Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions.
Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.
Institut f�r Mikrobiologie und Hygiene, Universit�tsklinikum Charit�Humboldt-Universit�t zu Berlin, Dorotheenstr. 96, D-10117 BerlinGermany.
Annette
A
Moter
Institut f�r Veterin�r-Pathologie der Freien Universit�t BerlinStra�e 518, Nr. 15, D-14163 BerlinGermany.
Gregor
G
Leist
Institut f�r Veterin�r-Pathologie der Freien Universit�t BerlinStra�e 518, Nr. 15, D-14163 BerlinGermany.
Roland
R
Rudolph
Institut f�r Mikrobiologie und Tierseuchen der Freien Universit�t BerlinLuisenstr. 56, D-10117 BerlinGermany.
Kirstin
K
Schrank
Department of Oral Biology, Yonsei University134 Shinchon-Dong, 120-752 SeoulKorea.
Bong-Kyu
BK
Choi
Technische Universit�t M�nchenLehrstuhl f�r Mikrobiologie, Arcisstr. 16, D-80290 M�nchenGermany.
Michael
M
Wagner
Institut f�r Mikrobiologie und Hygiene, Universit�tsklinikum Charit�Humboldt-Universit�t zu Berlin, Dorotheenstr. 96, D-10117 BerlinGermany.
Ulf B
UB
G Bel
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology* / Q000473:pathology; D003872:Dermatitis / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014208:Treponema / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D014211:Treponemal Infections / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S