Changshen Ning

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.

Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China

Cryptosporidium and Giardia infections are common causes of diarrhea worldwide. To better understand the transmission of human cryptosporidiosis and giardiasis in Henan, China, 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens were characterized at the species/genotype and subtype levels. Cryptosporidium specimens were analyzed by DNA sequencing of the small subunit rRNA and 60kDa glycoprotein genes. Among those genotyped, nine belonged to C. hominis and one C. felis, with the former belonging to three subtype families: Ia, Ib, and Id. The three Ib subtypes identified, IbA16G2, IbA19G2, and IbA20G2, were very different from the two common Ib subtypes (IbA9G3 and IbA10G2) found in other areas of the world. The distribution of Giardia duodenalis genotypes and subtypes was assessed by sequence analysis of the triosephosphate isomerase (tpi) gene. The assemblages A (eight belonging to A-I and four A-II) and B (belonging to six new subtypes) were found in 12 and six specimens, respectively. More systematic studies are needed to understand the transmission of Cryptosporidium and G. duodenalis in humans in China.

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.

Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus)

The Cryptosporidium in the small intestine of domestic mice (Mus musculus) was initially described as Cryptosporidium parvum. Recent genetic and biologic characterization of Cryptosporidium isolates indicate that domestic mice are infected with several morphologically indistinguishable intestinal Cryptosporidium parasites with different host specificities, including C. parvum sensu stricto, mouse genotype I, and mouse genotype II. In this study, the morphological, biological, and genetic characteristics of the Cryptosporidium mouse genotype I are described. As a full re-description of C. parvum was made in 1985 for isolates from calves and humans and the name C. parvum has been widely used for the parasite that is infectious to both ruminants and humans, the mouse genotype I is named as Cryptosporidium tyzzeri. Oocysts of the new species (4.64±0.05 μm ×4.19±0.06 μm, with a mean shape index of 1.11±0.02; n=69) are slightly smaller than those of the re-described C. parvum. The prepatent period was six and seven days, and the patent period was 24-28 and 28-29 days in neonatal and adult mice, respectively. Oocysts were not infectious to lambs and calves. Light, transmission electron and scanning electron microscopy studies of the new species showed the presence of developmental stages in the microvillar brush border of the jejunum and ileum of experimentally infected mice, with the infection most intensive in the ileum. It had nucleotide sequences significantly different from C. parvum at the small subunit rRNA, 70 kDa heat shock protein, oocyst wall protein, actin, and the 60 kDa glycoprotein genes. Based on the morphological, genetic, and biological data and in compliance of established Cryptosporidium species naming criteria, this geographically widespread parasite is named as a new species in honor of Ernest Edward Tyzzer, who pioneered Cryptosporidium research.

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.

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.

Predomination and New Genotypes of Enterocytozoon bieneusi in Captive Nonhuman Primates in Zoos in China: High Genetic Diversity and Zoonotic Significance

To appreciate the genetic diversity and zoonotic implications of Enterocytozoon bieneusi in nonhuman primates (NHPs) in zoos, we genotyped E. bieneusi in captive NHPs in seven zoos located at six major cities in China, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 496 fecal specimens from 36 NHP species under nine families were analyzed and E. bieneusi was detected in 148 (29.8%) specimens of 25 NHP species from six families, including Cercopithecidae (28.7%), Cebidae (38.0%), Aotidae (75.0%), Lemuridae (26.0%), Hylobatidae (50.0%) and Hominidae (16.2%) (P = 0.0605). The infection rates were 29.0%, 15.2%, 18.2%, 37.3%, 29.2%, 37.7% and 44.8% in Shijiazhuang Zoo, Wuhan Zoo, Taiyuan Zoo, Changsha Wild Animal Zoo, Beijing Zoo, Shanghai Zoo and Shanghai Wild Animal Park, respectively (P = 0.0146). A total of 25 ITS genotypes were found: 14 known (D, O, EbpC, EbpA, Type IV, Henan-IV, BEB6, BEB4, Peru8, PigEBITS5, EbpD, CM1, CM4 and CS-1) and 11 new (CM8 to CM18). Genotype D was the most prevalent one (40/148), followed by CM4 (20/148), CM1 (15/148), O (13/148), CM16 (13/148), EbpC (11/148). Of them, genotypes D, EbpC, CM4 and O were widely distributed in NHPs (seen in 9 to 12 species) whereas genotypes CM1 and CM16 were restricted to one to three NHP species. In phylogenetic analysis, 20 genotypes (121/148, 81.8%), excluding genotypes BEB4, BEB6, CM9, CM4 and CM18, belonged to group 1 with zoonotic potential. New genotype CM9 clustered in group 2 with BEB4 and BEB6. The remaining two genotypes CM4 and CM18 formed new cluster (group 9) in between two other genotypic clusters found in primates. The findings of high diversity in E. bieneusi genotypes and their zoonotic potentiality concluded the importance of captive NHPs as reservoir hosts for human microsporidiosis.

Cryptosporidium parvum IId family: clonal population and dispersal from Western Asia to other geographical regions

In this study, 111 Cryptosporidium parvum IId isolates from several species of animals in China, Sweden, and Egypt were subtyped by multilocus sequence typing (MLST). One to eleven subtypes were detected at each of the 12 microsatellite, minisatellite, and single nucleotide polymorphism (SNP) loci, forming 25 MLST subtypes. Host-adaptation and significant geographical segregation were both observed in the MLST subtypes. A clonal population structure was seen in C. parvum IId isolates from China and Sweden. Three ancestral lineages and the same RPGR sequence were shared by these isolates examined. Therefore, the present genetic observations including the higher nucleotide diversity of C. parvum IId GP60 sequences in Western Asia, as well as the unique distribution of IId subtypes (almost exclusively found in Asia, Europe, and Egypt) and in combination with the domestication history of cattle, sheep, and goats, indicated that C. parvum IId subtypes were probably dispersed from Western Asia to other geographical regions. More population genetic structure studies involving various C. parvum subtype families using high-resolution tools are needed to better elucidate the origin and dissemination of C. parvum in the world.