Molecular diversity of Giardia duodenalis, Cryptosporidium spp. and Blastocystis sp. in asymptomatic school children in Leganés, Madrid (Spain)

Abstract Background: The protozoa Giardia duodenalis, Cryptosporidium spp., the stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi are among the most frequent diarrheal pathogens affecting humans globally. This molecular epidemiological study assesses the frequency and molecular diversity of these intestinal parasites in schoolchildren in central Spain, complementing previously published data on risk and protective factors associated with parasite infection (Reh et al., Euro Surveill. 2019;24).Methods: Stool samples were collected from voluntary asymptomatic schoolchildren (4‒14 years) and their siblings (1‒16 years) attending 12 primary and secondary schools in Leganés (Madrid). Initial detection of pathogens was conducted by PCR-based methods targeting the small subunit (ssu) ribosomal RNA or the internal transcribed spacer (ITS) genes of these parasite species. Genotyping of G. duodenalis-positive samples was carried out by PCR and Sanger sequencing of appropriate markers including the glutamate dehydrogenase (gdh), the ß-giardin (bg), and triose phosphate isomerase (tpi) loci. For C. hominis/C. parvum-positive samples the 60-kDa glycoprotein (gp60) locus was used.Results: A total of 1,512 stool samples were analysed. Giardia duodenalis was the most prevalent pathogen (17.4%, 95% CI: 15.5‒19.4%), followed by Blastocystis sp. (13.0%, 95% CI: 11.4‒14.8%), and Cryptosporidium spp. (0.9%, 95% CI: 0.5%‒1.5%). Enterocytozoon bieneusi was not detected. Sequence analyses of the 24 G. duodenalis isolates genotyped at the gdh, bg, and/or tpi loci revealed the presence of sub-assemblages AII (16.6%, 4/24) and BIV (79.2%, 19/24). An additional sequence (4.2%, 1/24) represented an ambiguous BIII/BIV result. Analyses of the 14 Cryptosporidium sequences generated at the ssu rRNA allowed the identification of C. hominis (71.4%; 10/14) and C. parvum (21.4%; 3/14). An additional sequence (7.2%, 1/14) was only identified at the genus level. A total of 162 Blastocystis sp. isolates were successfully genotyped, revealing the presence of five subtypes including ST1 (22.8%; 37/162), ST2 (36.4%; 59/162), ST3 (21.6%; 35/162), ST4 (18.6%; 30/162), and ST8 (0.6%; 1/162).Conclusions: Giardia duodenalis sub-assemblage BIV, Cryptosporidium hominis and Blastocystis ST2 were the genetic variants of these parasite species more prevalent in the asymptomatic schoolchildren population investigated. These findings are very similar to those previously reported in clinical, symptomatic populations in Spain. Enterocytozoon bieneusi was absent in apparently healthy schoolchildren.

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Intestinal parasites in children hospitalized at the Central Hospital in Maputo, Mozambique

The Journal of Infection in Developing Countries ◽

10.3855/jidc.3916 ◽

2014 ◽

Vol 8 (06) ◽

pp. 786-789 ◽

Cited By ~ 11

Author(s):

Ana Maria Fonseca ◽

Natercia Fernandes ◽

Filipa S Ferreira ◽

Joana Gomes ◽

Sónia Centeno-Lima

Keyword(s):

Low Income ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Low Income Countries ◽

Central Hospital ◽

Concentration Technique ◽

Contagious Diseases ◽

Parasite Detection ◽

Stool Samples ◽

Direct Microscopic Observation

Introduction: Intestinal parasites are important contributors to the global disease burden, especially in children of low-income countries. The present study determined the frequency of intestinal parasites in children hospitalized at the diarrhea section of the Infectious-Contagious Diseases ward and at the Malnutrition ward of the Department of Pediatrics of the Maputo Central Hospital in Mozambique. Methodology: This pilot study conducted between February and March 2009 enrolled a total of 93 children between 1.5 and 48.2 months of age; 87.1% were younger than 24 months. Parasite detection in stool samples was achieved using direct microscopic observation and Ritchie’s concentration technique. Results: Infection with pathogenic intestinal parasites was detected in 16.1% (15/93) of the children. Giardia duodenalis and Trichuris trichiura were the most common parasites (6.5%, 6/93 each), followed by Ascaris lumbricoides (2.2%, 2/93). One case of mixed infection with A. lumbricoides plus T. trichiura was also detected. Conclusion: This study reinforces the importance of routinely examining stool samples for the diagnosis of intestinal parasites (including protozoa) in children hospitalized in endemic areas.

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Evaluation of intestinal parasites in patients with chronic spontaneous urticaria in a territory hospital in Turkey

The Journal of Infection in Developing Countries ◽

10.3855/jidc.11552 ◽

2019 ◽

Vol 13 (10) ◽

pp. 927-932

Author(s):

Sedat Vezir ◽

Filiz Kaya ◽

Emine Vezir ◽

Nermin Karaosmanoğlu ◽

Ali Kudret Adiloğlu

Keyword(s):

Patient Group ◽

Chronic Urticaria ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Adult Patients ◽

Chronic Spontaneous Urticaria ◽

Parasitic Infections ◽

Cutaneous Manifestations ◽

Blastocystis Sp ◽

Stool Samples

Introduction: Chronic spontaneous urticaria (CSU) which develops without a known stimulation is defined as the occurrence of spontaneous wheals, angioedema or both for longer than six weeks. Infections, autoimmunity, food intolerance and internal parasitic infections are supposed to be underlying causes of CSU. The aim of this study was to evaluate the intestinal parasites in children and adult patients diagnosed as CSU, to determine the frequency of parasites in chronic urticaria, and to compare these patients with healthy demographic control groups. Methodology: Seventy six children and 38 adult patients with CSU were examined in terms of parasitic infections. The patients whom parasites were detected received anti-parasitic therapy and the improvements in CSU symptoms were evaluated. Stool samples were examined with direct microscopic examination (native-lugol), stool concentration and trichrome staining methods. Results: In pediatric patient group, 18.4% (n = 14) of the stool samples were positive for Blastocystis sp., 2.6% (n = 2), Dientamoeba fragilis and 1.3% (n = 1), Giardia duodenalis. In adult patient group, Blastocystis sp. was detected in 18.4% (n = 7) of the stool samples. Anti-parasitic therapy yielded substantial improvement in urticaria symptoms in 57.1% of pediatric and 60.0% of adult patients. Conclusions: Blastocystis sp. and D. fragilis may play a role in chronic urticaria which seriously disrupts the patient's quality of life. Parasitic infections should not be neglected in patients with cutaneous manifestations.

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Molecular detection of Enterocytozoon bieneusi in farm-raised pigs in Hainan Province, China: infection rates, genotype distributions, and zoonotic potential

Parasite ◽

10.1051/parasite/2020009 ◽

2020 ◽

Vol 27 ◽

pp. 12 ◽

Cited By ~ 6

Author(s):

Huan-Huan Zhou ◽

Xin-Li Zheng ◽

Tian-Ming Ma ◽

Meng Qi ◽

Jing-Guo Zhou ◽

...

Keyword(s):

Its Region ◽

Small Subunit ◽

Enterocytozoon Bieneusi ◽

Human Infection ◽

Hainan Province ◽

Nested Polymerase Chain Reaction ◽

High Prevalence ◽

Stool Samples ◽

Group 10 ◽

High Degree

Enterocytozoon bieneusi is a zoonotic fungal pathogen with a high degree of host diversity that can parasitize many animals, including humans. Pigs may play an important role in the epidemiology of E. bieneusi as reservoir hosts. Nevertheless, the genotypes of E. bieneusi in pigs in China remain poorly understood. The aim of this study was to determine the prevalence of E. bieneusi infection amongst pigs raised on farms from four cities of Hainan Province, using nested polymerase chain reaction (PCR) of the partial small subunit of the ribosomal RNA gene, and to identify genotypes of E. bieneusi isolates based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Among 188 stool samples, E. bieneusi was detected in 46.8% (88/188). Eight genotypes including four known (EbpA, CS-4, MJ14, and CHG19) and four novel (HNP-I – HNP-IV) genotypes were identified. Using phylogenetic analysis, genotypes EbpA, CS4, CHG19, HNP-III, and HNP-IV were clustered into zoonotic Group 1, while the remaining three genotypes (MJ14, HNP-I, and HNP-II) clustered into Group 10. The high prevalence of zoonotic genotypes of E. bieneusi among pigs suggests that pig farming is a potential source of human infection. Additionally, this is the first identification of genotypes in Group 10 in pigs indicating unique epidemic features of E. bieneusi in pigs in Hainan Province, the southernmost part of China.

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Molecular characterization of Cryptosporidium spp. from patients with diarrhoea in Lusaka, Zambia

Parasite ◽

10.1051/parasite/2020050 ◽

2020 ◽

Vol 27 ◽

pp. 53

Author(s):

Namwiinga Rozaria Mulunda ◽

Kyoko Hayashida ◽

Junya Yamagishi ◽

Sandie Sianongo ◽

Gilbert Munsaka ◽

...

Keyword(s):

Age Distribution ◽

Small Subunit ◽

University Teaching ◽

Small Subunit Rrna ◽

African Countries ◽

Cryptosporidium Hominis ◽

Genetic Characteristics ◽

Transmission Cycle ◽

Stool Samples ◽

Sub Saharan

Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1–4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.

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Genotyping and subtyping ofGiardiaandCryptosporidiumisolates from commensal rodents in China

Parasitology ◽

10.1017/s0031182014001929 ◽

2015 ◽

Vol 142 (6) ◽

pp. 800-806 ◽

Cited By ~ 27

Author(s):

Z. ZHAO ◽

R. WANG ◽

W. ZHAO ◽

M. QI ◽

J. ZHAO ◽

...

Keyword(s):

Intestinal Parasites ◽

Giardia Duodenalis ◽

Small Subunit ◽

House Mice ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Gene Encoding ◽

Gp60 Gene ◽

Commensal Rodents ◽

Rrna Gene Sequencing

SUMMARYCryptosporidiumandGiardiaare two important zoonotic intestinal parasites responsible for diarrhoea in humans and other animals worldwide. Rodents, as reservoirs or carriers ofCryptosporidiumandGiardia, are abundant and globally widespread. In the present study, we collected 232 fecal specimens from commensal rodents captured in animal farms and farm neighbourhoods in China. We collected 33 Asian house rats, 168 brown rats and 31 house mice. 6·0% (14/232) and 8·2% (19/232) of these rodents were microscopy-positive forGiardiacysts andCryptosporidiumoocysts, respectively. All 14Giardiaisolates were identified asGiardia duodenalisassemblage G at a minimum of one or maximum of three gene loci (tpi, gdhandbg). By small subunit rRNA (SSU rRNA) gene sequencing,Cryptosporidium parvum(n= 12) andCryptosporidium muris(n= 7) were identified. The gp60 gene encoding the 60-kDa glycoprotein was successfully amplified and sequenced in nineC. parvumisolates, all of which belonged to the IIdA15G1 subtype. Observation of the same IIdA15G1 subtype in humans (previously) and in rodents (here) suggests that rodents infected withCryptosporidiumhave the potential to transmit cryptosporidiosis to humans.

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Parasitic Intestinal Protists of Zoonotic Relevance Detected in Pigs by Metabarcoding and Real-Time PCR

Microorganisms ◽

10.3390/microorganisms9061189 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1189

Author(s):

Christen Rune Stensvold ◽

Kateřina Jirků-Pomajbíková ◽

Katrine Wegener Tams ◽

Pikka Jokelainen ◽

Rebecca P. K. D. Berg ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Animal Health ◽

Giardia Duodenalis ◽

Enterocytozoon Bieneusi ◽

Health And Disease ◽

Generation Sequencing ◽

Entamoeba Polecki

Several parasite species are shared between humans and pigs. We explored the application of next-generation sequencing-based metabarcoding supplemented with real-time PCR to fecal DNAs from 259 samples from 116 pigs in Denmark to detect and differentiate single-celled intestinal parasites of zoonotic relevance. Enterocytozoon bieneusi, Balantioides coli, and Giardia duodenalis were observed in 34/37 (92%), 148/259 (57%), and 86/259 (33%) samples, respectively. Entamoeba polecki ST1, E. polecki ST3, and Entamoeba hartmanni were detected in 104/259 (40%), 161/259 (62%), and 8/259 (3%) samples, respectively. Metabarcoding and real-time PCR detected Cryptosporidium in 90/259 (35%) and 239/259 (92%) of the samples, respectively, with Cryptosporidium suis and Cryptosporidium scrofarum observed in nearly equal proportions. Blastocystis subtypes 1, 3, 5, and 15 were found in 72 (28%), 6 (2%), 176 (68%), and 36 (14%) of 259 samples, respectively. Iodamoeba was identified in 1/259 samples (<1%), while none of 37 tested samples was positive for Dientamoeba fragilis. Our results illustrate how metabarcoding exemplifies a ‘one-fits-many’ approach to detecting intestinal single-celled parasites in feces supplemented with real-time PCR for selected parasites. Using metabarcoding with pathogen-specific assays may help detect emerging and previously underdetected pathogens and further elucidate the role of micro-eukaryotic parasites in human and animal health and disease.

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Molecular characterization of three intestinal protozoans in hospitalized children with different disease backgrounds in Zhengzhou, central China

Parasites & Vectors ◽

10.1186/s13071-019-3800-5 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 6

Author(s):

Fuchang Yu ◽

Dongfang Li ◽

Yankai Chang ◽

Yayun Wu ◽

Zhenxin Guo ◽

...

Keyword(s):

Giardia Duodenalis ◽

Small Subunit ◽

Enterocytozoon Bieneusi ◽

Central China ◽

Rrna Genes ◽

Multilocus Genotype ◽

Genetic Characteristics ◽

Transmission Potential ◽

Intestinal Pathogens

Abstract Background Cryptosporidium spp. and Giardia duodenalis are major intestinal pathogens that can cause diarrheal diseases in humans, especially children. Enterocytozoon bieneusi is another parasite which can cause gastrointestinal tract disorders, with diarrhea being the main clinical symptom. However, few genetic studies of these parasites in pediatric inpatients in China have been published. Methods To assess the genetic characteristics and epidemiological status of these parasites, a total of 2284 fecal samples were collected from children in the pediatric departments of three hospitals in Zhengzhou, central China, and screened for these protozoans with PCR, based on the small subunit ribosomal RNA (SSU rRNA) genes of Cryptosporidium spp. and G. duodenalis and the internal transcribed spacer (ITS) of E. bieneusi. Results Six (0.26%), 14 (0.61%), and 27 (1.18%) of the samples were positive for Cryptosporidium spp., G. duodenalis and E. bieneusi, respectively. Of the 12 successfully sequenced G. duodenalis isolates, four were identified as assemblage A and eight as assemblage B. In subtype and multilocus genotype (MLG) analyses, C. parvum IIdA19G1 (n = 4) and two novel G. duodenalis MLGs belonging to subassemblage AII (n = 3) and BIV (n = 5) were successfully identified. The E. bieneusi isolates included genotypes D (n = 17), J (n = 2), PigEBITS7 (n = 1), BEB6 (n = 1), and CM8 (n = 1). This is the first report of C. parvum subtype IIdA19G1 in HIV-negative children and E. bieneusi genotype CM8 in humans. Conclusions The dominance of zoonotic C. parvum subtype IIdA19G1 indicates that this parasite is turning into zoonotic origin from human-to-human transmission. The phylogenetic analysis also revealed the zoonotic origins and anthroponotic transmission potential of G. duodenalis and E. bieneusi, suggesting more efforts must be made to minimize the threat these pathogens pose to public health.

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Identification of Cryptosporidium species, Enterocytozoon bieneusi genotypes, and Giardia duodenalis assemblages in birds in Henan, China

10.21203/rs.2.19784/v1 ◽

2019 ◽

Author(s):

Haiju Dong ◽

Ru Cheng ◽

Xinmiao Li ◽

Junqiang Li ◽

Yuancai Chen ◽

...

Keyword(s):

Migratory Birds ◽

Giardia Duodenalis ◽

Pcr Amplification ◽

Small Subunit ◽

Enterocytozoon Bieneusi ◽

Rrna Gene ◽

Fecal Samples ◽

Zoonotic Pathogens ◽

Its Gene ◽

Turtle Dove

Abstract BackgroundDomesticated, wild, and migratory birds have been known to transmit diseases such as diarrhea in humans and other animals, but studies specifically on the zoonotic pathogens Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in birds in Henan Province, China are lacking. Hence, this study sought to characterize the prevalence of these pathogens, and to identify the different species of Cryptosporidium and their phylogenetic relationships, the genotypes of E. bieneusi, and the assemblages of G. duodenalis, in birds in the province. MethodsFresh fecal samples were collected from birds in parks and pet shops in Henan, China and were screened for the presence of the pathogens using nest-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) gene. ResultsA total of 1,005 fecal samples were collected from 32 species of birds. 21 fecal samples (2.09%) were found positive for Cryptosporidium spp., 45 (4.48%) for E. bieneusi, and 33 (3.28%) for G. duodenalis. This study identified five Cryptosporidium species: C. baileyi (10 out of 21 fecal samples, 47.62%) in crested myna (Acridotheres cristatellus), Java sparrow (Lonchura oryzivora), Chinese hwamei (Garrulax canorus), common quail (Coturnix coturnix), and Chinese grosbeak (Eophona migratoria); C. galli (5/21, 23.81%) in Chinese blackbird (Turdus mandarinus), zebra finch (Taeniopygia guttata), and white-eyes (Zosterops sp.); C. andersoni (1/21, 4.76%) in a white-eye for the first time; C. meleagridis (4/21, 19.05%) in parrots and crested myna; and C. parvum (1/21, 4.76%) in a pigeon. Two E. bieneusi genotypes: Peru6 and PtEb I were found in pigeons and European turtle dove (Streptopelia turtur). The G. duodenalis assemblage E was detected in parrots, common hill myna, crested myna, Java sparrow, white-eyes, black-throated laughingthrush, and other birds. ConclusionsOur findings indicate that the aforementioned species of birds in Henan, China could be a source of zoonotic pathogens, such as C. meleagridis, C. andersoni, C. parvum, E. bieneusi genotype Peru6, and G. duodenalis assemblage E, that cause diseases in humans.

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Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method

Parasitology ◽

10.1017/s0031182000078318 ◽

1994 ◽

Vol 109 (3) ◽

pp. 281-289 ◽

Cited By ~ 41

Author(s):

T. Van Gool ◽

E. U. Canning ◽

H. Gilis ◽

M. A. Van Den Bergh Weerman ◽

J. K. M. Eeftinck Schattenkerk ◽

...

Keyword(s):

Intestinal Parasites ◽

Original Description ◽

Enterocytozoon Bieneusi ◽

Type I ◽

Aids Patients ◽

Cultivation System ◽

Antibiotic Solution ◽

Stool Samples ◽

First Time

SummaryTwo species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.

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