Longxian Zhang

Zoonotic Cryptosporidium Species and Enterocytozoon bieneusi Genotypes in HIV-Positive Patients on Antiretroviral Therapy

ABSTRACT Molecular diagnostic tools have been used increasingly in the characterization of the transmission of cryptosporidiosis and microsporidiosis in developing countries. However, few studies have examined the distribution of Cryptosporidium species and Enterocytozoon bieneusi genotypes in AIDS patients receiving antiretroviral therapy. In the present study, 683 HIV-positive patients in the National Free Antiretroviral Therapy Program in China and 683 matched HIV-negative controls were enrolled. Cryptosporidium species and subtypes and Enterocytozoon bieneusi genotypes were detected and differentiated by PCR and DNA sequencing. The infection rates were 1.5% and 0.15% for Cryptosporidium and 5.7% and 4.2% for E. bieneusi in HIV-positive and HIV-negative participants, respectively. The majority (8/11) of Cryptosporidium cases were infections by zoonotic species, including Cryptosporidium meleagridis (5), Cryptosporidium parvum (2), and Cryptosporidium suis (1). Prevalent E. bieneusi genotypes detected, including EbpC (39), D (12), and type IV (7), were also potentially zoonotic. The common occurrence of EbpC was a feature of E. bieneusi transmission not seen in other areas. Contact with animals was a risk factor for both cryptosporidiosis and microsporidiosis. The results suggest that zoonotic transmission was significant in the epidemiology of both diseases in rural AIDS patients in China.

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.

Characteristics of Cryptosporidium Transmission in Preweaned Dairy Cattle in Henan, China

ABSTRACT To estimate the prevalence and public health significance of cryptosporidiosis in preweaned calves in China, 801 fecal samples from eight farms in seven areas in Henan Province were examined for Cryptosporidium oocysts. The overall infection rate of Cryptosporidium was 21.5%, with the farm in Xinxiang having the highest prevalence (40%). No significant difference in infection rates was observed between seasons. Cryptosporidium spp. were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene and DNA sequencing of the 60-kDa glycoprotein (gp60) gene. The SSU rRNA-based PCR identified four Cryptosporidium species, including Cryptosporidium parvum (54/172), C. bovis (65/172), C. ryanae (19/172), and C. andersoni (12/172), and the occurrence of infections with mixed species (22/172). The earliest detection of C. bovis was in calves of 1 week of age, showing that the prepatent period was shorter than the previously stated 10 to 12 days. Infections with C. parvum peaked in summer, whereas C. bovis dominated in autumn and winter. There was no apparent difference in the age of cattle infected with either C. parvum or C. bovis. Sequencing analysis of the gp60 gene showed all 67 C. parvum samples belonged to subtype IIdA19G1. These findings suggested that the transmission of Cryptosporidium spp. in preweaned calves in Henan, China, appeared to be different from other areas both at genotype and subtype levels. Further molecular epidemiologic studies (including samples from both calves and humans) are needed to elucidate the transmission dynamics and public significance of C. parvum in cattle in China.

Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China.

ABSTRACT Enterocytozoon bieneusi is an important zoonotic pathogen. To assess the human-infective potential of E. bieneusi in nonhuman primates (NHPs), we examined the prevalence and genotype distribution of E. bieneusi in 23 NHP species by PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS). A total of 1,386 fecal specimens from NHPs from five provinces in China were examined, and E. bieneusi was detected in 158 (11.4%) specimens from five NHP species, including cynomolgus monkey (67.7%), rhesus macaque (8.8%), Japanese macaque (33.3%), white-headed langur (13.6%), and golden snub-nosed monkey (3.5%) (P < 0.0001). The infection rates were 70.2%, 21.5%, 8.5%, 7.5%, and 5.6% in Guangdong, Yunnan, Guangxi, Henan, and Sichuan Provinces, respectively (P < 0.0001). The prevalence was significantly higher in captive (13.7%) than in free-range (5.0%) animals (P < 0.0001). Altogether, 16 ITS genotypes were observed, including nine known genotypes (IV, D, Henan V, Peru8, PigEBITS7, EbpC, Peru11, BEB6, and I) and seven new genotypes (CM1 to CM7). The common genotypes included CM1, IV, and D, which were detected in 43, 31, and 30 specimens, respectively. Phylogenetic analysis revealed that seven known genotypes (but not BEB6 and I) and four new genotypes (CM1, CM2, CM3, and CM6) belonged to the previously described group 1 with zoonotic potential. Genotypes CM5 and CM7 clustered with group 2, whereas genotype CM4 did not belong to any of the previously proposed groups. It was concluded that humans and NHPs residing in the same geographical location shared the same E. bieneusi genotypes, indicating a potential role of these animals in the zoonotic transmission of E. bieneusi.

Cryptosporidium spp. in Wild, Laboratory, and Pet Rodents in China: Prevalence and Molecular Characterization

ABSTRACT To understand the prevalence of Cryptosporidium infection in rodents in China and to assess the potential role of rodents as a source for human cryptosporidiosis, 723 specimens from 18 rodent species were collected from four provinces of China and examined between August 2007 and December 2008 by microscopy after using Sheathers sugar flotation and modified acid-fast staining. Cryptosporidium oocysts were detected in 83 specimens, with an overall prevalence of 11.5%. Phodopus sungorus, Phodopus campbelli, and Rattus tanezumi were new reported hosts of Cryptosporidium. The genotypes and subtypes of Cryptosporidium strains in microscopy-positive specimens were further identified by PCR and sequence analysis of the small subunit rRNA and the 60-kDa glycoprotein (gp60) genes. In addition to Cryptosporidium parvum, C. muris, C. andersoni, C. wrairi, ferret genotype, and mouse genotype I, four new Cryptosporidium genotypes were identified, including the hamster genotype, chipmunk genotype III, and rat genotypes II and III. Mixed Cryptosporidium species/genotypes were found in 10.8% of Cryptosporidium-positive specimens. Sequence analysis of the gp60 gene showed that C. parvum strains in pet Siberian chipmunks and hamsters were all of the subtype IIdA15G1, which was found previously in a human isolate in The Netherlands and lambs in Spain. The gp60 sequences of C. wrairi and the Cryptosporidium ferret genotype and mouse genotype I were also obtained. These findings suggest that pet rodents may be potential reservoirs of zoonotic Cryptosporidium species and subtypes.

Cryptosporidium spp. in pet birds: Genetic diversity and potential public health significance

To characterize the prevalence and assess the zoonotic transmission burden of Cryptosporidium species/genotypes in pet birds in Henan, China, 434 fecal samples were acquired from 14 families of birds in pet shops. The overall prevalence of Cryptopsoridium was 8.1% (35/434) by the Sheathers sugar flotation technique. The Cryptosporidium-positive samples were analyzed by DNA sequence analysis of the small subunit (SSU) rRNA gene. Three Cryptosporidium species and two genotypes were identified, including C. baileyi (18/35 or 51.4%) in five red-billed leiothrixes (Leiothrix lutea), four white Java sparrows (Padda oryzivora), four common mynas (Acridotheres tristis), two zebra finches (Taeniopygia guttata), a crested Lark (Galerida cristata), a Gouldian finch (Chloebia gouldiae), and a black-billed magpie (Pica pica); Cryptosporidium meleagridis (3/35 or 8.6%) in a Bohemian waxwing (Bombycilla garrulus), a Rufous turtle dove (Streptopelia orientalis), and a fan-tailed pigeon (Columba livia); Cryptosporidium galli (5/35 or 14.3%) in four Bohemian waxwings (Bombycilla garrulus) and a silver-eared Mesia (Leiothrix argentauris); Cryptosporidium avian genotype III (3/35 or 8.6%) in two cockatiels (Nymphicus hollandicus) and a red-billed blue magpie (Urocissa erythrorhyncha); and Cryptosporidium avian genotype V (6/35 or 17.1%) in six cockatiels (Nymphicus hollandicus). Among the pet birds, 12 species represented new hosts for Cryptosporidum infections. The presence of C. meleagridis raises questions on potential zoonotic transmission of cryptosporidiosis from pet birds to humans.

Genetic Diversity in Enterocytozoon bieneusi Isolates from Dogs and Cats in China: Host Specificity and Public Health Implications

ABSTRACT To explore the genetic diversity, host specificity, and zoonotic potential of Enterocytozoon bieneusi, feces from 348 stray and pet dogs and 96 pet cats from different locations in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 15.5% of the dogs, including 20.5% of stray dogs and 11.7% of pet dogs, and in 11.5% of the pet cats. Higher infection rates were recorded in the >2-year and the 1- to 2-year age groups in dogs and cats, respectively. Altogether, 24 genotypes, including 11 known and 13 new, were detected in 65 infected animals. In 54 positive dogs, 18 genotypes, 9 known (PtEbIX, O, D, CM1, EbpA, Peru8, type IV, EbpC, and PigEBITS5) and 9 new (CD1 to CD9), were found. In contrast, 8 genotypes, 4 known (D, BEB6, I, and PtEbIX) and 4 new (CC1 to CC4), were identified in 11 infected cats. The dominant genotype in dogs was PtEbIX (26/54). Phylogenetic analysis revealed that 8 known genotypes (D, Peru8, type IV, CM1, EbpC, PigEBITS5, O, and EbpA) and 7 new genotypes (CD1 to CD4 and CC2 to CC4) were the members of zoonotic group 1, whereas genotypes CD7, CD8, and CD9 together with PtEbIX belonged to the dog-specific group, and genotypes CD6 and CC1 were placed in group 2 with BEB6 and I. Conversely, genotype CD5 clustered with CM4 without belonging to any previous groups. We conclude that zoonotic genotypes are common in dogs and cats, as are host-specific genotypes in dogs.

Distribution and Genetic Characterizations of Cryptosporidium spp. in Pre-Weaned Dairy Calves in Northeastern China’s Heilongjiang Province

Background Cryptosporidium spp. are common parasites of humans and animals. Farm animals, especially pre-weaned calves, are considered to be one of main animal reservoir hosts of Cryptosporidium in the transmission of human cryptosporidiosis. The aim of this study was to determine the distribution and genotypes of Cryptosporidium spp. in pre-weaned calves using molecular tools and to assess zoonotic transmission and elucidate the public health significance in northeastern China. Methodology/Principal Findings A total of 151 fecal specimens from pre-weaned calves were collected in Heilongjiang Province and were screened for Cryptosporidium by PCR. The average prevalence of Cryptosporidium was 47.68% (72/151). Cryptosporidium spp. were characterized by DNA sequencing of the small subunit (SSU) rRNA gene and the 60-kDa glycoprotein (gp60) gene. Based on the SSU rRNA gene, five Cryptosporidium spp. were identified, including C. bovis (n = 34), C. andersoni (n = 26), C. ryanae (n = 5), C. meleagridis (n = 5) and C. parvum (n = 2). The SSU rRNA nucleotide sequences were identical to each other, respectively, within C. ryanae, C. parvum, C. meleagridis and C. andersoni. Four types of C. bovis were found in the SSU rRNA gene, with two novel types. The gp60 gene was successfully sequenced in one C. parvum isolate and three C. meleagridis isolates, with IIdA19G1 for C. parvum and IIIeA22G2R1 for C. meleagridis. Conclusion/Significance Molecular analysis indicates that Cryptosporidium spp. are endemic in pre-weaned calves in Heilongjiang Province. The findings of C. parvum and C. meleagridis suggested the possibility of zoonotic transmission and public health significance. The transmission dynamics of C. parvum and C. meleagridis needed to be clarified by further molecular epidemiologic studies from humans and animals. Whether calves could act as the natural reservoirs of C. meleagridis needed to be confirmed by more systematic experimental infection studies.

Extended outbreak of cryptosporidiosis in a pediatric hospital, China.

Four Cryptosporidium spp. and 6 C. hominis subtypes were isolated from 102 of 6,284 patients in 3 pediatric hospitals in People’s Republic of China. A cryptosporidiosis outbreak was identified retrospectively. The outbreak lasted >1 year and affected 51.4% of patients in 1 hospital ward, where 2 C. hominis subtypes with different virulence were found.

Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world.