Confirmation of the Human-Pathogenic Microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in Water

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.     

Molecular Characterization of Microsporidia Indicates that Wild Mammals Harbor Host-Adapted Enterocytozoon spp. as well as Human-Pathogenic Enterocytozoon bieneusi

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.     

Molecular Detection of Enterocytozoon bieneusi and Identification of a Potentially Human-Pathogenic Genotype in Milk

Milk specimens from 180 dairy cows were examined for the presence of Enterocytozoon bieneusi, using molecular assays. Fifteen specimens were found to be positive, of which 3 were identical to the human E. bieneusi types, which suggests that some E. bieneusi isolates from milk can infect humans. Overall, dairy cows' milk may play a significant role in the transmission of E. bieneusi infections to humans.     

Identification of potentially human-pathogenic Enterocytozoon bieneusi genotypes in various birds

Enterocytozoon bieneusi was detected in 24 of 83 samples from birds of the orders Columbiformes, Passeriformes, and Psittaciformes. It was identical to or closely related to the Peru6 genotype, which was previously found in humans in Peru. Thus, various birds can be a significant source of environmental contamination by potentially human-pathogenic E. bieneusi.     

Identification of Potentially Human-Pathogenic Enterocytozoon bieneusi Genotypes in Various Birds

Enterocytozoon bieneusi was detected in 24 of 83 samples from birds of the orders Columbiformes, Passeriformes, and Psittaciformes. It was identical to or closely related to the Peru6 genotype, which was previously found in humans in Peru. Thus, various birds can be a significant source of environmental contamination by potentially human-pathogenic E. bieneusi.     

基于特异性PCR和荧光检测技术快速鉴定艾纳香

为建立快速准确的艾纳香分子鉴定方法。采取筛选艾纳香及其混伪品基因组DNA的提取方法,针对艾纳香特异性位点设计引物,优化PCR扩增条件,荧光检测扩增产物。结果表明碱裂解法更适于艾纳香基因组DNA的提取;叶绿体基因（tDNA）特异引物能特异性扩增艾纳香DNA,其扩增产物荧光检测呈绿色,混伪品无反应发生。试验结果显示该法简化了分子鉴定过程,省时节力,且结果准确可靠,可作为艾纳香植物和药材的鉴定方法。     

Population genetic analysis of Enterocytozoon bieneusi in humans

Genotyping based on sequence analysis of the ribosomal internal transcribed spacer has revealed significant genetic diversity in Enterocytozoonbieneusi. Thus far, the population genetics of E. bieneusi and its significance in the epidemiology of microsporidiosis have not been examined. In this study, a multilocus sequence typing of E. bieneusi in AIDS patients in Lima, Peru was conducted, using 72 specimens previously genotyped as A, D, IV, EbpC, WL11, Peru7, Peru8, Peru10 and Peru11 at the internal transcribed spacer locus. Altogether, 39 multilocus genotypes were identified among the 72 specimens. The observation of strong intragenic linkage disequilibria and limited genetic recombination among markers were indicative of an overall clonal population structure of E. bieneusi. Measures of pair-wise intergenic linkage disequilibria and a standardised index of association (IAS) based on allelic profile data further supported this conclusion. Both sequence-based and allelic profile-based phylogenetic analyses showed the presence of two genetically isolated groups in the study population, one (group 1) containing isolates of the anthroponotic internal transcribed spacer genotype A, and the other (group 2) containing isolates of multiple internal transcribed spacer genotypes (mainly genotypes D and IV) with zoonotic potential. The measurement of linkage disequilibria and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. The formation of the two sub-populations was confirmed by STRUCTURE and Wright's fixation index (FST) analyses. The data highlight the power of MLST in understanding the epidemiology of E. bieneusi.     

Enterocytozoon bieneusi Genotypes in Cattle on Farms Located within a Water Catchment Area

Enterocytozoon bieneusi is a microsporidian found in humans and other animals around the world. Investigations in some countries, such as the U.S., have indicated the importance of E. bieneusi as a zoonotic water‐ and food‐borne pathogen. However, there is scant epidemiological information on E. bieneusi in animals in many countries including Australia. Here, we conducted the first molecular epidemiological study of E. bieneusi in farmed cattle in Victoria, Australia, to assess whether these bovids are carriers of “zoonotic” genotypes of E. bieneusi. A total of 471 individual faecal samples were collected from calves of < 3 mo and of 3–9 mo of age. Genomic DNAs were extracted from individual faecal samples and then subjected to nested PCR‐based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA to identify E. bieneusi and define genotypes. Enterocytozoon bieneusi was detected in 49 of the 471 samples (10.4%). An analysis of ITS sequence data revealed three known genotypes (BEB4, I, and J) and three novel genotypes (designated TAR_fc1 to TAR_fc3). Phylogenetic analysis showed that genotypes BEB4, I, J, TAR_fc1, and TAR_fc2 clustered with genotypes identified previously in humans, indicating that cattle are carriers of E. bieneusi with zoonotic potential.     

Enterocytozoon bieneusi Genotypes and Infections in the Horses in Korea

Enterocytozoon bieneusi is a microsporidian pathogen. Recently, the equestrian population is increasing in Korea. The horse-related zoonotic pathogens, including E. bieneusi, are concerns of public health. A total of 1,200 horse fecal samples were collected from riding centers and breeding farms in Jeju Island and inland areas. Of the fecal samples 15 (1.3%) were PCR positive for E. bieneusi. Interestingly, all positive samples came from Jeju Island. Diarrhea and infection in foals were related. Two genotypes (horse1, horse2) were identified as possible zoonotic groups requiring continuous monitoring.     

Molecular Characterization of Enterocytozoon bieneusi in Wild Carnivores in Spain

Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty‐five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi‐positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1‐EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1‐EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human‐pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination.     

Frequent Occurrence of Mixed Enterocytozoon bieneusi Infections in Humans

Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.     

Molecular Detection,Multilocus Genotyping,and Population Genetics of Enterocytozoon bieneusi in Pigs in Southeastern China

Enterocytozoon bieneusi is an important opportunistic pathogen widely distributed in humans and animals that causes diarrhea or fatal diarrhea in immunocompromised hosts. To examine the infection status and molecular characteristics of E. bieneusi in pigs, 725 fecal samples were collected from pigs in six areas of Fujian Province. The E. bieneusi genotypes were identified based on the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene by nested PCR, and its population genetics were analyzed by multilocus sequence typing (MLST). The results showed that the infection rate of E. bieneusi was 24.4% (177/725), and 11 known genotypes (EbpC, EbpA, CHN‐RR2, KIN‐1, CHG7, CHS5, CM11, CHG23, G, PigEBITS, and D) and 2 novel genotypes (FJF and FJS) were identified. All the genotypes were found to be clustered into zoonotic Group 1. Moreover, 52 positive samples were successfully amplified at minisatellite and microsatellite loci and formed 48 distinct multilocus genotypes (MLGs). Further population structure analyses showed strong genetic linkage disequilibrium (LD) and several recombination events (Rm), indicating that E. bieneusi has a clonal population structure. This study is the first to investigate the prevalence and molecular characteristics of E. bieneusi in Fujian Province and could provide baseline data to control E. bieneusi infection in pigs and humans and deepen our understanding of the zoonotic risk of E. bieneusi and its distribution in China.     

PCR Methods for Identification and Specific Detection of Probiotic Lactic Acid Bacteria

Probiotics are defined as “microbes improving animal feed.” Three lactic acid bacteria, previously selected as probiotic for pig feeding, were identified by sequencing the variable V₁ region of the 16S rDNA after PCR amplification primed in the flanking constant region. A V_R region showing strong nucleotide differences between the three probiotic and the reference strains was delimited. Oligonucleotides specific for each strain were designed. A specific assay for probiotic detection was developed, based on a PCR reaction with three primers. Received: 7 December 1995 / Accepted: 30 January 1996