| To assess the potential for transmission of cryptosporidiosis, microsporidiosis, and cyclosporiasis between humans and non-human primates that are in close contact with humans, 235 fecal specimens from captive baboons in Kenya were analyzed for Cryptosporidium spp., Enterocytozoon bieneusi, and Cyclospora spp. by PCR and DNA sequence analysis of the small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region of the rRNA gene, and the SSU rRNA gene, respectively. Cryptosporidium hominis was identified in six (2.6%) of the specimens. DNA sequence analysis of the 60kDa glycoprotein (gp60) gene of five C. hominis specimens revealed the presence of three subtypes in subtype families, Ib, If and a novel subtype family named Ii. E. bieneusi was detected in 29 (12.3%) specimens, belonging to 10 genotypes (four known genotypes: A, D, Peru7 and Peru11; six new genotypes: KB-1 to KB-6) that formed two phylogenetic clusters. All the E. bieneusi genotypes were previously found in humans or were genetically related to those in humans. Only one species of Cyclospora, C. papionis, was identified in 42 (17.9%) specimens. Results of this study indicated that non-human primates in Kenya were infected with human-pathogenic Cryptosporidium genotypes and subtypes and E. bieneusi genotypes. Thus, cross-species transmission of cryptosporidiosis and microsporidiosis is possible between humans and non-human primates that are in close contact with each other.Then, we applied multilocus sequence typing (MLST) tool described by Feng[1] to type 32 E. bineusi-positive specimens (3 with DNA sequencing failure at the ITS locus were included). Using nested PCR, we obtained 24, 8, 15, and 21 positives at loci MS1, MS3, MS4, and MS7, respectively. At the MS1 locus, genotype classification was based on a combination of the number of trinucleotide TGC, TAA and TAC repeats in the microsatellite region and the SNP in the rest of the sequence, revealing the presence of 10 distinct genotypes in 24 specimens with a gene diversity of 0.88. At the MS3 locus, the TA indels and SNP were used to determine the genotypes, resulting in the generation of 5 distinct genotypes in 8 specimens with a gene diversity of 0.86. The MS4 locus has a 35 bp minisatellite repeat region and two regions of insertion-deletion polymorphisms (a 4 bp fragment of GGTA between positions 147 and 150 and a 10 bp fragment of TTTTTTTCTT between positions 358 and 367). Coupled with SNPs outside the repeat region, MS4 yielded a total of 5 genotypes in 15 specimens with a gene diversity of 0.70. The MS7 locus has a microsatellite region with trinucleotide TAA repeats and the SNP in the rest of the sequence. It produced 7 genotypes (Hd = 0.77) in 21 specimens. ITS genotyping have shown that there were 10 ITS genotypes in 29 specimens, including host-adapted and zoonotic genotypes. The absence of PCR amplification at the locus MS3 in most isolates of host-adapted ITS genotypes such as KB-6 implied MS3 had the ability of discriminating the host-adapted from zoonotic E. bieneusi. Phylogenetic analysis of the nucleotide sequences obtained at microsatellite and minisatellite loci produced genetic relationship similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most human-pathogenic E. bieneusi genotypes. Thus, the MLST tool was appropriate for high resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., public health significance of E. bieneusi in animals, and source of human E. bieneusi infections.Genotyping based on DNA sequence analysis of the ITS locus has revealed significant genetic diversity in E. bieneusi. Thus far, the population genetics of E. bieneusi and its significance in microsporidiosis epidemiology have not been examined. To better understand the extent of genetic heterogeneity within E. bieneusi and examine its genetic structure, a MLST study 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. Together with the ITS, the use of four other genetic markers (MS1, MS3, MS4, and MS7) enabled high resolution genotyping of E. bieneusi. By a combination of sequence length polymorphism and nucleotide substitutions, 39 multilocus genotypes (MLGs) were identified among the 72 specimens. The observation of strong intragenic linkage disequilibria (LD) and detection of limited genetic recombination among loci were highly indicative of an overall clonal population structure of E. bieneusi. Measures of pair-wise intergenic LD and standardized index of association (ISA) 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 ITS genotype A, and the other (group 2) containing isolates of multiple ITS genotypes (mainly genotypes D and IV) with zoonotic potential. The measurement of LD and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. Sub-structuring analysis using STRUCTURE resulted in the formation of two or three sub-populations depending on the setting of K values, and this was verified via measuring Wright's fixation index (FST) between sub-populations. In conclusion, the MLST analysis of five markers had adequate power of resolution in delineating the population genetic structure of E. bieneusi. The data highlight the power of high resolution MLST in understanding the epidemiology of E. bieneusi.Thereafter, we applied MLST to genotype 105 E. bieneusi specimens isolated from AIDS patients in Peru, Nigeria, and India and five specimens isolated from captive baboons in Kenya using a combination of the ITS marker and four microsatellite and minisatellite markers, of which 72 isolates from Peru have been analyzed before. We determined 65 MLGs in total 110 isolates based on combined sequence length and nucleotide polymorphism. When the isolates of various geographical areas were treated as a single total population, the measures of intragenic and intergenic LD coupled with the limited genetic recombination detected indicated that there was an overall clonal population structure in E. bieneusi. The phylogenetic analysis based on either multilocus gene sequence or allelic profile data as well as the substructural analysis using STRUCTURE all found no evidence to support geographic sub-structuring of the population. When geographic populations (India, Kenya, and Nigeria) were analyzed, the observation of strong and significant LD and detection of limited recombination were highly indicative of a clonal population structure in each population tested. Although there was no evidence of geographic sub-population structure variations, both phylogenetic analysis and substructural analysis revealed the presence of two major genetically isolated groups in total population, one (group 1) contained isolates representing all ITS genotype A. The other (group 2) harbored isolates of multiple ITS genotypes with zoonotic potential. The measurement of LD and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. The data further confirmed the existence of various genetic sub-populations in E. bieneusi, which should be helpful in determining the transmission patterns of human microsporidiosis. |
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