Molecular characterization and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in humans and domestic animals in Heilongjiang Province, China.

BACKGROUND: Cryptosporidiosis and giardiasis are significant parasitic diseases shared between humans and domestic animals. Due to the close contact between humans and domestic animals in rural areas, it is important to consider the potential transmission of zoonotic parasites from infected domestic animals to humans. This investigation aimed to determine the prevalence and molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in domestic animals and villagers. METHODS: A total of 116 fecal samples from villagers and 686 fecal samples from domestic animals in Heilongjiang Province, China, were analyzed for two parasites using nested polymerase chain reaction (PCR) targeting various genetic loci and DNA sequence analysis of the PCR products. RESULTS: By sequence analysis of the SSU rRNA gene, the prevalence of Cryptosporidium in humans was 0.9% (1/116), with one species of C. parvum (n = 1) detected; among domestic animals, the prevalence was 2.6% (18/686), with five species identified: C. suis (n = 7) and C. scrofarum (n = 7) in pigs, C. meleagridis (n = 1) in chickens, C. andersoni (n = 1) in cattle, and C. canis (n = 2) in foxes. C. parvum and C. canis were further subtyped as IIdA19G1 and XXa4 on the basis of gp60 gene. Regarding G. duodenalis, based on the SSU rRNA, bg, gdh, and tpi genes, the prevalence in domestic animals was 5.1% (31/608), with three assemblages identified: A (n = 1) in pigs, D (n = 1) in foxes, and E (n = 27) in geese, cattle, pigs, ducks, and sheep, along with mixed infection of A + E (n = 1) in one pig and B + E (n = 1) in one sheep. No G. duodenalis was detected in humans (0/116). CONCLUSIONS: The present results show that no overlap of subtypes between animals and villagers was found in Cryptosporidium spp. and G. duodenalis, indicating a minor role of domestic animals in infecting humans in this population. However, the presence of zoonotic protozoa in domestic animals highlights the need for special attention to high-risk individuals during close contact with domestic animals.

The frequency and subtype distribution of Blastocystis sp. in humans and domestic animals in households in Heilongjiang Province, China.

Blastocystis is an enteric protozoan able to infect humans and a large variety of animal hosts worldwide. It exhibits significant genetic diversity, with at least 17 subtypes (STs) identified to date, most of which have low host specificity. In the present study, a cross-sectional survey was conducted on Blastocystis infection among humans and domestic animals sharing habitats in northeastern China's Heilongjiang Province, in order to investigate the frequency and subtype distribution and to evaluate the risk of the zoonotic transmission of Blastocystis sp..Results show that the frequency was 10.5% (6/57), and three subtypes (ST1, ST2, and ST3) were found in humans; in animals, the frequency was 17.9% (46/257), and six subtypes (ST1, ST3, ST5, ST7, ST10, and ST26) were detected. A significant association between Blastocystis sp. infection and eating unwashed vegetables and fruits was found (P = 0.007). We found no effect on gender, age and season on Blastocystis sp. colonization. Phylogenetic analysis showed that Blastocystis ST7 sequences from chicken in two geographical locations formed two distinct clades. Alleles were identified using the Blastocystis 18S database, and a total of 10 different alleles were found in seven STs. Overlap of STs in humans and domestic animals (pig or cattle) was observed in Dongfanghong village. The findings of potentially zoonotic subtypes in domestic animals suggest that these animals may serve as reservoirs of human Blastocystis sp. infections. Multisectoral cooperation is needed to slow down the transmission of Blastocystis in domestic animals, minimize environmental contamination by Blastocystis cysts, and increase molecular epidemiological surveillance of Blastocystis sp. in humans and animals.

Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA.

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Giardia duodenalis in patients with diarrhea and various animals in northeastern China: prevalence and multilocus genetic characterization.

BACKGROUND: Giardia duodenalis is a common parasitic diarrheal agent in humans, especially in developing countries. The aim of this study was to investigate the prevalence and multilocus genetic characterization of G. duodenalis in patients with diarrhea and animals in northeastern China, and to assess the epidemiological role of animals in the transmission of human giardiasis. METHODS: A total of 1739 fecal specimens from 413 diarrheal patients and 1326 animals comprising 16 mammal species were collected in Heilongjiang Province of China and screened for G. duodenalis by PCR and sequencing of the SSU rRNA gene. All G. duodenalis-positive specimens were subtyped by PCR and sequencing of the bg, tpi, and gdh genes. To detect additional mixed infections of different assemblages, assemblage A/B/E-specific PCRs were performed to amplify the tpi gene. RESULTS: Sequence analysis of the SSU rRNA gene determined the prevalence of G. duodenalis (5.81%, 24/413) in diarrheal patients, with a peak in minors aged 5-17 years, and identified assemblages A and B. MLG-AII and MLG-B1 were obtained based on concatenated nucleotide sequences of the bg, tpi, and gdh genes, with MLG-AII being identical to a cat-derived isolate reported previously. By sequence analysis of the SSU rRNA gene, G. duodenalis was detected in 214 (16.14%) animals belonging to 11 mammal species, with the prevalence ranging from 1.69 to 53.85%, and assemblages A to G were identified. Sequence analysis of the bg, tpi, and gdh genes from 46 specimens produced 31 MLGs, including MLG-AI (n = 1), MLG-B2-B8 (n = 18), and MLG-E1-E23 (n = 27). CONCLUSIONS: The finding of G. duodenalis in diarrheal patients enhances consciousness of detecting G. duodenalis in clinical practice and emphasizes the importance of health education in local inhabitants, especially in the age group of 5-17 years. The identification of seven assemblages (A to G) and 33 MLGs reveals genetic heterogeneity of G. duodenalis in the investigated areas. Due to insufficient homology data on the zoonotic transmission of G. duodenalis, the precise epidemiological role that animals play in the transmission of human giardiasis needs to be assessed by more large-scale molecular epidemiological investigations of local humans and animals.

Prevalence and Genetic Characterization of Two Mitochondrial Gene Sequences of Strobilocercus Fasciolaris in the Livers of Brown Rats (Rattus norvegicus) in Heilongjiang Province in Northeastern China.

Rodents constitute the largest and most successful group of mammals worldwide. Brown rats (Rattus norvegicus) are one of the most common rodent species, and they serve as intermediate hosts of Hydatigera taeniaeformis. Although there have been a few studies reporting on the presence of the larval form of H. taeniaeformis (strobilocercus fasciolaris) in brown rats worldwide, little information is available on the genetic characterization of this parasite, with no molecular data from China. Therefore, from April 2014 to March 2016, this study was carried out to understand the prevalence and genetic characters of strobilocercus fasciolaris in brown rats captured in Heilongjiang Province in northeastern China. The livers of brown rats were collected and examined for the presence of cysts. Each cyst was identified based on morphological observation: the larvae with the naked eye and the scolexes under a microscope. The results were confirmed by polymerase chain reaction (PCR) and sequencing of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4) genes. At the investigated sites, 11.8% (13/110) of the brown rats were infected with strobilocercus fasciolaris. Based on sequence analysis, there were 10 and six haplotypes regarding the cox1 and the nad4 loci, with 24 and 42 polymorphic sites, respectively (degree of intraspecific variation: 0.3%-4.4% and 0.6%-4.7%, respectively). Twelve nucleotide sequences (six of the 10 at the cox1 locus and all six at the nad4 locus) have not previously been described. Base differences in three of the six novel cox1 gene sequences and five of the six novel nad4 gene sequences caused amino acid changes. Phylogenetic analyses of the cox1 and nad4 gene sequences based on neighbor-joining and Bayesian inference trees indicated that all the strobilocercus fasciolaris isolates belonged to Hydatigera taeniaeformis sensu stricto (s.s.). This is the first report on the genetic characterization of strobilocercus fasciolaris in brown rats in China. The findings of novel cox1 and nad4 nucleotide and amino acid sequences may reflect the region-specific genetic characterization of the parasite. The data will be useful to explore the biological and epidemiological significance of the intraspecific variation within H. taeniaeformis s.s.

Multilocus Sequence Typing of Enterocytozoon bieneusi Isolates From Various Mammal and Bird Species and Assessment of Population Structure and Substructure.

Enterocytozoon bieneusi is one of the most common intestinal pathogens in humans and animals. E. bieneusi has been confirmed to be complex microsporidian species. Approximately 500 ITS genotypes of E. bieneusi have been defined. With the establishment and application of multilocus sequencing typing and population genetic tools in E. bieneusi, the studies on these aspects have been carried out worldwide, but little information is available. To understand genetic variation of mini-/micro-satellites and the population structure and substructure of E. bieneusi in northeastern China, 305 E. bieneusi DNA specimens composed of 28 ITS genotypes were from 13 mammal species and five bird species in the investigated areas. They were characterized by nested-PCR amplification and sequencing at four mini-/micro-satellite loci (MS1, MS3, MS4, and MS7). At the MS1, MS3, MS4, and MS7 loci, 153 (50.16%), 131 (42.95%), 133 (43.61%), and 128 (41.97%) DNA specimens were amplified and sequenced successfully with 44, 17, 26, and 24 genotypes being identified, respectively. Multilocus genotypes (MLGs) showed a higher genetic diversity than ITS genotypes. 48 MLGs were produced out of 90 ITS-positive DNA specimens based on concatenated sequences of all the five genetic loci including ITS. Linkage disequilibrium (LD) and limited genetic recombination were observed by measuring LD using both multilocus sequences and allelic profile data, indicating an overall clonal population structure of E. bieneusi in the investigated areas. These data will aid in the longitudinal tracking of the attribution of source of infection/contamination and in elucidating transmission dynamics, and will provide valuable information for making efficient control strategies to intervene with and prevent occurrence of microsporidiosis caused by E. bieneusi among animals and transmission of E. bieneusi from animals to humans in the investigated areas. Phylogenetic and network analyses identified three different subgroups, revealing the presence of host-shaped segregation and the absence of geographical segregation in E. bieneusi population. Meanwhile, the MLGs from zoonotic ITS genotypes were observed to be basically separated from the MLGs from host-adapted ones. Assessment of substructure will have a reference effect on understanding of zoonotic or interspecies transmission of E. bieneusi and evolution direction from zoonotic genotypes to host-adapted genotypes.

First identification and genotyping of Enterocytozoon bieneusi in humans in Myanmar.

BACKGROUND: Intestinal pathogen infections are widespread among impoverished populations. Enterocytozoon bieneusi is the most common pathogen of intestinal microsporidian species in humans worldwide. However, no epidemiological information is available on E. bieneusi infection in humans in Myanmar. The present study comprised the first identification and genotyping of E. bieneusi in humans conducted in Myanmar. RESULTS: A total of 172 fecal specimens were collected from the Wa people (one each) in four villages of Pangsang Township of the Matman District of Shan State, Myanmar, and each participant completed a questionnaire. E. bieneusi was identified and genotyped using polymerase chain reaction (PCR) and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. The average prevalence of E. bieneusi was 8.72% (15/172), ranging from 3.85 to 13.89% by village. E. bieneusi infection was not related to any of the risk factors studied. Six genotypes were identified, comprising two known genotypes Peru6 (n = 10) and D (n = 1) and four novel genotypes (MMR23, MMR25, MMR86, and MMR87) (one each), and two people infected with genotype Peru6 were from the same family. A phylogenetic analysis based on a neighbor-joining tree of the ITS sequences of E. bieneusi indicated that all the six genotypes were clustered into group 1. CONCLUSIONS: This is the first identification and genotyping of E. bieneusi in humans in Myanmar. The observations that the two people infected with genotype Peru6 were from the same family, and that all six genotypes obtained in the present study fell into zoonotic group 1, showed the potential for anthropogenic and zoonotic transmissions. The present data argue for the importance of epidemiological control and prevention from medical sectors.

Prevalence and subtype distribution of Blastocystis in ethnic minority groups on both sides of the China-Myanmar border, and assessment of risk factors.

Blastocystis is one of the most common intestinal parasites in humans worldwide. To understand its prevalence and to explore the risk factors for Blastocystis in humans in developing countries, a molecular epidemiological investigation of Blastocystis was conducted in ethnic minority groups on both sides of the China-Myanmar border. A total of 461 fecal specimens were collected from 289 Yao people in China and 172 Wa people in Myanmar, together with a questionnaire for each participant. Based on sequence analysis of the partial small subunit of ribosomal RNA (SSU rRNA) gene (barcode region or 260 bp region), an average prevalence of 6.29% (29/461) was observed, with 4.50% (13/289) in Yao people and 9.30% (16/172) in Wa people. Twenty-two Blastocystis isolates were successfully subtyped by sequence analysis of the barcode region. Three subtypes were identified: ST1 (n = 7), ST3 (n = 13) and ST4 (n = 2). A statistical difference in the prevalence of Blastocystis was only observed between children (12.37%, 12/97) and adults (4.95%, 16/323), and between not washing hands (11.02%, 14/127) and washing hands (4.76%, 15/315) after using toilets, emphasizing the importance and necessity of health education in people in the investigated areas, especially in children.

First survey of Enterocytozoon bieneusi and dominant genotype Peru6 among ethnic minority groups in southwestern China's Yunnan Province and assessment of risk factors.

BACKGROUND: Enterocytozoon bieneusi is the most common microsporidian species causing diarrhea and other intestinal disorders in humans and animals. Like other infectious diseases, microsporidiosis usually disproportionately affects poor populations. In China, some ethnic minority areas remain poor. Currently, no information of E. bieneusi infection is available in minority populations. The present aims were to understand occurrence and genetic characterizations of E. bieneusi in ethnic minority groups from a poverty-stricken ethnic township in Yunnan Province, and to assess risk factors for E. bieneusi infection. METHODOLOGY/PRINCIPAL FINDINGS: 289 fecal specimens were collected from Yao people (one specimen each) with and without diarrhea, in Yunnan Province. E. bieneusi was identified and genotyped by PCR and sequence analysis of the ITS region of the rRNA gene. An average prevalence of 8.30% (24/289) was observed and four genotypes were identified-genotype Peru6 (n = 21) and three novel genotypes (one each). Genotype Peru6 was detected in two family members in each of three families. In a phylogenetic analysis, all of four genotypes fell into group 1 with zoonotic potential. The people owning individual pit toilets had a statistically higher prevalence of E. bieneusi (16.67%, 12/72) than those using public pit toilets (6.06%, 12/198). CONCLUSIONS/SIGNIFICANCE: This is the first report on occurrence and genetic characteristics of E. bieneusi in ethnic minority groups in China. Genotype Peru6 was found in humans in China for the first time and showed dominance in Yao people. The same genotype was found in some family members and all the genotypes fell into group 1, suggesting the possibility of anthroponotic and zoonotic transmissions. The majority (83.33%, 20/24) of E. bieneusi positive individuals did not present diarrhea. In any case, it is important to recognize their existence and the importance that asymptomatic individuals to E. bieneusi may have from an epidemiological point of view, as transmitters of this pathogen. The analysis of risk factors provides scientific evidence for the development of effective strategies for prevention and control of E. bieneusi infection.

A human case of Dioctophyma renale (giant kidney worm) accompanied by renal cancer and a retrospective study of dioctophymiasis.

Due to the rarity of human cases and the nonspecific clinical symptoms of dioctophymiasis, Dioctophyma renale infection is not well recognized and is easily neglected or misdiagnosed. Recently, we diagnosed a human case of dioctophymiasis accompanied by renal cancer. To enhance the understanding of human dioctophymiasis, this case is presented here, and a retrospective study of this disease was conducted based on relevant papers screened from PubMed and three Chinese databases. In the end, 32 papers describing 37 human cases of dioctophymiasis were assessed. These cases were distributed in ten countries of Asia, Europe, North America and Oceania, with the highest number in China (n = 22). The majority of the cases occurred in adults (91.9%, 34/37) and involved the kidneys (83.8%, 31/37). Ectopic parasitism mainly occurred in subcutaneous tissue (83.3%, 5/6). A proportion of 45.9% (17/37) of individuals had a history of eating raw or undercooked fish or frogs. The main clinical manifestations of human dioctophymiasis were loin pain (59.5%) and hematuria (59.5%). All the cases were diagnosed based on the morphological characteristics of eggs or adults in urine or tissue sections. Currently, there is no strictly defined therapeutic approach. This is the first retrospective analysis of human cases of dioctophymiasis. These review data will deepen our understanding of dioctophymiasis and help avoid misdiagnosis in clinical practice.

Distribution and genetic diversity of Blastocystis subtypes in various mammal and bird species in northeastern China.

BACKGROUND: Blastocystis is one of the most common intestinal parasites in humans and animals worldwide. At least 17 subtypes have been identified in mammals and birds. In China, although some studies have reported the occurrence of Blastocystis in humans and animals, our understanding of the role of animals in the transmission of human blastocystosis is only superficial due to a paucity of available molecular data. The aim of the present study was to understand infection rates of Blastocystis and the distribution and genetic diversity of subtypes in various mammal and bird species in northeastern China, as well as to assess the zoonotic potential of Blastocystis isolates. METHODS: A total of 1265 fresh fecal specimens (1080 from ten mammal species and 185 from eight bird species) were collected in Heilongjiang, Liaoning and Jilin provinces of China. Each specimen was examined for the presence of Blastocystis by PCR amplification and sequence analysis of the partial SSU rRNA gene. RESULTS: Fifty-four specimens (4.3%) were positive for Blastocystis. Birds (7.0%) had a higher infection rate of Blastocystis than mammals (3.8%). Blastocystis was found in seven mammal species, reindeer (6.7%), sika deer (14.6%), racoon dogs (7.5%), Arctic foxes (1.9%), dogs (2.9%), rats (3.7%) and rabbits (3.3%), as well as three bird species, pigeons (2.1%), chickens (13.0%) and red crowned cranes (14.0%). Eight subtypes were identified including ST1 (n = 5), ST3 (n = 3), ST4 (n = 13), ST6 (n = 8), ST7 (n = 6), ST10 (n = 13), ST13 (n = 4) and ST14 (n = 2). 64.8% (35/54) of Blastocystis isolates belonged to potentially zoonotic subtypes. CONCLUSIONS: To our knowledge, this is the first report of Blastocystis in reindeer (ST10 and ST13), rabbits (ST4), racoon dogs (ST3) and Arctic foxes (ST1, ST4 and ST7). The findings of potentially zoonotic subtypes suggest that the animals infected with Blastocystis might pose a threat to human health. These data will improve our understanding of the host range and genetic diversity of Blastocystis, and also help develop efficient control strategies to intervene with and prevent the occurrence of human blastocystosis in the investigated areas.

Multilocus sequence typing and population genetic structure of Cryptosporidium cuniculus in rabbits in Heilongjiang Province, China.

Multilocus sequence typing (MLST) tools have been used widely to characterize population genetic structure of some Cryptosporidium species. To understand MLST subtypes and population genetic structure of Cryptosporidium cuniculus from rabbits in Heilongjiang Province, China, 34 C. cuniculus DNA specimens were collected including VbA21 (n = 6), VbA28 (n = 2), VbA29 (n = 18) and VbA32 (n = 8). They were analyzed by nested-PCR amplification and sequencing at seven microsatellite, minisatellite and polymorphic loci including CP47, CP56, ML2, DZ-HRGP, MSC6-5, MSC6-7 and RPGR. The CP47, CP56, MSC6-5 and MSC6-7 loci were monomorphic. The remaining loci were polymorphic, with two, three and two subtypes being found at ML2, DZ-HRGP and RPGR loci, respectively. Six MLST subtypes were obtained based on sequence information of 29 DNA specimens successfully amplified at all eight loci including gp60 locus. Linkage disequilibrium (LD) analysis showed a clonal population structure of C. cuniculus in the investigated areas. STRUCTURE, neighbor-joining and network analyses indicated the presence of two distinct groups, corresponding to VbA21 subtype and VbA28, VbA29 and VbA32 subtypes. This is the first report of MLST analysis of C. cuniculus. A clonal population structure of C. cuniculus suggested the prevalence of C. cuniculus in Heilongjiang Province is not attributed to the introduction of rabbits. Thus, prevention and control strategies should be focused on making stricter measures to avoid occurrence of cross-transmission and re-infection among rabbit individuals. Based on the previous findings of VbA21 subtype only in rabbits and VbA28, VbA29 and VbA32 subtypes both in rabbits and humans, the results of subpopulation analyses might be used to assess zoonotic potential of C. cuniculus subtypes in Vb family. These data will be helpful to explore source attribution of infection/contamination of C. cuniculus and understand its transmission dynamics in humans and rabbits in the investigated areas.

Molecular characterizations of Cryptosporidium spp. and Enterocytozoon bieneusi in brown rats (Rattus norvegicus) from Heilongjiang Province, China.

BACKGROUND: Cryptosporidium spp. and Enterocytozoon bieneusi are prevalent zoonotic pathogens responsible for the high burden of diarrheal diseases worldwide. Rodents are globally overpopulated and are known as reservoirs or carriers of a variety of zoonotic pathogens including Cryptosporidium spp. and E. bieneusi. However, few data are available on genetic characterizations of both pathogens in rodents in China. The aim of the present work was to determine the prevalence and genetic characterizations of Cryptosporidium spp. and E. bieneusi in brown rats (Rattus norvegicus) from Heilongjiang, China. METHODS: A total of 242 wild brown rats were captured in Heilongjiang Province of China. A fresh fecal specimen was collected directly from the intestinal and rectal content of each brown rat. All the fecal specimens were examined for the presence of Cryptosporidium spp. and E. bieneusi by PCR and sequencing of the partial small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) region of the rRNA gene of the two pathogens, respectively. RESULTS: The infection rate was 9.1% (22/242) for Cryptosporidium spp. and 7.9% (19/242) for E. bieneusi. Sequence analysis confirmed the presence of C. ubiquitum (1/22, 4.5%) and three genotypes of Cryptosporidium, including Cryptosporidium rat genotype I (14/22, 63.6%), Cryptosporidium rat genotype IV (6/22, 27.3%) and Cryptosporidium suis-like genotype (1/22, 4.5%). Meanwhile, two E. bieneusi genotypes were identified, including D (17/19, 89.5%) and Peru6 (2/19, 10.5%). CONCLUSIONS: To the best of our knowledge, Enterocytozoon bieneusi genotype Peru6 was identified in rodents for the first time globally and Cryptosporidium rat genotype I and Cryptosporidium rat genotype IV were found in rats in China for the first time. The finding of zoonotic C. ubiquitum and C. suis-like genotype, as well as E. bieneusi genotypes, suggests that brown rats pose a threat to human health. It is necessary to control brown rat population in the investigated areas and improve local people's awareness of the transmission risk of the two pathogens from brown rats to humans.

Subtype distribution and genetic characterizations of Blastocystis in pigs, cattle, sheep and goats in northeastern China's Heilongjiang Province.

Blastocystis is a common protozoan found in the surveys of human and animal fecal specimens. Extensive genetic diversity has been observed within the genus Blastocystis. At least 17 subtypes (ST) have been identified in mammals and birds, nine of which (ST1 to ST9) have been identified in humans. In China, although there have been a few reports on Blastocystis infection in humans and many animal species, no epidemiological data are available in either humans or animals in northeastern China's Heilongjiang Province. To determine infection rates and subtype distributions of Blastocystis in pigs, cattle, sheep and goats, to understand genetic characterizations and to assess zoonotic possibility of Blastocystis isolates, 337 fecal specimens from livestock (68 from pigs, 147 from cattle, 109 from sheep and 13 from goats) were collected in northeastern China's Heilongjiang Province. Each of them was detected for Blastocystis by PCR amplification of the partial SSU rRNA gene. An average infection rate of Blastocystis was 7.7% (26/337), and the highest infection rate was found in 9.5% (14/147) in cattle, followed by 8.8% (6/68) in pigs and 5.5% (6/109) in sheep. There was an absence of Blastocystis in goats. Five Blastocystis subtypes were identified: ST5 (n=6) in pigs; ST3 (n=2), ST10 (n=10) and ST14 (n=2) in cattle; ST1 (n=1), ST5 (n=1), ST10 (n=3) and ST14 (n=1) in sheep. 38.5% (10/26) of Blastocystis isolates belong to potentially zoonotic subtypes based on the previous findings of ST1, ST3 and ST5 in humans. This is the first report of Blastocystis in pigs, cattle and sheep in northeastern China's Heilongjiang Province. ST1, ST5 and ST14 were identified in sheep for the first time. Due to the low infection rate of Blastocystis and the small percentage of potentially zoonotic subtypes in these livestock, there is a minimal risk of zoonotic transmission of Blastocystis.

Subtype analysis of zoonotic pathogen Cryptosporidium skunk genotype.

Cryptosporidium skunk genotype is a zoonotic pathogen commonly identified in surface water. Thus far, no subtyping tool exists for characterizing its transmission in humans and animals and transport in environment. In this study, a subtyping tool based on the 60kDa glycoprotein (gp60) gene previously developed for Cryptosporidium chipmunk genotype I was used in the characterization of Cryptosporidium skunk genotype in animal and storm runoff samples from a watershed in New York. Altogether, 17 positive samples from this watershed and 5 human and animal specimens from other areas were analyzed. We identified 14 subtypes of Cryptosporidium skunk genotype, 11 of which were seen in the watershed. In phylogenetic analysis, these subtypes belonged to 4 subtype families (XVIa, XVIb, XVIc, and XVId). No host-adapted subtypes were identified and the two subtypes in humans were genetically similar to some in raccoons, otters, and storm runoff samples from the watershed. The characteristics of gp60 protein sequences of the Cryptosporidium skunk genotype are similar to those of other Cryptosporidium species, but only its XVIb subtype family has a putative furin cleavage site. This subtyping tool might be useful in characterizing Cryptosporidium skunk genotype in clinical and environmental samples.

Subtyping of Cryptosporidium cuniculus and genotyping of Enterocytozoon bieneusi in rabbits in two farms in Heilongjiang Province, China.

Cryptosporidium spp. and Enterocytozoon bieneusi are two prevalent opportunistic pathogens in humans and animals. Currently, few data are available on genetic characterization of both pathogens in rabbits in China. The aim of the present study was to understand prevalence and genetic characterization of Cryptosporidium spp. and E. bieneusi in rabbits. We collected 215 fecal samples from 150 Rex rabbits and 65 New Zealand White rabbits on two different farms in Heilongjiang Province, China. Cryptosporidium spp. and E. bieneusi were tested by polymerase chain reaction (PCR) and sequencing the partial small subunit of ribosomal DNA (SSU rDNA) and the internal transcribed spacer (ITS) region of rDNA, respectively. Cryptosporidium was detected in 3.3% (5/150) of Rex rabbits and 29.2% (19/65) of New Zealand White rabbits. All the 24 Cryptosporidium isolates were identified as C. cuniculus. Enterocytozoon bieneusi was only found in 14.7% (22/150) of Rex rabbits. Five known genotypes: CHN-RD1 (n = 12), D (n = 3), Type IV (n = 2), Peru6 (n = 1), and I (n = 1), and three novel ones CHN-RR1 to CHN-RR3 (one each) were detected. By analyzing the 60-kDa glycoprotein (gp60) gene sequences of C. cuniculus isolates, three subtypes were obtained: VbA28 (n = 2), VbA29 (n = 16), and VbA32 (n = 3). All these three C. cuniculus subtypes were reported previously in humans. Four known E. bieneusi genotypes have been found to be present in humans. The three novel ones fell into zoonotic group 1. The results suggest zoonotic potential of C. cuniculus and E. bieneusi isolates in rabbits.

Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China.

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea in humans and animals. However, epidemiological data on E. bieneusi infections in birds are relatively scare worldwide, especially in China. To understand the prevalence and genetic diversity of E. bieneusi in birds and to assess the zoonotic potential of bird-derived E. bieneusi isolates, 194 fecal specimens from Gruidae, Anatidae and Columbidae in Heilongjiang Province, China, were analyzed by PCR and sequencing of the single internal transcribed spacer region of the rRNA gene. The average prevalence of E. bieneusi was 22.2%, with 12.5% for Gruidae, 15.9% for Anatidae and 44.0% for Columbidae. Altogether seven genotypes of E. bieneusi were identified, including four known genotypes-Peru6 (n=29), BEB6 (n=5), D (n=3) and EbpA (n=1)-and three novel genotypes named CHN-B1 (n=1), CHN-B2 (n=3) and CHN-B3 (n=1). All the known genotypes obtained here were previously detected in humans. All the novel genotypes were clustered into the zoonotic group 1 in phylogenetic analysis. The results indicate that these birds may play a potential role in the transmission of E. bieneusi to humans.

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China.

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.

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.