infects a wide range of mammalian hosts. differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some isolates experienced heterogeneous copies of the SWP-1 gene with numerous numbers of repeats. Intragenotypic variance was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate in clinical samples. is usually a common microsporidian parasite that infects numerous mammals, such as rabbits, rats, mice, horses, foxes, cats, dogs, muskrats, leopards, baboons, and humans (7, 13). Recent characterization of the internal transcribed spacer (ITS) of the rRNA gene has recognized three genotypes of based on the number of GTTT repeats present: a genotype or strain I from rabbits made up of three repeats, a genotype or strain II from mice made up of two repeats, and a genotype or strain III from dogs made up of four repeats (9). Thus far, both genotype or strain I and genotype or strain III genotypes of have been found in humans, indicating that human infection can be of zoonotic origin (6, 8, 18, 20, 21). The number of human isolates characterized so far, however, is very small (13). Currently, genotyping of entails mostly DNA sequencing of ITS, which is not practical in most diagnostic laboratories because of the technical demands and high cost. Thus, there is a need for the development of simpler genotyping tools that are more cost-effective and can be performed in most diagnostic laboratories. This will allow the genotyping of in large numbers of laboratories and better characterization of the epidemiology of human infection. Recently, genes coding for the polar tube protein (PTP) and spore wall protein I (SWP-1) of were reported (2, 5). Because the gene has long central repeats of 78 bp in PTP and 15 and 36 bp in SWP-1 and the number of repeats in repetitive proteins tends to vary in other parasites such as spp., we examined the sequence diversity of PTP and SWP-1 genes among numerous isolates of isolates used in this study included seven isolates from humans and one isolate from a rabbit (Table ?(Table1).1). Three previously characterized reference strains, strain I from a rabbit, strain II from a mouse, and strain III from a dog (9) were also used as controls. Most isolates were managed in E6 and HLF cell cultures after inoculation with biopsy samples, with the exception of isolates CDC:V428A, CDC:V449A, and 3275, for which infected tissues Rabbit Polyclonal to SGK (phospho-Ser422) were directly used in molecular analysis. TABLE 1 isolates used in this study and genotyping results from analyses of repetitive genes DNA extraction. DNA was extracted from biopsy specimens or cultured parasites using a previously explained method (10). Briefly, tissues or cultured organisms were washed twice in phosphate-buffered saline before they were placed in 300 l of lysis buffer made up of 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 mM dithiothreitol, 2 mg of proteinase K per ml, and 250 U of lyticase (Sigma Co., St. Louis, Mo.). After 15 min of incubation, mechanical disruption was performed with 0.5-mm-diameter glass beads (Biospec Products, Bartlesville, Okla.) and a mini-bead beater (Biospec Products) at 5,000 rpm for 3 min. The suspension was then incubated at 37C for 18 h. After addition of 150 l of 2% sodium dodecyl sulfate and 20 l of proteinase K (20 mg/ml), the incubation was continued at 50C for 72 h. DNA was extracted with phenol-chloroform and precipitated with ethanol. Nucleic acid from each sample was resuspended BAPTA in 50 l of distilled water and stored at ?20C before being used in PCR. Sequence analysis of the ITS. The biopsy specimens or cultured parasites were genotyped by sequence analysis of the ITS of the rRNA gene (24). Briefly, the 3 end of the small subunit rRNA, ITS, and the 5 end of the large subunit rRNA were amplified from extracted DNA by PCR using BAPTA primers ss530f [5-GTGCCAGC(C/A)GCTGGCAC-3] and ls212r1 [5-GTT(G/A)GTTTCTTTTCCTC-3]. The PCR product was sequenced BAPTA in both directions on an ABI377 autosequencer (Applied Biosystems, Foster City, Calif.). genotype was determined by the number of the GTTT repeats present.