Enterocytozoon Bieneusi Infection Research Articles

Meta-analysis of the global prevalence and risk factors of Enterocytozoon bieneusi infection in pigs from 1999 to 2021

Enterocytozoon bieneusi (E. bieneusi), which is one of the most common microsporidia, has been identified as an important obligate intracellular pathogen that commonly colonizes in a variety of animal species and humans worldwide, including humans. In this study, the statistical analyses of E. bieneusi infection and prevalence were performed to clarify the relationship between different genotypes in different countries. The databases Chinese National Knowledge Infrastructure (CNKI), VIP Chinese Journal Database, Wanfang Data, PubMed, Web of Science and ScienceDirect were used for data collection. The research data were subjected to subgroup, univariate regression, and correlation, to reveal factors related to the high prevalence of E. bieneusi. A total of, 34 of the 498 articles published before April 2022 met the inclusion criteria. The global prevalence of E. bieneusi in pigs was 37.69% (5175/12672). The prevalence of E. bieneusi in nursery pigs was 58.87% (588/946). In developing countries and Asia, the highest prevalence of E. bieneusi in pigs were 37.62% (4752/11645) and 40.14% (4715/11345), respectively. Moreover, humans and pigs have been found to be infected with the same genotype of E. bieneusi in some cases, as evidenced by the consolidation of genotype information. The results showed that pigs are susceptible to E. bieneusi during the nursery period. The prevalence of E. bieneusi is high in developing countries, and its genotype prevalence varies in each country. Thus, it is essential to strengthen the health inspection of vulnerable groups and customs quarantine inspection.

Global prevalence and risk factors of Enterocytozoon bieneusi infection in humans: a systematic review and meta-analysis.

Enterocytozoon bieneusi is one of the most important zoonotic pathogens. In this study, we present a systematic review and meta-analysis of the prevalence of human E. bieneusi infection in endemic regions and analyze the various potential risk factors. A total of 75 studies were included. Among 31,644 individuals tested, 2,291 (6.59%) were E. bieneusi-positive. The highest prevalence of E. bieneusi in the male population was 5.50%. The prevalence of E. bieneusi in different age groups was varied, with 10.97% in teenagers. The prevalence of E. bieneusi in asymptomatic patients (6.49%) is significantly lower than that in HIV-infected patients (11.49%), and in patients with diarrheal symptoms (16.45%). Rural areas had a higher rate (7.58%) than urban ones. The prevalence of E. bieneusi in humans was the highest (6.42%) at altitudes<10m. Moreover, the temperate zone marine climate (13.55%) had the highest prevalence. A total of 69genotypes of E. bieneusi have been found in humans. This is the first global study regarding E. bieneusi prevalence in humans. Not only people with low immunity (such as the elderly, children, people with HIV, etc.), but also people in Europe in temperate marine climates should exercise caution to prevent infection with E. bieneusi during contact process with animals.

Enterocytozoon bieneusi Infection in Livestock from selected farms in Lagos, Nigeria

Abstract Microsporidia are enteric intracellular pathogens infecting animals and humans. It causes life-threatening diarrhoea in humans. Microsporidia species have been isolated from several domestic and wild animals, however, data on microsporidia infection in animals in Nigeria is still very scanty. This study was conducted to clarify and improve our knowledge of the possibility of the zoonotic origin of Enterocytozoon species in the environment. Two hundred and fifty (250) animal faecal samples from various farm animals were examined for their frequency of microsporidia spores using Weber’s chromotrope stain and Indirect Fluorescence Antibody Test (IFAT). Enterocytozoon bienuesi were detected in 6 (24.0%) turkeys, 2 (8.0 %) goats, 3(12.0 %) cattle, and 4(16.0%) rams. No reactivity was detected with other species of microsporidia by IFAT. This study confirms the presence of E. bienuesi in the animals examined and shows that animals can be a significant source of zoonotic transmission in the environment.

HIV/AIDS in Combination with Enterocytozoon bieneusi Infection

Acquired immunodeficiency syndrome (AIDS) is a significant public health problem caused by infection with the human immunodeficiency virus (HIV). The most common cause of death in AIDS patients is opportunistic infection with various microorganisms. Intestinal protozoa, such as Enterocytozoon bieneusi, are prevalent yet difficult to treat. This review will provide an overview of HIV/AIDS mixed with Enterocytozoon bieneusi infection by discussing an introduction, epidemiology, detection methodology, and treatment strategies.

Molecular detection and characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi infections in dromedary camels (Camelus dromedaries) in Egypt.

Few studies have investigated the occurrence of microeukaryotic gut parasites in dromedary camels in Egypt, and the majority of these investigations are based on microscopic analysis of fecal material. Herein, we assessed the occurrence, molecular diversity, and zoonotic potential of protozoan (Cryptosporidium spp. and Giardia duodenalis) and microsporidian (Enterocytozoon bieneusi) pathogens in individual fecal samples (n = 102) of dromedary camels with (n = 26) and without (n = 76) diarrhea from Aswan Governorate, Upper Egypt. Other factors possibly associated with an increased risk of infection (geographical origin, sex, age, and physical condition) were also analyzed. The SSU rRNA or ITS genes were targeted by molecular (PCR and Sanger sequencing) techniques for pathogen detection and species identification. The most abundant species detected was G. duodenalis (3.9%, 4/102; 95% CI: 1.1-9.7), followed by Cryptosporidium spp. (2.9%, 3/102; 95% CI: 0.6-8.4). All samples tested negative for the presence of E. bieneusi. Sequence analysis data confirmed the presence of zoonotic C. parvum (66.7%, 2/3) and cattle-adapted C. bovis (33.3%, 1/3). These Cryptosporidium isolates, as well as the four Giardia-positive isolates, were unable to be amplified at adequate genotyping markers (Cryptosporidium: gp60; Giardia: gdh, bg, and tpi). Camels younger than 2 years old were significantly more likely to harbor Cryptosporidium infections. This connection was not statistically significant, although two of the three cryptosporidiosis cases were detected in camels with diarrhea. The spread of G. duodenalis infections was unaffected by any risk variables studied. This is the first report of C. parvum and C. bovis in Egyptian camels. The finding of zoonotic C. parvum has public health implications since camels may function as sources of oocyst pollution in the environment and potentially infect livestock and humans. Although preliminary, this study provides useful baseline data on the epidemiology of diarrhea-causing microeukaryotic parasites in Egypt. Further research is required to confirm and expand our findings in other animal populations and geographical regions of the country.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n=1058) and farmed (n=324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

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 Detection of Cryptosporidium spp. and Enterocytozoon bieneusi Infection in Wild Rodents From Six Provinces in China.

Enterocytozoon (E.) bieneusi and Cryptosporidium spp. are the most important zoonotic enteric pathogens associated with diarrheal diseases in animals and humans. However, it is still not known whether E. bieneusi and Cryptosporidium spp. are carried by wild rodents in Shanxi, Guangxi, Zhejiang, Shandong, and Inner Mongolia, China. In the present study, a total of 536 feces samples were collected from Rattus (R.) norvegicus, Mus musculus, Spermophilus (S.) dauricus, and Lasiopodomys brandti in six provinces of China, and were detected by PCR amplification of the SSU rRNA gene of Cryptosporidium spp. and ITS gene of E. bieneusi from June 2017 to November 2020. Among 536 wild rodents, 62 (11.6%) and 18 (3.4%) samples were detected as E. bieneusi- and Cryptosporidium spp.-positive, respectively. Differential prevalence rates of E. bieneusi and Cryptosporidium spp. were found in different regions. E. bieneusi was more prevalent in R. norvegicus, whereas Cryptosporidium spp. was more frequently identified in S. dauricus. Sequence analysis indicated that three known Cryptosporidium species/genotypes (Cryptosporidium viatorum, Cryptosporidium felis, and Cryptosporidium sp. rat genotype II/III) and two uncertain Cryptosporidium species (Cryptosporidium sp. novel1 and Cryptosporidium sp. novel2) were present in the investigated wild rodents. Meanwhile, 5 known E. bieneusi genotypes (XJP-II, EbpC, EbpA, D, and NCF7) and 11 novel E. bieneusi genotypes (ZJR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were also observed. This is the first report for existence of E. bieneusi and Cryptosporidium spp. in wild rodents in Shanxi, Guangxi, Zhejiang, and Shandong, China. The present study also demonstrated the existence of E. bieneusi and Cryptosporidium spp. in S. dauricus worldwide for the first time. This study not only provided the basic data for the distribution of E. bieneusi and Cryptosporidium genotypes/species, but also expanded the host range of the two parasites. Moreover, the zoonotic E. bieneusi and Cryptosporidium species/genotypes were identified in the present study, suggesting wild rodents are a potential source of human infections.

Zoonotic Microsporidia in Wild Lagomorphs in Southern Spain.

Simple SummaryA cross-sectional study was carried out to assess the presence of zoonotic microsporidia in organ meats of European wild rabbits and Iberian hares consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific polymerase chain reactions (PCRs) for the detection of microsporidia DNA. We confirmed the presence of Enterocytozoon bieneusi in three wild rabbits and Encephalitozoon intestinalis in one wild rabbit and three Iberian hares. However, none of the 462 sampled wild lagomorphs showed Encephalitozoon hellem nor Encephalitozoon cuniculi infection. This is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0–1.7%) but not in hares (0.0%; 95% CI: 0.0–4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0–0.8%) and three Iberian hares (3.8%; 95% CI: 0.0–8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0–0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors’ knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.

Zoonotic potential of Enterocytozoon bieneusi in pre-weaned Korean native calves

BackgroundEnterocytozoon bieneusi is the most common microsporidian species infecting humans and various animals worldwide. To date, there has been limited information on the prevalence of infection and genotypes of E. bieneusi in cattle in the Republic of Korea. Therefore, this study investigated the prevalence and genotypes of E. bieneusi circulating in pre-weaned Korean native calves and determined the age pattern of E. bieneusi infection and the relationship between E. bieneusi infection and diarrhea.MethodsThe prevalence of E. bieneusi infection in pre-weaned Korean native calves was screened by polymerase chain reaction. PCR-positive products were sequenced to determine the genotype of E. bieneusi. A Chi-square analysis was used to compare the association between diarrhea and the infection rate of E. bieneusi in each age range or for all ages.ResultsPCR and sequencing analysis revealed an overall prevalence (16.9%, 53/314) of E. bieneusi in pre-weaned calves. The prevalence of E. bieneusi was highest in September (36.2%), followed by March (28.3%). Enterocytozoon bieneusi infection was associated with diarrhea in calves (χ2 = 5.82, P = 0.016). Our results also indicated that E. bieneusi infection was significantly associated with calf age (χ2 = 11.61, P = 0.003), and the prevalence of E. bieneusi infection was significantly higher in calves aged 21–40 days-old (odds ratio: 2.90, 95% confidence interval: 1.54–5.45; P = 0.001) than in those aged 1–20 days-old. Interestingly, the association between E. bieneusi infection and diarrhea was observed only in calves aged 1–20 days-old (χ2 = 5.82, P = 0.010). We identified three known genotypes, BEB4 (n = 12), BEB8 (n = 21) and J (n = 16), and three novel genotypes, BEB8-like (n = 21), KCALF1 (n = 1) and KCALF2 (n = 1). The genotype BEB8 was the most prevalent among all age groups. All genotypes identified in this study exhibited zoonotic potential.ConclusionsTo our knowledge, this is the first report of the genotype BEB4 in pre-weaned Korean native calves. Zoonotic E. bieneusi infection was prevalent in pre-weaned calves, indicating that cattle may play an important role as a reservoir host for E. bieneusi transmission to humans.

Molecular detection of porcine Enterocytozoon bieneusi infection in Peninsular Malaysia and epidemiological risk factors associated with potentially zoonotic genotypes.

Enterocytozoon bieneusi is an emerging opportunistic pathogen infecting humans, and both domestic and wild pigs are known to harbour zoonotic genotypes. There remains a paucity of information on the prevalence and epidemiology of this enteropathogen in Southeast Asia. The present study was undertaken to determine the molecular prevalence and risk factors associated with E. bieneusi infection among commercially farmed pigs in Malaysia. Faecal samples were collected from 450 pigs from 15 different farms and subjected to nested PCR amplification of the ribosomal internal transcribed spacer (ITS) gene of E. bieneusi. Phylogenetic analysis involved 28 nucleotide sequences of the ITS region of E. bieneusi. An interviewer-administered questionnaire provided information on the animal hosts, farm management systems and environmental factors and was statistically analysed to determine the risk factors for infection. The prevalence of E. bieneusi infection was relatively high (40.7%). The highest prevalence (51.3%) was recorded among the piglets, while the adults showed the lowest level of infection (31.3%). Multivariate analysis indicated that age of the pigs, distance of the farm from human settlement and farm management system were significant risk factors of infection. Three genotypes (EbpA, EbpC and Henan-III) detected among the pigs are potentially zoonotic. The high prevalence of E. bieneusi among locally reared pigs, the presence of zoonotic genotypes and the spatial distribution of pig farms and human settlements warrant further investigation on the possibility of zoonotic transmission.

Screening of opportunistic Encephalitozoon intestinalis and Enterocytozoon bieneusi in immunocompromised patients in Slovakia

In recent years new infectious diseases, i.e. emerging or re-emerging diseases, have been coming to the forefront. Currently, microsporidia, considered to be a major cause of emerging and opportunistic infections particularly in immunocompromised individuals, are also included in this group. Therefore, the aim of our study was to map the prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi infection in a group of patients and to compare it with the occurrence of specific antigens in immunocompetent people. Detection of spores of both pathogens in faecal samples was performed by an immunofluorescence test using species-specific monoclonal antibodies. Positivity to E. intestinalis in 91 examined immunosuppressed patients reached 33% (30/91), while only 4.3% (3/70) of the control group samples were found to be positive (relative risk 7.7, p < 0.001). In case of E. bieneusi 14.3% (13/91) of immunocompromised patients were positive, as were 5.7% (4/70) of people from the control group (relative risk 2.5, p = 0.095). In case of development of any opportunistic infection, the infection is detected and removed in most cases at an early stage. The incidence of clinically manifested microsporidiosis in patients with immunodeficiency is rare as they are under constant medical supervision. However, we must not forget about opportunistic infections, and in case of any non-specific symptoms it is necessary to exclude or confirm the diagnosis for immediate treatment.

Molecular detection and genotyping of Enterocytozoon bieneusi in family pet dogs obtained from different routes in Japan

The present study determined the prevalence of Enterocytozoon (E.) bieneusi infection and genotyped the isolates in family pet dogs obtained from three different routes in Japan. Fresh fecal samples were collected from 597 family pet dogs. A nested polymerase chain reaction (PCR) assay targeting the internal transcribed spacer (ITS) region of ribosomal DNA was employed for detection of E. bieneusi, and the obtained positive PCR amplicons were sequenced for genotyping. The overall prevalence of E. bieneusi in family pet dogs was 4.4%. Significant differences in prevalence were found among the categories of age (<1 year: 8.3% vs. ≥1 year: 3.4%), obtained route (from private owner: 3.2% vs. from pet shop: 3.9% vs. from breeding kennel: 14.3%), and living regions (ranged 0–10.3%). However, no significant differences were observed in the prevalence of living condition (indoor: 4.6% vs. outdoor: 2.0%), and fecal condition (formed: 4.5% vs. soft: 4.7% vs. diarrhea: 0%). Of the 26 sequenced samples, all were identified as genotype PtEb IX, which belongs to the dog-specific genotype. In conclusion, although E. bieneusi infections are relatively common in family pet dogs in Japan, the risk of zoonotic transmission from dogs to humans is likely to be low.

Molecular Phylodiagnosis of Enterocytozoon bieneusi and Encephalitozoon intestinalis in Children with Cancer: Microsporidia in Malignancies as an Emerging Opportunistic Infection.

Patients with cancer undergoing chemotherapy in a children's hospital in Northwestern Iran were studied; 132 stool samples were collected and stained by the Weber and Ryan-blue modified trichrome staining techniques. The extracted DNA samples were evaluated by the nested polymerase chain reaction (PCR) method. All positive isolates were sequenced for genotyping and phylogenetic analysis. A total of 17 (12.8%) samples were microscopically positive for microsporidia infection, whereas only 14 (10.6%) cases were positive based on nested PCR results. In the positive samples detected with nested PCR, the frequency of Enterocytozoon bieneusi and Encephalitozoon intestinalis infections was 71.4% (n = 10) and 28.6% (n = 4), respectively. After sequencing and phylogenetic analysis, the genotype of E. bieneusi was type D and the sequences of the isolated species were similar to those of the registered ones. E. bieneusi is a major contributor to microsporidiosis in young immunocompromised patients in Iran. Microsporidia species are well-detected when confirmatory techniques such as molecular methods are in agreement with staining. So, to ensure this, a suggestion has been made to introduce a certain diagnostic test for microsporidiosis.

Prevalence and genetic diversity of Enterocytozoon bieneusi in sheep in China

BackgroundEnterocytozoon bieneusi is a common species of microsporidia that not only influences human health but also threatens animal productive performance and value. However, there have been no systematic studies of the prevalence of E. bieneusi in sheep in China.ResultsA total of 953 fecal specimens were collected from sheep from 11 provinces across five regions of China and analyzed for E. bieneusi by nested PCR targeting the ribosomal internal transcribed spacer (ITS). Enterocytozoon bieneusi infections were detected in four regions, with an overall infection rate of 20.4% (194/953). The highest infection rate was detected in pre-weaned lambs (25.0%), followed by post-weaned lambs (22.2%) and adult sheep (14.6%). Enterocytozoon bieneusi was found in nine of the 11 tested provinces, with infection rates between 2.9–51.7%. Eleven genotypes were identified based on ITS analysis, including seven known genotypes (BEB6, CHG1, CHG3, CHS7, CHS8, COS-I and NESH5) and four novel genotypes (CHHLJS1, CHHLJS2, CHNXS1 and CHXJS1). All 11 genotypes were clustered into group 2, and the zoonotic genotype BEB6 was the dominant genotype (n = 129, 66.5%) in sheep.ConclusionThe prevalence of E. bieneusi was studied in five regions representing most areas where sheep are bred in China. This is the first report of E. bieneusi infection in sheep for seven Chinese provinces. Geographical differences were detected in the distribution of E. bieneusi genotypes, but no differences were found among sheep in different age groups. The zoonotic genotype BEB6 was the dominant genotype, indicating that sheep are a potential source of zoonotic microsporidiosis in China. These results improve our knowledge of the epidemiology of E. bieneusi in sheep in China.

Prevalence, genotypes, and risk factors of Enterocytozoon bieneusi in Asiatic black bear (Ursus thibetanus) 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.

Molecular characterization of Enterocytozoon bieneusi isolates in laboratory macaques in north China: zoonotic concerns.

The significance of wild and zoo nonhuman primates (NHPs) as potential sources of human Enterocytozoon bieneusi infections has been increasingly appreciated, while the role of laboratory NHPs in zoonotic transmission of microsporidiosis remains elusive. In this study, the infection rate, genetic characteristic, and zoonotic potential of E. bieneusi were investigated for 205 laboratory macaques in Beijing, north China. The parasite was identified in 37 (18.0%) animals by nested PCR and sequencing of the ribosomal internal transcribed spacer (ITS), with an infection rate of 25.6% in Macaca fascicularis (34/133) and 4.2% in Macaca mulatta (3/72). The differences in infection rate between the two species of macaques and between young macaques aged ≤5 years (29.6%, 32/108) and adults aged >5 years (5.2%, 5/97) were significant (p<0.01). Analysis of the ITS sequence polymorphisms recognized eight known genotypes (CC4, CM1, CM2, D, Peru8, Peru11, Type IV, and WL21) and two new genotypes (named as CMB1 and CMB2), with well-known human-pathogenic genotypes (D, Peru8, Peru11, and Type IV) most frequently detected. The rest genotypes (CC4, CM1, CM2, WL21, CMB1, and CMB2) were clustered into zoonotic group 1 in phylogenetic analysis. The high diversity and widespread presence of the human-pathogenic or group 1 E. bieneusi genotypes in laboratory NHPs, notably M. fascicularis and the young animals, suggest potential of zoonotic transmission. These findings imply that laboratory rhesus macaques could be significant reservoirs for human microsporidiosis.

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.

Why the International Workshops on Opportunistic Protists?

After human immunodeficiency virus (HIV)/AIDS was recognized in the 1980s as a pandemic impacting the United States and Western Europe, it was discovered that in many cases, several microbes rather than HIV itself were the direct cause of mortality. Given the importance of these microbes in AIDS, immunodeficiency-associated diseases (IDADs) and opportunistic infections (OIs) caused by several eukaryotic protists have received the attention of biologists and clinicians. These organisms, however, have been particularly difficult to work on or manipulate experimentally. They represent diverse taxa such as microfungi (Pneumocystis, microsporidians, Candida, Histoplasma), apicomplexans (Cryptosporidium, Cyclospora, Toxoplasma), and other unicellular eukaryotes (free-living amebae, Blastocystis) but present similar challenges to investigators. The difficulties faced with most of these organisms are primarily based on the lack of in vitro axenic mass culture methods that would allow rapid organism proliferation and indefinite subcultivation. Various immunodeficient animal models and cocultivation methods with animal cell lines have been developed, but these alternatives have not eliminated the challenges of low organism yields, serial subcultivation, and facile genetic manipulations. Hence, despite “brute force,” expensive, labor-intensive, and creative research approaches, there remain many unanswered questions. The infectious forms of some of these organisms are not yet fully understood (e.g., Pneumocystis) or there remains uncertainty about the species or genotypes that are able to infect humans (e.g., Cryptosporidium, Enterocytozoon). While these OIs are pathogenic in severely immunocompromised hosts, they are also able to infect those who are immunocompetent. Only limited data are available on the impact of these infections in immunocompetent hosts and the potential roles of these hosts in spreading these pathogens in host populations (e.g., Pneumocystis, microsporidia). There are still limited reproducible animal models, which hinders the understanding of the pathophysiological mechanisms in these infections. The lack of effective drugs against these eukaryotic OIs remains a major concern, and pharmaceutical drug development is still limited. For example, there are only a few effective drugs against Pneumocystis pneumonia (PcP) and some have serious side effects. Also, no safe drugs that efficiently clear Cryptosporidium, Enterocytozoon bieneusi, Toxoplasma, or amebic encephalitis infections are available.

First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic

Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people. The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E. bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity . Spores were detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR) identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.