Development and application of oligonucleotide probes for in situ detection of thermotolerant Campylobacter in chicken faecal and liver samples.
Based on Campylobacter 16S- and 23S-rRNA sequence data oligonucleotide probes specific for thermotolerant campylobacters and for members of the genus Campylobacter have been developed. The 16S-rRNA-targeted probe CAMP 653, recommended for a comprehensive detection of members of the genus Campylobacter, specifically detected all Campylobacter strains used in this study. Detection of thermotolerant species has been achieved by the 23S-rRNA-targeted probe CAJECO1427. Optimal hybridisation conditions have been derived for both probes from melting profiles of fluorescence-labelled probe-target hybrids recorded in fluorescence in situ hybridisation experiments (FISH). The FISH assay was evaluated both by spiking poultry faecal samples with Campylobacter jejuni and by detecting Campylobacter in naturally colonized chickens. C. jejuni was reliably detected at levels of 10(6) cfu/g faeces after a 3- h enrichment step in Blood Preston Selective broth. Low level contaminations (<or=10(2) cfu/g) were reliably identified after 24 h of enrichment. By screening cloacal swab samples obtained from birds from a poultry slaughterhouse, thermotolerant Campylobacter spp. were even detected without pre-enrichment. Moreover, the in situ assay was applied on poultry liver samples. The probes allowed a direct, specific detection of thermotolerant Campylobacter in cryosectioned liver samples. Campylobacter spp. detection by FISH using highly specific probes looks promising to become a future monitoring system in a logistic poultry slaughter concept.
Schmid
Michael W
MW
GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Bodenökologie, Abteilung Rhizosphärenbiologie, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany.
Lehner
Angelika
A
Stephan
Roger
R
Schleifer
Karl-Heinz
KH
Meier
Harald
H
D000818:Animals; D002167:Campylobacter / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial; D005243:Feces / Q000382:microbiology*; D005516:Food Microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008099:Liver / Q000382:microbiology*; D018613:Microscopy, Confocal; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S