Bohumil Sak

Subtyping Cryptosporidium ubiquitum, a Zoonotic Pathogen Emerging in Humans

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.

Cryptosporidium Pig Genotype II in Immunocompetent Man

To the Editor: Protozoan parasites from the genus Cryptosporidium have been described as a cause of diarrheal disease in immunodeficient and immunocompetent humans worldwide. Although C. hominis and C. parvum (cattle genotype) cause most infections, humans can be infected by several other Cryptosporidium species or genotypes: C. meleagridis; C. felis; C. canis; C. suis; C. muris; C. andersoni; C. hominis monkey genotype; C. parvum (mouse genotype); and Cryptosporidium rabbit genotype, deer genotype, skunk genotype, horse genotype, and chipmunk genotype I (1–4). Wild and domestic animals are sources of infection for humans (and other animals) and important contributors to contamination of food and drinking water; many nonhuman Cryptosporidium species or genotypes are detected in untreated water (5). We examined the diversity of Cryptosporidium spp. in immunocompetent persons in South Bohemia in the Czech Republic. Diarrheal fecal samples (n = 457) from 203 anonymous immunocompetent patients <69 years of age with suspected cryptosporidiosis (at least 2 samples/patient/3-day period) were obtained from local health departments and public hospitals in South Bohemia during 2005–2007. Samples were examined for Cryptosporidium oocysts by using aniline-carbol-methyl violet staining and light microscopy at × 1,000 magnification (6). The microscopically positive samples were confirmed by DNA sequencing of the small subunit (SSU) rRNA gene. Total DNA was extracted from 200–300 mg stool by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions, after previous homogenization and disruption of oocysts with the Mini-BeadBeater (Biospec Products, Bartlesville, OK, USA). An ≈830-bp fragment of the SSU rRNA gene was amplified by nested PCR according to Jiang et al. (7). Purified PCR products were sequenced in both directions on an ABI3130 sequencer analyzer (Applied Biosystems, Foster City, CA, USA) by using the secondary PCR primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were assembled by using Chromas Pro (www.technelysium.com.au/chromas.html) and aligned with reference sequences using ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX). The BLAST server (www.ncbi.nlm.nih.gov/BLAST) was used for DNA database searches. The SSU rRNA gene partial sequences of the 7 patient isolates have been submitted to GenBank (Table). Table Cryptosporidium genotypes identified by using sequencing of partial sequences of the small subunit rRNA gene in the stool samples of immunocompetent humans, Czech Republic Of the 203 patients, 7 (3.4%) (6 children and 1 adult) had positive results for Cryptosporidium spp. Moreover, all samples obtained from these persons during the 3-day period were Cryptosporidium spp. positive; partial sequences of the Cryptosporidium SSU rRNA gene were obtained from all positive samples identifying 3 different species or genotypes of Cryptosporidium. Five were C. parvum (bovine genotype), 1 was C. hominis, and 1 contained the Cryptosporidium pig genotype II (Table). Cryptosporidium pig genotype II was found in stool samples from a 29-year-old man who also was infected with Giardia intestinalis (assemblage A) (data not shown). Only C. parvum (bovine genotype), C. hominis, and Cryptosporidium rabbit genotype have been implicated in waterborne outbreaks of cryptosporidiosis in humans. Further studies are needed to determine the potential of other cryptosporidia of animal origin. Recent genetic and biologic characterization studies have identified 2 distinct host-adapted cryptosporidia in pigs, C. suis and Cryptosporidium pig genotype II. Furthermore, both above-mentioned cryptosporidia have been identified in untreated water (8). Pigs could be sources of Cryptosporidium water and food pollution and a consequent risk to public health. Although human infection with C. suis has been previously described (9), human infection with Cryptosporidium pig genotype II has been never reported. This genotype was found in diarrheal stool of 1 adult patient in this study. However, onset of diarrhea could have been caused by co-infection with G. intestinalis (assemblage A), which recently also has been described in pigs (10). Contact with infected animals and ingestion of contaminated food or water could be the source of both Cryptosporidium and Giardia infection in the Cryptosporidium pig genotype II–positive patient. The passage of oocysts can be excluded because of the number of oocysts detected in repeat samples (Table). Moreover, identification of the infection in an immunocompetent patient underlines the zoonotic potential of this pig genotype and possible presence of risk factors in rural areas with poor water treatment or inadequate biosecurity in pig units. Further evidence of the zoonotic potential of this Cryptosporidium genotype is needed to show its pathogenic potential in immunocompetent patients as a cause of gastroenteritis (in the absence of Giardia spp. and other established enteropathogens) and to demonstrate invasive tissue stages. The use of molecular techniques to identify Cryptosporidium spp. probably will show more zoonotic species or genotypes in humans.

Unapparent Microsporidial Infection among Immunocompetent Humans in the Czech Republic

ABSTRACT In the present population-based study, we determined the prevalences of the most common human-pathogenic microsporidia, Encephalitozoon spp. and Enterocytozoon bieneusi, in asymptomatic healthy people living in the Czech Republic. A total of 382 males and females (ages, 1 to 84 years) living in the Czech Republic, of whom 265 were Czech nationals and 117 were foreign students, were included in a study testing for the presence of microsporidia by use of coprology and molecular methods. Single-species infections with Enterocytozoon bieneusi or an Encephalitozoon sp. were detected for 9 and 136 individuals, respectively. Moreover, coinfections were detected for 14 individuals. Four genotypes of 3 human-pathogenic Encephalitozoon spp. and 7 E. bieneusi genotypes, including 3 novel genotypes, were detected. Some of these were reported in humans for the first time. The highest prevalence was recorded for individuals older than 50 years and for loose, unformed stool samples. These findings clearly show that exposure to microsporidia is common among immunocompetent people and that microsporidiosis is not linked to any clinical manifestation in healthy populations.

Latent microsporidial infection in immunocompetent individuals - a longitudinal study.

Background Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved. Aims To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis. Methods Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22–56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination. Results Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded. Conclusions These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.

First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic.

Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people. The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E. bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity. Spores were detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR) identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.

Sources of potentially infectious human microsporidia: Molecular characterisation of microsporidia isolates from exotic birds in the Czech Republic, prevalence study and importance of birds in epidemiology of the human microsporidial infections

A total of 287 faecal specimens of captive exotic birds from the orders Psittaciformes, Passeriformes and Columbiformes were randomly collected from Bohemian pet stores, avian breeders and avian keepers and were screened for the presence of human pathogenic microsporidia by polymerase chain reaction (PCR). Microsporidial DNA was identified in 115 faecal samples (40.1%). Single-species infection was detected in 36 birds (12.5%) for Enterocytozoon bieneusi, 36 birds (12.5%) for Encephalitozoon cuniculi and 18 birds (6.3%) for Encephalitozoon hellem. No Encephalitozoon intestinalis positive samples were identified. Moreover, co-infections were detected in 25 birds: E. bieneusi together with E. cuniculi in 14 animals (4.9%) or E. hellem in 11 cases (3.8%). E. hellem was present in 1A (5.2%) and three (0.3%) genotypes, E. cuniculi in I (2.4%), II (8.0%) and III (0.7%) genotypes and E. bieneusi in A (8.4%) and EbpA (10.8%) genotypes. Several of these genotypes have never been recorded in birds before. The results of this report suggest the low host specificity of E. bieneusi, E. hellem and E. cuniculi and describe 44 new avian hosts.

Prevalence and age-related infection of Cryptosporidium suis, C. muris and Cryptosporidium pig genotype II in pigs on a farm complex in the Czech Republic.

A total of 413 pig faecal samples were collected from pre-weaners (119), starters (131), pre-growers (123) and sows (40) from a farm with a closed breeding system segmented into two breeding complexes and a growing complex in the region of Vysocina, Czech Republic and screened for the presence of Cryptosporidium using staining methods and genotyping (SSU rRNA). Cryptosporidium oocysts were detected by microscopy in the faeces of 21.1% of the samples (87/413). Sequence analyses and RFLP identified C. suis in 44, Cryptosporidium pig genotype II in 23 and C. muris in 2 samples. No mixed infections were found. Pigs under 7 weeks of age were infected with C. suis only. Cryptosporidium pig genotype II was found in animals from 7 weeks of age. No relationship was found between diarrhoea and any Cryptosporidium infection in any of the different age groups (P<0.05). The pre-weaned pigs shed significantly more Cryptosporidium oocysts than older pigs and it was associated with C. suis infection.

The first report on natural Enterocytozoon bieneusi and Encephalitozoon spp. infections in wild East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) in a hybrid zone across the Czech Republic–Germany border

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.

Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa)

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.

Molecular characterization of Cryptosporidium isolates from pigs at slaughterhouses in South Bohemia, Czech Republic

A total of 123 fecal samples of slaughtered finisher pigs and 21 sows from 14 farms were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method. Positive samples were molecularly characterized by direct sequencing of partial small subunit ribosomal RNA (SSU rRNA) and GP60 partial genes and polymerase chain reaction restriction fragment length polymorphism of SSU rRNA. Cryptosporidium oocysts were microscopically identified in 36 finishers (29%) and two sows (10%). Twenty-one mono-infections of Cryptosporidium pig genotype II and 15 mixed-infection of Cryptosporidium pig genotype II and Cryptosporidium suis in finishers were found. Both sows were infected with the Cryptosporidium parvum subgenotype IIaA16G1R1, which is reported from pigs for the first time.