GI Flora and Arthritis Pattern in Spondyloarthropathy

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GI Flora and Arthritis Pattern in Spondyloarthropathy

Materials and Methods

Skeletal Analysis

The articular surfaces of skeletons of 79 free-ranging Pan troglodytes troglodytes, 26 free-ranging Pan troglodytes schweinfurthii, 99 Gorilla gorilla, and 38 Gorilla beringei adults (as determined by fused epiphyses) were examined. Presence of erosions or fusion was noted. Pan troglodytes schweinfurthii and G. beringei were curated at the Musée Royal de l'Afrique Centrale, Tervuren, Belgium. Pan troglodytes troglodytes and G. gorilla were curated at the Department of Anthropology at the Cleveland Museum of Natural History, the Department of Mammalogy at the National Museum of Natural History (Smithsonian, Washington, DC), and the Department of Mammalogy at the Museum of Comparative Zoology (Harvard, Cambridge, MA), but represent repositories of a single collection effort from the French Cameroons, the Central African Republic of Congo, Gabon, and Nigeria with ranges from the mouth of the Congo River into Cameroon. Pan troglodytes schweinfurthii and G. beringei were from the Democratic Republic of Congo, north of the Congo River.

Sample Collection and Processing

Feces of Eastern and Western gorillas collected in their normal ranges were assessed for 16S rRNA from existing banks of specimens originally collected for simian immunodeficiency disease studies. Fecal samples were from great ape scat (identified to origin by experienced trackers in the field and confirmed by mtDNA analysis) previously collected from Cameroon, the Central African Republic, and the Democratic Republic of the Congo. The fecal samples were derived from a different sample than that used in the skeletal analysis.

The QIAamp DNA Stool Kit (Qiagen, Valencia, CA) was utilized to extract thawed scat by spin-column filtration, and the highly variable V6 region of 16S rDNA region spanned by primers 926F (5'-aaactYaaaKgaattgacgg-3') and 1492R (5'-tacggYtaccttgttacgactt-3') was amplified. The 50 μL reaction cell utilized 1.25 U Taq (GE Healthcare, Chalfont, Buckinghamshire, UK), 0.25 μL 10 mM dNTP mix (MBI Fermentas, Hanover, MD), 1.5 μL 10 mg/mL bovine serum albumin (New England Biolabs, Ipswich, MA), 0.5 μL of each 10 μM primer, and 40 ng template DNA. Three-minute incubation at 95°C was followed by twenty-five 30-second cycles of 95°C, 45 seconds at 55°C, and 90 seconds at 72°C, with a10-minute final extension at 72°C and purification on MinElute PCR columns (Qiagen, Valencia, CA). The 454 Life Sciences/Roche (Branford, CT) software was used to convert pyrosequencing flowgrams to sequence reads (starting at position 851–857 and end-trimmed with LUCY and assigned based on identifying bar-code sequences, utilizing RDP classifier); χ and Fisher exact tests were utilized to assess significance of scat bacterial organism DNA content correlation with ape species/subspecies and locale.

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