Treponema spirochetes have been found in many species of animals in close association with their host, with distinct species colonizing genitalia, gastrointestinal tracts and oral cavity. Treponema spirochetes can co-exist as harmless commensals (e.g., T. refringens, T. minutum), symbionts of the intestinal tract (e.g., T. bryantii of ruminants, T. primitia from termites), pathogens (T. pallidum spp.) or as part of a pathogenic complex of bacteria (T. denticola, T. vincentii, and others from the oral cavity) [20, 22]. Additionally, several different phylogenetic groups of Treponema species have been isolated or identified in digital dermatitis lesions, with similarities to T. denticola, T. phagedenis, T. vincentii, T. medium, and the proposed new species T. brennaborense and T. pedis[16, 23–27]. Four Treponema spirochetes were isolated from DD lesions on an Iowa dairy, and the characterization presented here demonstrates that they are highly similar to the T. phagedenis type strain. Despite classification as the same genus, these organisms occupy not just different hosts (bovine vs. human), but also very different anatomical locations (dermis adjacent to heel bulb and dewclaw vs. genitalia). There most likely exists some overlap of microenvironment within these anatomical locations (low oxygen availability, epithelial cell layers, etc.) as both the DD isolates and T. phagedenis have similar growth characteristics and nutrient requirements.
Other pathogenic organisms such as Mycobacterium intracellulare, Yersinia species and Bacillus species have identical 16 s rRNA gene sequences and are highly genetically similar based on DNA-DNA hybridization . However, they exhibit distinct “ecophysiological” properties based on virulence phenotypes or host ranges. Some are distinct species, Y. pestis and Y. pseduotuberculosis for example, while others are merely different serovars within the species, such as M. intracellulare. Some pathogens are separated from other genetically identical species by acquisition of a plasmid conferring pathogenic properties. Evaluation of the draft contigs of T. phagedenis and the DD isolates do not give any indication of acquisition of a plasmid that would have conferred the expansion of host range or conversion into a more virulent organism.
These studies herein led us to develop a growth medium reduced in complexity so that the individual nutrients and growth factors of previously isolated spirochetes could be further evaluated. While the list of components appear similar to fastidious anaerobe broth used by many groups [17, 29], the quantities of several components are greatly reduced. Systematic studies on essential nutrients and environmental growth factors of the non-pallidum treponemes are scarce  and consist of a few incomplete lists in such reference texts as Bergey’s Manual of Systematic Bacteriology and The Prokaryotes [18, 21]. A recently published report showed that isolate 1A achieved log phase growth in 3 to 5 days of culture in a rich media similar to fastidious anaerobe broth  consistent with our results in both media types.
We have defined temperature tolerances, pH tolerances and essential growth requirements (serum and VFAs) of isolate 4A. It was very interesting that an organism isolated from the hoof of a cow was tolerant to and preferred higher temperatures (up to 40°C). The hoof temperature of a dairy cow ranges from 21 to 23°C . The hoof surface temperature was found to increase in cases of DD, sole ulcers, or other hoof diseases , and thus could create a more favorable environment for treponemal growth.
Further insight into the Iowa DD isolates physiology was sought by evaluation of substrate utilization and enzymatic activity of the treponeme isolates. By understanding growth requirements and nutritional capabilities of these isolates, we can begin to piece together the microenvironment necessary for optimal survival and growth of the treponemes. As in the case of human periodontal disease, one bacterial colonizer may provide the nutritional substrates for secondary colonizers and tissue destructive bacteria . There were little differences between T. phagedenis and the DD isolates on the basis of enzymatic activity or substrate utilization, mainly regarding mannitol and trehalose. While there were slight differences in enzymatic profiles, these are generally not sufficient for the separation into different species. For example, T. phagedenis biovar Reiter is able to hydrolyze esculin but biovar Kazan does not . As the complete sequences of both T. phagedenis and these DD isolates become available, these small biochemical differences may be explained by alterations in the genome consistent with host adaptation.
Past studies have evaluated the similarity of DD Treponema isolates based on sequencing of 16S ribosomal regions, 16-23S intergenic spacer regions or conserved flagellin genes (i.e., flaB2). Previously published work has shown that the T. phagedenis-like isolates 9–3301, 7–2009, 2–1498 from California, and 1A and 4A from Iowa, have >99% identical 16S-23S rRNA gene sequence and intergenic spacer regions clustered into the same phylotype based on product length polymorphisms . Although a completed genome for any T. phagedenis isolate is not available, comparison of assembled contigs for isolate 4A revealed a high degree of similarity throughout the genome. Differences in the number of genes identified (3251 in isolate 4A and 2799 genes in F0421) most likely reflect a difference in sequencing coverage and completeness of the resulting contigs. Performance of in silico DDH using isolate 4A and F0421 further supports classification of the bovine lesion isolates as T. phagedenis.