:10.1371/journal.pone.0071840 · 3.53 Impact Factor
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    ABSTRACT: From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa) 29 randomly selected localities (both forest areas and enclosures) across the Central European countries of Austria, the Czech Republic, Poland, and the Slovak Republic were investigated. Cryptosporidium oocysts were microscopicaly detected in 11 out of 460 faecal samples examined using aniline-carbol-methyl violet staining. Sixty-one Cryptosporidium infections, including the 11 infections that were detected by microscopy, were detected using genus- or species-specific nested PCR amplification of SSU rDNA. This represents a 5.5 fold greater sensitivity for PCR relative to microscopy. Combining genus- and species-specific PCR tools significantly changes the perspective on the occurrence of Cryptosporidium spp. in wild boars. While RFLP and direct sequencing of genus specific PCR-amplified products revealed 56 C. suis (20) and C. scrofarum (36) monoinfections and only 5 mixed infections of these species, species-specific molecular tools showed 44 monoinfections and 17 mixed infections with these species. PCR analysis of the gp60 gene did not reveal any other Cryptosporidium infections. Similar to domestic pigs, C. scrofarum was detected as a dominant species infecting adult Eurasian wild boars (Sus scrofa). Cryptosporidium infected wild boars did not show signs of clinical disease. This report is perhaps the most comprehensive survey of cryptosporidial infection in wild boars.
    Veterinary Parasitology 07/2013; 197(3-4). DOI:10.1016/j.vetpar.2013.07.003 · 2.55 Impact Factor
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    ABSTRACT: Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24×3.69μm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49×3.90μm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.
    International Journal for Parasitology 06/2013; 43(10):805-817. DOI:10.1016/j.ijpara.2013.04.007 · 3.40 Impact Factor