Prevalence and molecular characteristics of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in cattle in Korea

Enterocytozoon bieneusi is the most common microsporidium associated with AIDS patients. Moreover, its detection in increasing numbers of immunocompetent patients has made it an emerging pathogen. This organism was also identified in a wide range of animals, and the zoonotic potential of human infections is of particular interest. In this study, 538 fecal samples from cattle in Korea were analyzed for the presence of E. bieneusi by PCR. Approximately 15% were found to be positive, with higher rates being detected over the summer months. The internal transcribed spacer (ITS) regions of the rRNA gene of ten E. bieneusi positive samples were amplified using nested PCR and sequenced. Genetic polymorphisms, which were represented by six distinct genotypes (CEbA–CEbF), were found among the E. bieneusi isolates. Five isolates from this study had identical ribosomal ITS to the previously known E. bieneusi genotype ITSs in cattle and other animals. Four isolates were previously unreported but were quite similar to the previously known genotypes of E. bieneusi from cattle and other animals. One isolate was identical to the human E. bieneusi type D, which indicated some E. bieneusi isolates from cattle in the country may be of public health importance. To the best of my knowledge, this is the first report of E. bieneusi study in cattle in Asia.     

<Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in mature dairy cattle on farms in the eastern United States

Fecal specimens were obtained from mature milking cows on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Polymerase chain reaction (PCR)-positive specimens for Enterocytozoon bieneusi were found in 24 of 541 cows examined (4.4%) and on 12 of 14 farms. The prevalence of E. bieneusi varied considerably from farm to farm, with the lowest prevalence (2.3%) on FL-2 and the highest prevalence (12.5%) on VT-2. None of the cows exhibited signs of diarrhea. All PCR-positive specimens were sequenced to determine the genotype of E. bieneusi. Five genotypes were identified. Three were identified as cattle-specific genotypes previously reported as BEB1, BEB2, and BEB4, and two new genotypes, BEB 6 and BEB7, were found. None have been reported to infect humans.     

Occurrence and genetic diversity of Enterocytozoon bieneusi (Microsporidia) in owned and sheltered dogs and cats in Northern Spain

Enterocytozoon bieneusi is an obligate intracellular protist-like fungi parasite that infects numerous mammal hosts including humans, raising concerns of zoonotic transmission. There is little information available on the presence and diversity of E. bieneusi genotypes in companion animals. Here, we determined the occurrence and genetic diversity of E. bieneusi in domestic dogs and cats from Northern Spain. A total of 336 genomic DNA samples extracted from canine (n = 237) and feline (n = 99) faecal specimens were retrospectively investigated. The presence of E. bieneusi was assessed by PCR of the rRNA internal transcribed spacer (ITS) gene. The parasite was detected in 3.0% (3/99) and 0.8% (2/237) of the cats and dogs examined, respectively. All three feline positive samples were from stray cats living in an urban setting, whereas the two canine samples were from owned dogs living in rural areas. Sequence analysis revealed the presence of two genotypes in dogs, BEB6 and PtEb IX, and two genotypes in cats, D and Peru11. The identification of Peru11 in a cat and BEB6 in a dog constitutes the first report of those genotypes in such hosts as well as first report in Spain. This is also the first evidence of genotype D in cats and PtEb IX in dogs in Spain. Three out of the four genotypes, BEB6, D and Peru11, have been previously reported as human pathogens and are potentially zoonotic indicating that dogs and cats need to be considered potential sources of human infection and environmental contamination.

     

Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States

Fecal specimens were obtained from 12–24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.     

Molecular characterization of<Emphasis Type="Italic"> Enterocytozoon bieneusi</Emphasis> in cattle indicates that only some isolates have zoonotic potential

In this study, 338 fecal samples were analyzed for Enterocytozoon bieneusi from cattle farms in Florida, Maryland, Michigan, New York, North Carolina, Pennsylvania, Virginia, and Portugal. The internal transcribed spacer region (392 bp) of the rRNA gene of E. bieneusi was amplified using a nested PCR protocol. Thirty-two E. bieneusi-PCR positive samples were sequenced. A high degree of genetic polymorphism, represented by five distinct genotypes (BEB1–BEB5), was found among the E. bieneusi isolates from cattle. Most of the isolates formed a distinct cluster consisting of only the four cattle genotypes, but six isolates of a genotype clustered together with E. bieneusi genotypes from humans and other domestic animals. Therefore, only some E. bieneusi isolates from cattle may be of public health importance.An erratum to this article can be found at     

Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes

As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9 % (29/91) was observed in deer, with 32.6 % (28/86) for sika deer, and 20 % (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n?=?20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n?=?5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.     

Infection patterns,clinical significance,and genetic characteristics of Enterocytozoon bieneusi and Giardia duodenalis in dairy cattle in Jiangsu,China

The infection patterns and clinical significance of Enterocytozoon bieneusi and Giardia duodenalis in dairy cattle remain poorly investigated despite their common occurrence. Data on the genetic diversity are also needed to understand the transmission and human-infective potential of the two pathogens. In this study, fecal specimens from 1366 dairy cattle on a large farm were examined for the presence and genotype distribution of E. bieneusi and G. duodenalis by PCR and DNA sequencing. The overall infection rates of E. bieneusi and G. duodenalis were 13.0% and 20.6%, respectively. Pre-weaned calves had significantly higher infection rates of both pathogens than post-weaned and adult cattle (P < 0.001), with peak occurrence of the pathogens in animals of 7–12 weeks. In both pre- and post-weaned calves, animals with diarrhea were 2.1–3.0 times more likely to be infected with either pathogen than those without diarrhea (P < 0.01). The E. bieneusi identified belonged to five genotypes, including J (n = 138), I (n = 21), BEB4 (n = 10), Type IV (n = 1), and a novel genotype CHC17 (n = 1). Genotype J was the dominant one in all age groups, whereas genotype I was only identified in calves of 6–11 weeks. Genotyping of G. duodenalis at three genetic loci identified assemblage E (n = 278), assemblage A (n = 2), and concurrence of the two (n = 1). Altogether, 13, 7 and 10 subtypes of assemblage E were detected at the bg, gdh, and tpi loci, respectively, forming 65 multilocus genotypes. The formation of two major clusters of MLGs in eBURST analysis indicated that intra-assemblage genetic recombination of two dominant MLGs could have led to the high genetic heterogeneity within assemblage E on a single farm. Results of this study provide much needed data on the pathogenicity of E. bieneusi and G. duodenalis in pre- and post-weaned calves. The clinical significance of the two pathogens in dairy cattle warrants further investigations.

     

Prevalence,genotypes, and risk factors of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in Asiatic black bear (<Emphasis Type="Italic">Ursus thibetanus</Emphasis>) in Yunnan Province,Southwestern China

Microsporidiosis is an important zoonotic disease, even leading to severe diarrhea. However, no information about prevalence and genotypes of Enterocytozoon bieneusi infection in Asiatic black bears in southwestern China is available. The objectives of the present study were to investigate the prevalence of E. bieneusi and to characterize their genotypes using the nested PCR amplification of the internal transcribed spacer region of the ribosomal RNA gene cluster and multilocus sequence typing (MLST). The overall prevalence of E. bieneusi was 19.75% (80/405) and the rate of E. bieneusi in Xishuangbanna (33.33%) was significantly higher than that in any other regions (Honghe, 17.65%; Dehong, 13.04%; Kunming, 0; P?=?0.01). Sequence analysis revealed that 4 known genotypes (D, n?=?2; SC02, n?=?10; SC01, n?=?5; and CHB1, n?=?4) and 13 novel genotypes (designed MJ1–MJ13) were identified. When 17, 5, 14, and 34 sequences at loci MS1, MS3, MS4, and MS7 via MLST analyses, representing 4, 4, 5, and 10 genotypes, respectively, were completed, one multilocus genotype (MLG novel-ABB1) was identified. This is the first report of E. bieneusi in Asiatic black bear in Yunnan province, Southwestern China. The results indicated the potential zoonotic risk of this parasite through the Asiatic black bear in this region and provided foundation data for preventing and controlling E. bieneusi infection of many other animals and humans in these regions.     

A longitudinal study of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in dairy cattle

Feces from each of 30 Holstein cattle on a Maryland dairy farm were examined at weekly, bimonthly, and then monthly intervals from 1 week to 24 months of age for the presence of Enterocytozoon bieneusi. DNA was extracted from spores cleaned of fecal debris, and a two-step nested PCR protocol was used to amplify a fragment of the internal transcriber spacer region of the rRNA gene. All PCR-positive specimens were sequenced to determine the genotype of E. bieneusi. The overall prevalence was 24% (239/990) with a lower prevalence in pre-weaned calves (less than 8 weeks of age; 11.7%) and heifers (13–24 months of age) than post-weaned calves (3–12 months of age; 44.4%). Over the course of 24 months, the cumulative prevalence of E. bieneusi was 100% since all 30 calves shed spores at some time during the study. One or more of three genotypes of E. bieneusi, J, I, and BEB4, were detected in all 30 animals. Genotype I was detected in all 30 cattle between 1 week and 22 months of age with some cattle remaining infected as long as 17 months. At 4 months of age, 28 cattle were infected with genotype I. Genotype BEB4 was detected briefly in seven cattle, most between 15 and 20 months of age. Genotype J was detected in eight cattle, all between 16 and 24 months of age. This longitudinal study strongly supports the findings of point prevalence, multiple farm studies in which genotypes J, I, and BEB 4 were found. These genotypes appear to be cattle specific and have not been found in humans or other animals.     

Transmission and Serial Propagation of Enterocytozoon bieneusi from Humans and Rhesus Macaques in Gnotobiotic Piglets

For over a decade Enterocytozoon bieneusi infections in people with AIDS have been linked with chronic diarrhea and wasting. The slow scientific progress in treating these infections is attributed to the inability of investigators to cultivate the parasite, which has also precluded evaluation of effective therapies. We report here successful serial transmissions of E. bieneusi from patients with AIDS and from macaques with AIDS to immunosuppressed gnotobiotic piglets. One infected piglet was still excreting spores at necropsy 50 days after an oral challenge. Spores in feces were detected microscopically by trichrome stain and by PCR and within enterocytes by in situ hybridization and immunohistochemistry. E. bieneusi infection induced no symptoms. The development of an animal model for E. bieneusi will open up new opportunities for investigating this parasite.     

Prevalence and genotypes of Enterocytozoon bieneusi in weaned beef calves on cow-calf operations in the USA

To determine the prevalence and genotype distribution of Enterocytozoon bieneusi in weaned beef calves in the USA, fecal samples were collected from 819 calves (6–18 months of age) from 49 operations. Feces were sieved and subjected to density gradient centrifugation to remove fecal debris and to concentrate spores. DNA extracted from each sample was subjected to the polymerase chain reaction (PCR) to amplify the complete internal transcriber spacer (ITS). All PCR-positive specimens were sequenced to determine the genotype(s) present. Overall, E. bieneusi was detected in 34.8% of the 819 fecal samples. The highest prevalence was found in the Midwest region (42.7%) followed by the South (35.8%) and the West (23.2%). The prevalence of E. bieneusi varied considerably from operation to operation (0–100%). A prevalence of 100% was observed in three operations, one in the Midwest and two in the South; E. bieneusi was not found in six operations, three in the South and three in the West. Sequence analysis revealed the presence of six genotypes, four previously reported (I, J, BEB4, and type IV) and two novel genotypes (BEB8 and BEB9). Mixed infections were identified in five specimens, three contained I and BEB4 and two contained J and BEB4. Most of the positive calves (238 of 285) harbored genotypes with zoonotic potential including I (59), J (108), BEB4 (65), type IV (1), mixed I/BEB4 (3), and mixed J/BEB4 (2).     

Evidence for the existence of genetically distinct strains of Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most frequently found microsporidium in human infections. In all, 3 distinct genotypes were detected in 12 stool samples from 8 patients with acquired immunodeficiency syndrome (AIDS). A total of 9 polymorphic sites were found in the 243-bp-long internal transcribed spacer (ITS) of the rDNA gene, whereas none was found in 241 bp of adjacent rRNA coding regions. The genotype was stable in samples taken during 11 weeks of infection from one of the patients. The existence of and the ability to discriminate among strains of E. bieneusi are important prerequisites for elucidation of the hitherto unknown reservoirs of this pathogen and the mode of its transmission and may explain its pathogenicity. Received: 18 March 1997 / Accepted: 10 April 1997     

High diversity of human-pathogenic Enterocytozoon bieneusi genotypes in swine in northeast China

Despite the advances in defining Enterocytozoon bieneusi genotypes worldwide, rare genotypic surveys have been documented on this ubiquitous pathogenic protozoan in mammals in China, especially the role of pigs in zoonotic transmission of microsporidiosis remains unclear. In this study, the distribution of E. bieneusi genotypes in 113 duodenal mucosal specimens of pigs with acute diarrhea from 15 cities in northeast China was determined by PCR and DNA sequence analysis of the ribosomal internal transcribed spacer. The organism was detected in 51 (45.1 %) pigs from 12 cities, with infection rates of the nursery pigs (21/33, 63.6 %) significantly higher than the preweaned (25/61, 41.0 %; P?<?0.05) and the growing (5/19, 26.3 %; P?<?0.01) ones. E. bieneusi belongs to nine known human-pathogenic genotypes (D, EbpA, EbpC, EbpD, H, Henan-I, Henan-III, Henan-IV, and O) and eight new genotypes (CS-1 to CS-8). Genotypes D, EbpA, EbpC, EbpD, Henan-I, Henan-III, and Henan-IV have been found in human infections and D, EbpA, EbpC, and EbpD in wastewater in central China. The new genotypes were genetically clustered into a group of existing E. bieneusi genotypes with zoonotic potential. Considering the discovery of a high prevalence and wide genetic diversity of E. bieneusi zoonotic strains in pigs in northeast China and the co-occurrence of seven known genotypes in pigs and humans and four in pigs and wastewater, pigs probably served as a reservoir for human microsporidiosis and an important source of water contamination in China.     

Intestinal Enterocytozoon bieneusi microsporidiosis in an HIV-infected patient: diagnosis by ileo-colonoscopic biopsies and long-term follow up

Summary A 39-year-old patient with acquired immunodeficiency syndrome was diagnosed as having intestinal Enterocytozoon bieneusi microsporidiosis after persistent watery diarrhea for 30 months and a 16-kg weight loss. Microsporidian parasites were found by light and electron microscopy in tissue specimens of the duodenum, jejunum, and terminal ileum, and by light microscopic examination of stool specimens. When duodenal tissue sections obtained 16 months previously were reviewed retrospectively, E. bieneusi was also found. Until now, diagnosis of intestinal microsporidiosis has been based on examination of bioptic specimens of the upper small intestine because the sensitivity of new coprodiagnostic techniques has not been determined. Our findings of ileal microsporidiosis show that examination of the terminal ileum and ileal biopsy collection in tandem with colonoscopy is indicated for patients infected with human immunodeficiency virus and suffering from unexplained chronic diarrhea. The long-term course of our patient demonstrates that E. bieneusi, although not necessarily life threatening, can cause protracted debilitating diarrhea and wasting in severely immunodeficient patients.Abbreviations Aids acquired immunodeficiency syndrome - HIV human immunodeficiency virus     

Interspecies transmission of <Emphasis Type="Italic">Enterozytozoon bieneusi</Emphasis> supported by observations in laboratory animals and phylogeny

Enterocytozoon bieneusi is emerging as an important cause of chronic diarrhoea in AIDS patients. Its reservoirs and transmission patterns are unknown. In this study, we have examined E. bieneusi sequences from four Rhesus macaques of different origin, which were kept at one animal facility. The sequences were identical in all animals, which suggested that infection had occurred within the facility. Full sequence agreement of E. bieneusi from macaques was found with an E. bieneusi genotype that occurs frequently in humans. To clarify, the relevance of possible inter-species transmission from man to macaque, a phylogenetic analysis was conducted including all sequences of E. bieneusi deposited in GenBank. The hitherto used system of diverse nomenclatures could be reduced to an outlier group and three main lineages, one of which could be further sub-divided into five subgroups. Based in this phylogeny, an association of parasites and host species could be observed for main lineages 2 and 3, as well as for most of the subgroups of main lineage 1. For confirmation, the phylogeny of main lineage 1 was reconstructed with an alternative method of distance estimation, yielding essentially the same parasite–host associations. Zoonotic potential of E. bieneusi is thus supported on a phylogenetic basis.     

Prevalence and diversity of Encephalitozoon spp. and Enterocytozoon bieneusi in wild boars (Sus scrofa) in Central Europe

From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1–6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.     

Infectivity of<Emphasis Type="Italic"> Cryptosporidium parvum</Emphasis> genotype I in conventionally reared piglets and lambs

Parasites of the genus Cryptosporidium are intracellular parasites that occur throughout the animal kingdom and have been reported in many species of mammals, including human. Most infections in humans are caused by two C. parvum genotypes, genotype I and genotype II; these are the human and the bovine (zoonotic) genotypes, respectively. Successful experimental infection of Cryptosporidium parvum genotype I "human genotype" is described in four conventionally reared piglets and in a lamb. The inoculum was originally obtained from two diarrheic children, and the Cryptosporidium genotypes were determined by PCR and rDNA sequencing. The infective dose was between 10(6) and 2 x 10(6) oocysts. No clinical signs were observed in the infected animals, except in a piglet that showed watery diarrhea. The oocyst shedding period in positive animals ranged between 4 and 10 days. Histopathologic examination of the gastrointestinal tract of two positive piglets revealed shortening of the villi and denudation of the villous tips of the jejunum. In one piglet, the colon mucosa revealed numerous Cryptosporidium oocysts. The storage time of the inocula (< or =3 weeks in PBS at 4 degrees C) and the age of the animal (newborn) were important for the successful induction of infection.     

Determination of Types of Enterocytozoon bieneusi Strains Isolated from Patients with Intestinal Microsporidiosis

To determine the types of Enterocytozoon bieneusi strains associated with intestinal microsporidiosis, we developed a rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of E. bieneusi. RFLPs generated with two restriction enzymes (NlaIII and Fnu4HI) in PCR-amplified ITS products were used to classify strains into different lineages. This approach was successfully used to differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we found four genetically unrelated lineages, showing the genetic diversity of E. bieneusi. Type I strains of E. bieneusi were found in a majority of the samples, accounting for 51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III, and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases, respectively. All strains of E. bieneusi we have tested so far fall into one of four different lineages, and this study shows that human intestinal microsporidiosis is most often associated with type I strains. PCR-RFLP analysis of the ITS region of E. bieneusi should be useful for epidemiological studies.Microsporidia are obligate intracellular protozoan parasites that can infect vertebrates as well as invertebrates. Over 1,000 species of microsporidia have been described, but reports of human infections were rare before the AIDS epidemic. Since then, microsporidia have been recognized as opportunistic pathogens in patients with AIDS. Different species of microsporidia have been shown to infect humans, and among these species, Enterocytozoon bieneusi (7) is by far the most frequently identified. E. bieneusi infection is responsible for chronic diarrhea and wasting in immunocompromised patients, such as patients with AIDS and organ transplant recipients (2, 15, 17, 23). Substantial variation in the progression and severity of disease is observed among cases of intestinal microsporidiosis, and these differences are presumably due to several factors, including host immune defenses and phenotypic differences in parasite strains (23).However, modes of transmission and sources of human infection by E. bieneusi are largely unknown (11, 23). Epidemiologic investigations of a number of infectious diseases have shown that amplification methods could be extremely valuable tools for laboratory-based investigations (20). This approach has proven successful in defining different strains of Encephalitozoon cuniculi, a zoonotic microsporidian that can infect humans and a wide variety of other mammals (6, 8, 10, 14). At least three strains of this species have been defined, on the basis of the number of 5′-GTTTT-3′ repeats present in the internal transcribed spacer (ITS) region of the ribosomal DNA. We wished to study the genetic diversity of E. bieneusi strains by a similar approach. In this report, we describe the development of an amplification-based assay for genetic analysis of the ITS region of E. bieneusi that should be useful for epidemiological studies.     

Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.Enterocytozoon bieneusi is an opportunistic organism causing diarrhea in human immunodeficiency virus (HIV)-positive patients, in whom it has a prevalence of 2 to 50% (5). The infection not only has been reported to occur in immunocompromised hosts but also has been found in healthy individuals (14, 20). This organism can infect a broad range of animals (4, 12, 16, 18, 22, 23). Genotypes of E. bieneusi in humans and animals are differentiated using the polymorphisms of the internal transcribed spacer (ITS) sequence of the rRNA gene (4, 11). To date, at least 70 ITS genotypes have been reported to infect humans and animals (2, 6). The zoonotic nature of E. bieneusi was confirmed because ITS genotypes found in domestic and wild animals had been reported to occur in immunosuppressed hosts (22). In Thailand, we reported genotypes E, O, and PigEBITS 7, which have previously been identified in pigs (3, 4) and in Thai HIV-infected patients (9). This study aimed to identify the ITS genotypes of E. bieneusi in pigs and humans who worked in or lived near pig farms to investigate the transmission of E. bieneusi among these host species.A cross-sectional study of E. bieneusi infection was conducted in a community in Nakorn Pathom Province, Central Thailand, January 2005. This community is composed primarily of four pig farms, a residential area, and a school. The residential area, but not the school, was near the pig farms. Fecal specimens were collected from school children and those who were living in this community, including pig farmers. Fecal specimens were also collected from pigs of four farms and examined for E. bieneusi. The study was approved by the Ethics Committee of the Royal Thai Army, Medical Department. Informed consent was obtained from each adult individual and from parents of school children before enrollment in the study.Fecal specimens from pigs and humans were examined for microsporidian spores using gram-chromotrope staining under light microscopy (13). DNA was prepared from water-ethyl acetate-concentrated stool specimens using FTA filter paper (Whatman Bioscience, United Kingdom) as previously described (21). Amplification of the ITS region of the small-subunit rRNA gene was performed using primers under conditions described by Katzwinkel-Wladarsch et al. (8). For specimens with PCR-negative results, PCR amplification was repeated at least twice. DNA sequencing was conducted by Macrogen Inc., Seoul, Republic of Korea. Nucleotide sequences were determined using the Sequencher program (Gene Codes Corporation, Inc.), and multiple alignment was performed using Clustal X 1.83 for Windows (24). The genotype of each specimen was confirmed by determining the homology of the sequenced PCR product with the published sequence.A total of 268 pig fecal samples were collected. Pigs aged between 21 days and 22 months were examined for E. bieneusi infection. Microsporidial spores were found in 0.7% of pig fecal samples using gram-chromotrope staining, while a greater prevalence of E. bieneusi infection, 15.7%, was detected by PCR. The prevalences of E. bieneusi infection among the four farms and different age groups are presented in Table ​Table1.1. A significantly higher prevalence of E. bieneusi was found in pigs aged 2 to 3.9 months than in pigs of other age groups (chi-square test, P < 0.001). Multivariate analysis confirmed that pigs aged 2 to 3.9 months had a 5.3-times-greater risk of infection than pigs in other age groups (95% confidence interval, 2.6 to 10.6; P < 0.001). Of these 42 E. bieneusi-positive samples, 21 (50%) were successfully characterized by sequencing analysis, and the organism was identified as being of genotypes E (12 samples [57.1%]), O (8 samples [38.1%]), and H (1 sample [4.8%]).

TABLE 1.

Prevalence of E. bieneusi positivity in pig specimens as determined by PCR

Monoclonal Antibodies against Enterocytozoon bieneusi of Human Origin

Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.