Vol. 22, No. 3

INFECTION AND IMMUNITY, Dec. 1978, p. 736-739 0019-9567/78/0022-0736$02.00/0 Copyright © 1978 American Society for Microbiology

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Genetics of Treponema: Characterization of Treponema hyodysenteriae and Its Relationship to Treponema pallidum R. M. MIAO,'* A. HOWARD FIELDSTEEL,' AND D. L. HARRIS2 Life Sciences Division, SRI International, Menlo Park, California 94025,' and Veterinary Medical Research Institute, College of Veterinary Medicine, Iowa State University, Ames, Iowa 500112

Received for publication 15 August 1978

Saturation reassociation assays with "2'I-labeled treponemal DNAs show that Treponema hyodysenteriae is genetically unrelated to T. pallidum (Nichols), T. phagedenis biotype Reiter, and T. refringens biotype Noguchi. Pathogenic and, nonpathogenic isolates of T. hyodysenteriae exhibited 28% sequence homology and had an extremely low guanine-plus-cytosine content (25.8%).

Previous studies have shown that pathogenic Treponema pallidum (Nichols) is genetically distinct from biotypes of the cultivable nonpathogenic human treponemes T. phagedenis and T. refringens (9). None of the pathogenic human Treponema has been successfully cultivated in vitro. The only pathogenic treponeme that has proven cultivable is T. hyodysenteriae (5), which is a causative factor of swine dysentery (1, 2). Both pathogenic and nonpathogenic isolates of T. hyodysenteriae have been cultivated. It was, therefore, of interest to determine the genetic relationship between T. pallidum (Nichols), pathogenic and nonpathogenic isolates of T. hyodysenteriae, as well as that between T. hyodysenteriae, T. phagedenis, and T. refringens. The results of these studies show that pathogenic and nonpathogenic T. hyodysenteriae isolates were genetically related to each other but were unrelated to T. pallidum or the cultivable human treponemes. MATERIALS AND METHODS Chemicals. Nuclease S1 (EC 3.1.4) was obtained from Miles Laboratories (Elkhart, Ind.) and was stored at -20'C. Salmon sperm DNA was from Calbiochem (La Jolla, Calif.), and Clostridium perfringens DNA was from Worthington Biochemical Corp. (Freehold, N.J.). Phosphate-buffered saline, standard saline citrate, and phosphate buffer were as previously described (9). Treponemes. Virulent T. pallidum (Nichols) was obtained originally from James N. Miller, University of California, Los Angeles. It was propagated and purified as previously described (9). Two pathogenic isolates (B204, A-1) and two nonpathogenic isolates (B256, 4/71) of T. hyodysenteriae were used (5). These isolates were grown in an artificial medium and used between in vitro passage 11 and 14 (4). The source, cultivation, and other characteristics of the T. hyodysenteriae isolates have been reported (5). The nonpathogenic cultivable treponemes T. phagedenis biotype

Reiter and T. refringens biotype Noguchi were grown as previously described by us (9). DNA purification. DNAs were purified by the procedure of Marmur (6) and dissolved in 0.085% NaCl. For radiolabeling and reassociation assays, DNAs were alkaline sheared to 150,000 single-strand molecular weight (8). Preparation of ['25I]DNA. ['25I]DNA was prepared as previously described (9). All preparations were rendered acid soluble (>95%) by treatment with 1,600 U of S1 per ml for 2 h at 37°C and were insensitive to ribonuclease A. Saturation-reassociation assays. DNA-DNA saturation-reassociation assays and the analysis of reaction products by treatment with S1 were as previously described (9), except that reassociation was allowed to occur at 55°C. The percent reassociation was calculated, in counts per minute, as the ratio of S1resistant radioactivity to the total counts per minute in each sample. The total radioactivity per sample was 1,500 to 3,000 cpm. Determination of G+C content. DNA base compositions were determined from thermal denaturation profiles of double-stranded DNA dissolved in standard saline citrate (7). Assays were performed with a GCA/McPherson model EU-707K spectrophotometer recorder with a P120 electronic temperature programmer. The temperature was raised 18°C/h. Each assay included an internal standard of Clostridium perfringens DNA taken as a standard of 26.5% guanine-pluscytosine content (G+C) (7). Base compositions were calculated from thermal denaturation (Tm) values by using the relationship of 0.41°C/1% G+C.

RESULTS DNA sequence homology. The extent of DNA sequence homology among the various treponemes was determined by DNA-DNA saturation - reassociation assays. T. pallidum ['25I]DNA was used as a probe with unlabeled DNA from T. hyodysenteriae isolates B204, A1, B256, and 4/71. There was no reassociation (95% reassociation occurred with unlabeled totally homologous DNA (data not shown). When DNAs from B204, A-1, and B256 were used as 125I-labeled probes, no reassociation (95% reassociation of [125I]DNA in all cases. The extent of DNA sequence homology among the swine treponemes was then determined. The extent of reassociation of B204 [1251I]DNA in the presence of DNA from B204, A-1, B256, 4/71, or salmon sperm is shown in Fig. 1. The level of reassociation in the presence of totally homologous DNA was 100%, whereas reassociation with salmon sperm DNA was

Genetics of Treponema: characterization of Treponema hyodysenteriae and its relationship to Treponema pallidum.

Vol. 22, No. 3 INFECTION AND IMMUNITY, Dec. 1978, p. 736-739 0019-9567/78/0022-0736$02.00/0 Copyright © 1978 American Society for Microbiology Print...
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