Yujuan Shen

Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China.

BackgroundCryptosporidium spp., Enterocytozoon spp., Giardia spp. and Cyclospora spp. are important intestinal protozoan parasites causing diarrhea in humans, livestocks and wildlife and have a significant impact on public health. No reports exist about simultaneous prevalence rates or genotyping data of these four parasites in outpatients from China.MethodsFecal specimens from 252 diarrhea patients in a pediatric clinic (n = 169) and an intestinal clinic (n = 83) of a hospital in Shanghai, China, were collected between October 2012 and March 2013. All samples were examined for the presence of the four parasites by using molecular methods.ResultsIn total, 76/252 (30.16%) patients were positive for at least one intestinal parasite, of which Cryptosporidium spp., Enterocytozoon bieneusi and Giardia intestinalis were detected by nested PCR in 34 (13.49%), 34 (13.49%) and 17 (6.75%) of the fecal specimens, respectively. Sequence analysis showed that all Cryptosporidium-positive specimens were C. andersoni and that most G. intestinalis- positive patients were infected by assemblage C, which is usually found in canids, while only one sample was from assemblage B. Eight patients were co-infected with Cryptosporidium spp. and Enterocytozoon, while one was co-infected with Cryptosporidium and Giardia.ConclusionsThe patients infected with Cryptosporidium and Enterocytozoon bieneusi had higher infection rates in winter than in spring in this area. Data indicated that C. andersoni is the fourth major Cryptosporidium species infecting humans in addition to C. hominis, C. parvum and C. meleagridis. Our study also revealed a short-term outbreak of cryptosporidiosis and microsporidiosis and sporadic cases of giardiasis that occurred among humans in Shanghai, China.

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.

Genetic characterizations of Giardia duodenalis in sheep and goats in Heilongjiang Province, China and possibility of zoonotic transmission.

Background Giardia duodenalis is a widespread intestinal protozoan of both humans and mammals. To date, few epidemiological studies have assessed the potential and importance of zoonotic transmission; and the human giardiasis burden attributable to G. duodenalis of animal origin is unclear. No information about occurrence and genotyping data of sheep and goat giardiasis is available in China. The aim of the present study was to determine prevalence and distribution of G. duodenalis in sheep and goats in Heilongjiang Province, China, and to characterize G. duodenalis isolates and assess the possibility of zoonotic transmission. Methodology/Principal Findings A total of 678 fecal specimens were collected from sheep and goats on six farms ranging in age from one month to four years in Heilongjiang Province, China. The average prevalence of G. duodenalis infection was 5.0% (34/678) by microscopy after Lugols iodine staining, with 5.6% (30/539) for the sheep versus 2.9% (4/139) for the goats. Molecular analysis was conducted on 34 G. duodenalis isolates based on the triosephosphate isomerase (tpi) gene. 29 tpi gene sequences were successfully obtained and identified as assemblages A (n = 4), B (n = 2) and E (n = 23). High heterogeneity was observed within assemblage E at the tpi locus, with five novel subtypes found out of seven subtypes. Two subtypes of assemblage A were detected, including subtype AI (n = 3) and a novel subtype (designated as subtype AIV) (n = 1). Two assemblage B isolates were identical to each other in the tpi gene sequences. Conclusions/Significance This is the first report of G. duodenalis infections in sheep and goats in China. The present data revealed the unique endemicity on prevalence, distribution and genetic characterization of G. duodenalis in sheep and goats in Heilongjiang Province. The findings of assemblages A and B in sheep and goats implied the potential of zoonotic transmission.

Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission.

Background Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China. Methodology/Principal Findings 378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheathers sugar flotation technique and Lugols iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples. Conclusions/Significance The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.

High Prevalence of Enterocytozoon bieneusi in Asymptomatic Pigs and Assessment of Zoonotic Risk at the Genotype Level

ABSTRACT Enterocytozoon bieneusi is an emerging and clinically significant enteric parasite infecting humans and animals and can cause life-threatening diarrhea in immunocompromised people. Pigs are considered to be one of the main reservoir hosts of E. bieneusi based on their high prevalence rates and zoonotic genotypes in pigs. As an opportunistic pathogen, E. bieneusi infection of pigs can be inapparent, which leads to neglect in detecting this parasite in pigs and assessing the epidemiological role of pigs in the transmission of human microsporidiosis. In the present study, 95 healthy pigs aged 2 or 3 months were randomly selected from three areas in Heilongjiang Province, China. E. bieneusi isolates were identified and genotyped based on the small-subunit (SSU) rRNA and internal transcribed spacer (ITS) regions of the rRNA gene by PCR and sequencing. A high prevalence of E. bieneusi was observed, 83.2% (79/95) at the SSU rRNA locus versus 89.5% (85/95) at the ITS locus. Ten ITS genotypes were obtained, comprising six known genotypes—EbpA (n = 30), D (n = 19), H (n = 18), O (n = 11), CS-1 (n = 1), and LW1 (n = 1)—and four novel genotypes named HLJ-I to HLJ-IV; 70.6% (60/85) of E. bieneusi genotypes were zoonotic (genotypes EbpA, D, and O). The findings of a high prevalence of E. bieneusi in pigs and a large percentage of zoonotic genotypes indicate that pigs may play a role in the transmission of E. bieneusi to humans and may become an important source of water contamination in our investigated areas.

Cryptosporidium andersoni as a novel predominant Cryptosporidium species in outpatients with diarrhea in Jiangsu Province, China

BackgroundCryptosporidium hominis and C. parvum are usually considered to be the major pathogens responsible for human cryptosporidiosis. However, there have been few studies regarding the molecular epidemiology of Cryptosporidium in human infections in China. Here we investigated Cryptosporidium infection in patients with diarrhea, in Danyang Hospital of Jiangsu Province, China, at the genotype level.MethodsA total of 232 stool specimens were collected from outpatients with diarrhea in Danyang Hospital of Jiangsu Province, China, from February 2012 to January 2013. Each specimen was stained from direct fecal smears and examined for Cryptosporidium using modified acid fast staining and microscopy. Moreover, genomic DNA of each fecal sample was screened for the presence of Cryptosporidium with nested PCR, which was genotyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA).ResultsThe average infection rate of Cryptosporidium was 1.3% (3/232) by microscopy and subjected to PCR amplification of the SSU rRNA gene of Cryptosporidium, with 9.91% (23/232) being positive for Cryptosporidium with a significant peak in autumn. Based on the SSU rRNA gene, two Cryptosporidium spp. were identified, including C. andersoni (n =21) and C. hominis (n =2). Two types of C. andersoni, designated as A370+ and A370- , were found in the SSU rRNA gene in our present study, which was 100% homologous to C. andersoni infections derived from dairy calves and goats, respectively. The clinical questionnaires showed no significant difference in age, gender and frequency of diarrhea, but duration of diarrhea was shorter for C. andersoni than that of C. hominis (mean, 2 vs. 4 days; p <0.01).ConclusionsC. andersoni is the dominant species in Danyang City of Jiangsu Province. The fact that SSU rRNA sequences of C. andersoni obtained from human stools exhibited 100% homologous to those derived from dairy calves and goats supported that C. andersoni infection might be attributable to animal origin. The difference in the duration of diarrhea of C. andersoni and C. hominis indicated that different Cryptosporidium species might cause different clinical manifestations.

Enterocytozoon bieneusi in Dairy Cattle in the Northeast of China: Genetic Diversity of ITS Gene and Evaluation of Zoonotic Transmission Potential

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans. It has been found in a wide range of animals and is considered an important zoonotic pathogen. To date, little information is available on the role that cattle play in the epidemiology of human microsporidiosis caused by E. bieneusi in China. In this study, 133 fecal specimens from dairy cattle were collected in Heilongjiang Province, China. Enterocytozoon bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene, with 30.1% positive. Nine ITS genotypes were identified: six known genotypes—O (n = 26), EbpA (n = 2), I (n = 2), J (n = 2), D (n = 1) and BEB4 (n = 1)—and three novel genotypes named as CC‐I to CC‐III (two each). Genotype O was identified in cattle for the first time. The observation of all the six known genotypes here reported previously in humans, and also the fact of all the three novel genotypes (CHN‐DC1 to CHN‐DC3) falling into zoonotic group 1, indicate the possibility of cattle in the transmission of E. bieneusi to humans.

Molecular identification and distribution of Cryptosporidium and Giardia duodenalis in raw urban wastewater in Harbin, China.

Contamination of the water supply by protozoa often causes outbreaks of cryptosporidiosis and giardiasis. The goals of the present study was to investigate the level of Cryptosporidium and Giardia duodenalis in wastewater from wastewater treatment plants in Harbin, China, and to understand the endemic transmission characteristics of cryptosporidiosis and giardiasis. Forty-eight domestic wastewater specimens from the two wastewater treatment plants in Harbin City were collected from April 2009 to March 2010. Cryptosporidium spp. and G. duodenalis assemblages were identified by PCR and sequencing of the 18S ribosomal RNA and the triosephosphate isomerase genes, respectively. In total, 15 wastewater specimens were PCR positive for Cryptosporidium and 23 were PCR positive for G. duodenalis. The prevalence of contamination with G. duodenalis (47.9%) was higher than that of Cryptosporidium (31.3%). Molecular identification showed the presence of two Cryptosporidium spp. (14 belonging to Cryptosporidium andersoni and one belonging to Cryptosporidium ubiquitum) and two G. duodenalis assemblages (18 belonging to assemblage AII and six belonging to assemblage B). In addition, eight specimens contained both Cryptosporidium and G. duodenalis, and one specimen contained G. duodenalis assemblages AII and B. These results suggested humans might be the primary source of G. duodenalis contamination in wastewater in the studied area. In contrast, a low prevalence of C. ubiquitum suggested a reduced risk of human cryptosporidiosis caused by C. ubiquitum via waterborne route. This work provides basic experimental data needed for local wastewater treatment plants to develop protective strategies for water safety and to eliminate waterborne parasites.

Prevalence and molecular identification of Cryptosporidium spp. in pigs in Henan, China

The distribution and public health significance of Cryptosporidium species/genotypes in pigs differ among geographic areas and studies. To characterize the prevalence of cryptosporidiosis in pigs in Henan, China, a total of 1,350 fecal samples from 14 farms in ten prefectures in Henan Province were examined. The overall prevalence of Cryptosporidium was 8.2% (111/1,350), with the highest infection rate (79/383 or 20.6%) in 1–2-month-old piglets and the lowest infection rates in 3–6-month-old pigs. Cryptosporidium-positive samples from 108 animals were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the small subunit rRNA gene, and 35 were further analyzed by DNA sequencing of the PCR products. Two Cryptosporidium species/genotype were identified, including Cryptosporidium suis (94/108) and the Cryptosporidium pig genotype II (14/108). C. suis infection was more common in younger piglets whereas the pig genotype II was relatively common in older pigs. These findings suggest that pigs are not a major source of zoonotic Cryptosporidium in the study area.

Genetic Characterization of Human-Derived Hydatid Cysts of Echinococcus granulosus Sensu Lato in Heilongjiang Province and the First Report of G7 Genotype of E. canadensis in Humans in China

Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1–G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.