Identification of a Novel, Invasive, Not-Yet-Cultivated Treponema sp. in the Large Intestine of Pigs by PCR Amplification of the 16S rRNA Gene

Abstract

Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii T (90.1%). The spirochete, here named "Candidatus Treponema suis," was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.Helical-shaped bacteria are commonly present in the gastrointestinal tract of animals and humans. Intestinal spirochetes of pigs include several species of the genus Brachyspira, of which Brachyspira hyodysenteriae and Brachyspira pilosicoli are important pathogens causing swine dysentery, a severe mucohemorrhagic colitis, and spirochetal colitis, respectively (14, 15). Culturing of Brachyspira as well as Treponema species is fastidious and not always successful. Although ultrastructural differentiation is possible, the various spirochetes are quite similar (typically 0.2 to 0.4 m in width and 4 to 12 m long) (11), and the size is not a useful criterion for distinction in histopathological specimens. Alternative methods for species identification include fluorescent in situ hybridization (FISH) with specific oligonucleotide probes targeting 16S or 23S rRNA of the porcine pathogens B. hyodysenteriae and B. pilosicoli (2). Here, a novel technique, laser capture microdissection (LCM)-FISH (7), was used to identify an unknown spirochete and to establish its spatial distribution in the pig colonic tissue on archival tissue samples originating from a study by Fossi et al. (4). The bacterial cells were visualized by FISH with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted microcolony. The PCR was done directly on the captured cell material, and the dissected bacterial cells were subsequently identified by analyses of the amplified 16S rRNA gene sequence.Twenty-three formalin-fixed paraffin-embedded biopsy specimens of pig colon were obtained from the National Veterinary and Food Research Institute, Seinäjoki Unit, PB 60101, Seinä-joki, Finland. The samples originated from the previously mentioned study included two sets of eight piglets challenged with one of two B. pilosicoli strains (Br1622 and P43/6/8 T , respectively) and eight uninfected control pigs (4). However, upon both histopathological and in situ hybridization examination of all 23 pigs, an unknown species of spirochetes rather than the challenged bacteria was found to have invaded the colonic mucosa of several of the pigs, including the control pigs (4). For laser capture microdissection (LCM), 3-m-thick sections of the samples were mounted on 0.17-mm PALM POL-Membrane Slides (P.A.L.M. Microlaser

Spatial organization of intestinal microbiota in the mouse ascending colon

“…To capture microbes from both the interfold and digesta regions, we used LCM. We, along with others, have used LCM for analysis of host-gene expression (Wong et al, 2000;Stappenbeck et al, 2002) and to isolate microbial DNA from specified host cells De Hertogh et al, 2006;Molbak et al, 2006). Although the interfold region measures on the order of 100 mm in greatest dimension, the high resolution of LCM (B5 mm) permits easy and precise sample collection.…”

Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs

“…Estimates of variation in bacterial colonization patterns within and between treatment groups were made based on pairwise comparisons of T-RFs using the Dice similarity coefficients (SD) calculation in the Bionumerics software package as described previously (33). Generally, there is a direct relationship between the SD and the similarity of two compared pigs, with two identical T-RFLP profiles having a similarity coefficient of 1.00 (100% similarity).…”

Virulence Factors of the Oral Spirochete Treponema denticola

“…The numbers of both cultivated and uncultivated Treponema phylotypes reported in recent years have rapidly increased, mainly due to the mass of data obtained from efforts to sequence genes encoding 16S rRNA (Choi et al, 1994;Wyss et al, 2004;Demirkan et al, 2006;Molbak et al, 2006;Nordhoff et al, 2008a;Pringle et al, 2008Pringle et al, , 2009Evans et al, 2009;Sayers et al, 2009;Yano et al, 2009). There are currently 49 species of oral Treponema listed on the Human Oral Microbiome Database (Dewhirst et al, 2010), the best-characterized being T. denticola, Treponema amylovorum, T. lecithinolyticum, Treponema maltophilum, Treponema medium, Treponema parvum, …”