Molecular investigation of Cryptosporidium in farmed chickens in Hubei Province, China, identifies 'zoonotic' subtypes of C. meleagridis.
ABSTRACT: BACKGROUND:Cryptosporidium is a key genus of parasitic protists that infect humans and other vertebrates (mammals and birds). Birds are typically infected with C. avium, C. baileyi, C. galli and/or C. meleagridis, the latter of which is recognised as being zoonotic. Stimulated by the previous finding of C. meleagridis subtypes IIIbA21G1R1, IIIbA22G1R1 and IIIbA26G1R1 in diarrhoeic children in Wuhan city and environs in Hubei Province, China, we performed a molecular epidemiological survey to explore whether these or similar subtypes might occur in farmed chickens in this province. METHODS:PCR-coupled sequencing analyses of regions in the small subunit (SSU) of the nuclear ribosomal RNA and 60 kDa glycoprotein (gp60) genes were utilised to characterise Cryptosporidium in faecal samples from chickens (n = 471) from 14 farms from six distinct regions in Hubei Province. RESULTS:Cryptosporidium baileyi (33/471; 7.0%) and C. meleagridis (15/471; 3.2%) were identified in chickens on eight farms in five of the six distinct geographical regions. No significant age-associated difference in the prevalence of C. baileyi was evident, whereas the prevalence of C. meleagridis was significantly higher in younger (? 4 months) than in older chickens (> 4 months). For C. meleagridis, two subtype families, IIIb and IIIe, were defined; some of the subtypes (i.e. IIIbA26G1R1b and IIIbA22G1R1c) characterised here matched those identified previously in diarrhoeic children in Wuhan. CONCLUSIONS:This is the first molecular study reporting the genetic identity and prevalence of C. baileyi and C. meleagridis in chickens in Hubei. The findings suggest that C. meleagridis subtypes IIIbA26G1R1b and IIIbA22G1R1c are cross-transmissible between chickens and humans, raising awareness about the significance of birds as potential reservoirs of zoonotic variants of Cryptosporidium. Future studies might focus on investigating the prevalence of 'zoonotic' subtypes of Cryptosporidium meleagridis in various species of wild and domesticated birds, and on comparing them with those found in humans in China and other countries.
Project description:This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.
Project description:Cryptosporidium is an opportunistic parasite that has been reported in >30 avian hosts worldwide, however, there is no information regarding Cryptosporidium spp. in poultry in Bangladesh. Accordingly, we investigated the prevalence of Cryptosporidium spp. in poultry at open live bird markets in Bangladesh. A total of 197 samples were randomly collected from poultry at open live bird markets in Bangladesh and screened for the detection of Cryptosporidium. Initial microscopic examination revealed Cryptosporidium spp. was observed in 19.8% (39/197) of the poultry specimens. Subsequent nested PCR targeting the 18S rRNA gene revealed that 15.7% (31/197) of the samples were Cryptosporidium positive. Of these 31 samples, 17 were Cryptosporidium baileyi (8.7%), 12 were Cryptosporidium meleagridis (6.0%), and 2 were Cryptosporidium parvum (1.0%). Nucleotide sequence analysis of the GP60 gene of the C. meleagridis revealed that two subtypes (IIIbA21G1R1 and IIIbA23G1R1), which were found in broiler, native and sonali chickens and a pigeon, matched those previously reported in humans and poultry. We identified two novel subtypes (IIIbA21G2R1 and IIIbA20G2R1) in sonali chickens, a broiler chicken and a layer chicken. We also amplified the GP60 gene of C. parvum and found two subtypes (IIaA11G2R1 and IIaA13G2R1) in a sonali and a broiler chicken that were previously reported in calf. These findings suggest that poultry can be a source of cryptosporidial infections for humans and animals in Bangladesh. This is the first molecular investigation of Cryptosporidium genotypes and subtypes in poultry at open live bird markets in Bangladesh.
Project description:Cryptosporidium is the most common protozoan that can infect a wide range of animals, including mammals and birds. Avian Cryptosporidium spp. can cause enteric and respiratory diseases which can be fatal in birds and some species are zoonotic. Companion birds have the potential as reservoir due to their close contact with humans. Pet shops are the major source of companion birds. However, few reports are available regarding Cryptosporidium spp. infection among companion birds kept in pet shops. The present study reports the prevalence and molecular characteristics of Cryptosporidium spp. among companion birds kept in pet shops in Japan. A total of 265 fresh fecal samples were obtained from birds kept in 4 pet shops; these birds belonged to 41 species in 3 bird orders. A nested polymerase chain reaction (PCR) assay targeting the small subunit rRNA gene was employed for the detection of Cryptosporidium spp. A total of 24 samples (9.1%) were positive, and Cryptosporidium spp. were detected from all pet shops. The prevalence of Cryptosporidium spp. in each of the bird orders was 6.5% (10/153) in Psittaciformes, 14.4% (13/90) in Passeriformes, and 4.5% (1/22) in Galliformes. Based on sequence analysis, 13 (54.2%) isolates were classified to C. galli, 8 (33.3%) were avian genotype III, and the remaining 3 (12.5%) were C. baileyi. No infection with zoonotic C. meleagridis and no coinfection with multiple Cryptosporidium spp. and/or genotypes were observed. The zoonotic potential of Cryptosporidium spp. infecting companion birds kept in pet shops in Japan is likely to be low.
Project description:Molecular epidemiology of cryptosporidiosis is an active research area in China. The use of genotyping and subtyping tools in prevalence studies has led to the identification of unique characteristics of Cryptosporidium infections in humans and animals. Human cryptosporidiosis in China is exemplified by the high diversity of Cryptosporidium spp. at species and subtype levels, with dominant C. hominis and C. parvum subtypes being rarely detected in other countries. Similarly, preweaned dairy calves, lambs, and goat kids are mostly infected with non-pathogenic Cryptosporidium species (C. bovis in calves and C. xiaoi in lambs and goat kids), with C. parvum starting to appear in dairy calves as a consequence of concentrated animal feeding operations. The latter Cryptosporidium species is dominated by IId subtypes, with IIa subtypes largely absent from the country. Unlike elsewhere, rodents in China appear to be commonly infected with C. parvum IId subtypes, with identical subtypes being found in these animals, calves, other livestock, and humans. In addition to cattle, pigs and chickens appear to be significant contributors to Cryptosporidium contamination in drinking water sources, as reflected by the frequent detection of C. suis, C. baileyi, and C. meleagridis in water samples. Chinese scientists have also made significant contributions to the development of new molecular epidemiological tools for Cryptosporidium spp. and improvements in our understanding of the mechanism involved in the emergence of hyper-transmissible and virulent C. hominis and C. parvum subtypes. Despite this progress, coordinated research efforts should be made to address changes in Cryptosporidium transmission because of rapid economic development in China and to prevent the introduction and spread of virulent and zoonotic Cryptosporidium species and subtypes in farm animals.
Project description:Cryptosporidium spp., Giardia spp. and microsporidia are important intestinal protozoa responsible for diarrhea in humans and other mammals. China is a major chicken-raising country, and studies on these protozoa in chickens have important public health significance. Here, we investigated the prevalence and genetic characterization of these parasites in chickens from Ezhou City, Hubei Province, China. In total, 206 stool specimens were collected from chickens in four villages of Ezhou between July 2014 and February 2015. Genomic DNA of each specimen was tested by nested PCR based on the Cryptosporidium small subunit rRNA gene, the Giardia intestinalis triose phosphate isomerase gene, and the internal transcribed spacer of the Enterocytozoon bieneusi rRNA gene, respectively. The public health significance of G. intestinalis and E. bieneusi identified in our study was evaluated via phylogenetic analysis. The infection rates were determined to be 2.43% (5/206), 8.25% (17/206), and 1.94% (4/206) for Cryptosporidium, G. intestinalis, and E. bieneusi, respectively. One sample showed coinfection with G. intestinalis and E. bieneusi. Meanwhile, sequence analysis of the PCR-positive samples showed that the Cryptosporidium was C. baileyi, G. intestinalis was assemblage C, and E. bieneusi was genotype D and novel genotype EZ0008. This is the first report of zoonotic G. intestinalis assemblage C in chickens in the world, and the first report of zoonotic E. bieneusi genotype D in chickens in China. These findings indicate new transmission dynamics and molecular epizootiology.
Project description:Cryptosporidium is one of the most important parasites in poultry, and this pathogen can infect more than 30 avian species. The present study investigated the infection rate of Cryptosporidium among broiler chicken flocks. A total of 385 fecal samples from broiler chickens in 7 regions of Zhejiang Province collected from November 2010 to January 2012 were examined by microscopy. Thirty-eight (10%) samples were positive for Cryptosporidium infection, and 3 genotypes (Cryptosporidium baileyi, Cryptosporidium meleagridis, and avian genotype II) were identified by PCR and sequencing. A phylogenetic tree of the isolates was analyzed. These results suggest that cryptosporidiosis is widespread in poultry in Zhejiang Province, and is a potential threat to public health as well as the economy. This is the first report about the infection rate and molecular characterization of Cryptosporidium in broiler chickens in Zhejiang.
Project description:<h4>Background</h4>Despite their wide occurrence, cryptosporidiosis and giardiasis are considered neglected diseases by the World Health Organization. The epidemiology of these diseases and microsporidiosis in humans in developing countries is poorly understood. The high concentration of pathogens in raw sewage makes the characterization of the transmission of these pathogens simple through the genotype and subtype analysis of a small number of samples.<h4>Methodology/principal findings</h4>The distribution of genotypes and subtypes of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in 386 samples of combined sewer systems from Shanghai, Nanjing and Wuhan and the sewer system in Qingdao in China was determined using PCR-sequencing tools. Eimeria spp. were also genotyped to assess the contribution of domestic animals to Cryptosporidium spp., G. duodenalis, and E. bieneusi in wastewater. The high occurrence of Cryptosporidium spp. (56.2%), G. duodenalis (82.6%), E. bieneusi (87.6%), and Eimeria/Cyclospora (80.3%) made the source attribution possible. As expected, several human-pathogenic species/genotypes, including Cryptosporidium hominis, Cryptosporidium meleagridis, G. duodenalis sub-assemblage A-II, and E. bieneusi genotype D, were the dominant parasites in wastewater. In addition to humans, the common presence of Cryptosporidium spp. and Eimeria spp. from rodents indicated that rodents might have contributed to the occurrence of E. bieneusi genotype D in samples. Likewise, the finding of Eimeria spp. and Cryptosporidium baileyi from birds indicated that C. meleagridis might be of both human and bird origins.<h4>Conclusions/significance</h4>The distribution of Cryptosporidium species, G. duodenalis genotypes and subtypes, and E. bieneusi genotypes in urban wastewater indicates that anthroponotic transmission appeared to be important in epidemiology of cryptosporidiosis, giardiasis, and microsporidiosis in the study areas. The finding of different distributions of subtypes between Shanghai and Wuhan was indicative of possible differences in the source of C. hominis among different areas in China.
Project description:Genome sequencing has greatly contributed to our understanding of parasitic protozoa. This is particularly the case for Cryptosporidium species (phylum Apicomplexa) which are difficult to propagate. Because of their polymorphic nature, simple sequence repeats have been used extensively as genotypic markers to differentiate between isolates, but no global analysis of amino acid repeats in Cryptosporidium genomes has been reported. Taking advantage of several newly sequenced Cryptosporidium genomes, a comparative analysis of single-amino-acid repeats (SAARs) in seven species was undertaken. This analysis revealed a striking difference between the SAAR profile of the gastric and intestinal species which infect mammals and one species which infects birds. In average, total SAAR length in gastric species is only 25% of the cumulative SAAR length in the genome of Cryptosporidium parvum, Cryptosporidium hominis and Cryptosporidium meleagridis, species infectious to humans. The SAAR profile in the avian parasite Cryptosporidium baileyi stands out due to the presence of long asparagine repeats. Cryptosporidium baileyi proteins with repeats ?20 residues are significantly enriched in regulatory functions. As postulated for the related apicomplexan species Plasmodium falciparum, these observations suggest that Cryptosporidium SAARs evolve in response to selective pressure. The putative selective mechanisms driving SAAR evolution in Cryptosporidium species are unknown.
Project description:To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.
Project description:<h4>Background</h4>In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays.<h4>Methodology/principal findings</h4>Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1-10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67-55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79-11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16-7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58-0.95).<h4>Conclusions/significance</h4>This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites.