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Background Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are common enteric pathogens in humans and animals. Data on the transmission of these pathogens are scarce from Guangdong, China, which has a subtropical monsoon climate and is the epicenter for many emerging infectious diseases. This study was conducted to better understand the prevalence and identity of the three pathogens in pre-weaned dairy calves in Guangdong. Methods The occurrence and genetic identity of three pathogens were analyzed by polymerase chain reaction. PCR-positive products were sequenced to determine the species and genotypes. A Chi-square test was used to compare the prevalence of pathogens among sampling dates, age groups, or clinical signs. Results The detection rates of Cryptosporidium spp., G. duodenalis and E. bieneusi were 24.0% (93/388), 74.2% (288/388) and 15.7% (61/388), respectively. Three Cryptosporidium species were detected, including C. bovis ( n = 73), C. parvum ( n = 12) and C. ryanae ( n = 7); one animal had concurrence of C. bovis and C. parvum . C. parvum was the dominant species during the first two weeks of life, whereas C. bovis and C. ryanae were mostly seen at 3–9 weeks of age. Sequence analysis identified the C. parvum as subtype IIdA19G1. Assemblage E ( n = 282), assemblage A ( n = 1), and concurrence of A and E ( n = 5) were identified among G. duodenalis -positive animals using multilocus genotyping (MLG). Altogether, 15, 10 and 17 subtypes of assemblage E were observed at the bg , gdh and tpi loci, respectively, forming 49 assemblage E MLGs. The highest detection rate of G. duodenalis was found in winter. Sequence analysis identified genotypes J ( n = 57), D ( n = 3) and one concurrence of J and D among E. bieneusi- positive animals. The detection rate of E. bieneusi was significantly higher in spring (38.0%; 41/108) than in summer (7.2%; 8/111) and winter (7.1%; 12/169). Conclusions These results indicate a common occurrence of C. parvum subtype IIdA19G1, G. duodenalis assemblage E, and E. bieneusi genotype J in pre-weaned dairy calves in Guangdong. More studies are needed to understand the unique genetic characteristics and zoonotic potential of the three enteric pathogens in the province.

Background Bamboo rats are widely farmed in southern China for meat, but their potential in transmitting pathogens to humans and other farm animals remains unclear. Methods To understand the transmission of Cryptosporidium spp. in these animals, 709 fecal samples were collected in this study from Chinese bamboo rats ( Rhizomys sinensis ) on nine farms in Jiangxi, Guangxi and Hainan provinces, China. They were analyzed for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. Cryptosporidium parvum , C. parvum -like and C. ubiquitum -like genotypes identified were subtyped by sequence analysis of the 60 kDa glycoprotein ( gp60 ) gene. Results Altogether, Cryptosporidium spp. were detected in 209 (29.5%) samples. The detection rate in samples from animals under two months of age (70.0%,105/150) was significantly higher than in samples from animals above 2 months (18.6%, 104/559; χ 2  = 150.27, df  = 1, P  < 0.0001). Four Cryptosporidium species/genotypes were identified: C. parvum ( n  = 78); C. occultus ( n  = 1); a new genotype that is genetically related to C. ubiquitum ( n  = 85); and another new genotype that is genetically related to C. parvum ( n  = 44). Among them, C. parvum (27,610 ± 71,911 oocysts/gram of feces) and the C. parvum -like genotype (38,679 ± 82,811 oocysts/gram of feces) had higher oocyst shedding intensity than the C. ubiquitum -like genotype (2470 ± 7017 oocysts/gram of feces) and the C. occultus (1012 oocysts/gram of feces). The C. parvum identified belonged to three subtypes in two rare subtype families, including IIpA9 ( n  = 43), IIpA6 ( n  = 6) and IIoA15G1 ( n  = 9), while the C. parvum -like and C. ubiquitum -like genotypes generated very divergent gp60 sequences. Conclusions Results of the present study suggest that bamboo rats on the study farms were infected with diverse Cryptosporidium species and divergent C. parvum subtypes, which probably had originated from their native habitats. As similar C. parvum subtypes have been recently detected in humans and farmed macaques, attentions should be paid to the potential role of these new farm animals in the transmission of zoonotic pathogens.

Background Avian cryptosporidiosis is a common parasitic disease that is caused by five species, which are well characterised at the molecular and biological level, and more than 18 genotypes for which we have limited information. In this study, we determined the occurrence and molecular characteristics of Cryptosporidium spp. in farmed ostriches in the Czech Republic. Methods The occurrence and genetic identity of Cryptosporidium spp. were analysed by microscopy and PCR/sequencing of the small subunit rRNA, actin , HSP70 and gp60 genes. Cryptosporidium avian genotype II was examined from naturally and experimentally infected hosts and measured using differential interference contrast. The localisation of the life-cycle stages was studied by electron microscopy and histologically. Infectivity of Cryptosporidium avian genotype II for cockatiels ( Nymphicus hollandicus (Kerr)), chickens ( Gallus gallus f. domestica (L.)), geese ( Anser anser f. domestica (L.)), SCID and BALB/c mice ( Mus musculus L.) was verified. Results A total of 204 individual faecal samples were examined for Cryptosporidium spp. using differential staining and PCR/sequencing. Phylogenetic analysis of small subunit rRNA, actin , HSP70 and gp60 gene sequences showed the presence of Cryptosporidium avian genotype II ( n  = 7) and C. ubiquitum Fayer, Santín & Macarisin, 2010 IXa ( n  = 5). Only ostriches infected with Cryptosporidium avian genotype II shed oocysts that were detectable by microscopy. Oocysts were purified from a pooled sample of four birds, characterised morphometrically and used in experimental infections to determine biological characteristics. Oocysts of Cryptosporidium avian genotype II measure on average 6.13 × 5.15 μm, and are indistinguishable by size from C. baileyi Current, Upton & Haynes, 1986 and C. avium Holubová, Sak, Horčičková, Hlásková, Květoňová, Menchaca, McEvoy & Kváč, 2016. Cryptosporidium avian genotype II was experimentally infectious for geese, chickens and cockatiels, with a prepatent period of four, seven and eight days post-infection, respectively. The infection intensity ranged from 1000 to 16,000 oocysts per gram. None of the naturally or experimentally infected birds developed clinical signs in the present study. Conclusions The molecular and biological characteristics of Cryptosporidium avian genotype II, described here, support the establishment of a new species, Cryptosporidium ornithophilus n. sp.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Background Cryptosporidium felis , a host-specific protozoan with possible zoonotic potential, is a common parasite of cats ( Felis catus ). To date, there have been few studies on the molecular subtyping of C . felis worldwide, and no have been conducted in Central Europe. The aim of this study was to analyse the prevalence and genetic variability of C . felis in domestic and stray cats in Central Europe, particularly in the Czech Republic, Poland and Slovakia. Methods Faecal samples were collected from domestic and stray cats. The presence of Cryptosporidium spp. was analysed by light microscopy with aniline-carbol-methyl violet staining and polymerase chain reaction (PCR)/sequencing of the small subunit rRNA gene (18S rDNA). All PCR-positive samples were further subtyped using PCR and sequencing of the 60 kDa glycoprotein gene ( gp60 ). A chi-square test and odds ratio (OR) analysis were used to compare infection rates and assess the risk of C . felis infection between domestic and stray cats. Results A total of 711 faecal samples were collected – 350 from domestic cats and 361 from stray cats. The overall infection rate of Cryptosporidium spp. was 4.5% (32/711), with stray cats being significantly more frequently infected (6.7%) than domestic cats (2.3%). Oocysts of Cryptosporidium spp. were not detected microscopically in any of the samples. There were no significant differences between the infection rates in the three countries. All isolates were identified as C . felis , and analysis of the gp60 gene revealed five different subtypes, all belonging to the XIXa subtype family of C . felis . These subtypes formed five well-supported phylogenetic clusters, none of which had been previously reported worldwide. Only one subtype was found in domestic cats, whereas all five subtypes were found in stray cats. The subtypes identified in stray cats showed a clear geographical distribution in the study region. Conclusions The results of this study extend our knowledge of the genetic variability of C . felis and indicate a possible geographical distribution of the detected subtypes. The significance of the observed genetic variability in terms of geographical distribution, host specificity and zoonotic potential remains unclear and requires further investigation. Graphical Abstract

We identified an unusual subtype of a Cryptosporidium sp. horse genotype as the cause of cryptosporidiosis in a 13-year-old girl in Poland who was undergoing immunosuppressive treatment for juvenile rheumatoid arthritis and Crohn's disease. The same subtype was identified in a horse the girl had ridden.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

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. 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. 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. 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. 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.

Abstract Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

The diversity and biology of Cryptosporidium that is specific for rats (Rattus spp.) are not well studied. We examined the occurrence and genetic diversity of Cryptosporidium spp. in wild brown rats (Rattus norvegicus) by microscopy and polymerase chain reaction (PCR)/sequencing targeting the small subunit rDNA (SSU), actin and HSP70 genes. Out of 343 faecal samples tested, none were positive by microscopy and 55 were positive by PCR. Sequence analysis of SSU gene revealed the presence of Cryptosporidium muris (n = 4), C. andersoni (n = 3), C. ryanae (n = 1), C. occultus (n = 3), Cryptosporidium rat genotype I (n = 23), Cryptosporidium rat genotype IV (n = 16) and novel Cryptosporidium rat genotype V (n = 5). Spherical oocysts of Cryptosporidium rat genotype I obtained from naturally-infected rats, measuring 4.4–5.4 μm × 4.3–5.1 μm, were infectious to the laboratory rats, but not to the BALB/c mice (Mus musculus) nor Mongolian gerbils (Meriones unguiculatus). The prepatent period was 3 days post infection and the patent period was longer than 30 days. Naturally- and experimentally-infected rats showed no clinical signs of disease. Percentage of nucleotide similarities at the SSU, actin, HSP70 loci between C. ratti n. sp. and the rat derived C. occultus and Cryptosporidium rat genotype II, III, IV, and V ranged from 91.0 to 98.1%. These genetic variations were similar or greater than that observed between closely related species, i.e. C. parvum and C. erinacei (93.2–99.5%). Our morphological, genetic and biological data support the establishment of Cryptosporidium rat genotype I as a new species, Cryptosporidium ratti n. sp.