Journal: Applied and Environmental Microbiology

Article Title: Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

doi: 10.1128/aem.69.6.3435-3447.2003

Figure Lengend Snippet: FIG. 1. Flow diagram of the experiments conducted in each objective. (A) In the first objective, an internal control (IC) and primers for the genus Campylobacter, C. coli, C. jejuni, C. fetus, and C. hyointestinalis were developed. The presentation of two taxon names in the same box (on the right) represents multiplex reactions. Boxes with thick walls represent nested PCR. Names in the box labeled “Test taxa” represent taxa that were used to determine the specificities of PCRs; numbers in parentheses following the taxon names represent the numbers of isolates tested. (B) In the second objective, bovine feces were inoculated with various concentrations of C. coli, C. fetus, C. hyointestinalis, and C. jejuni (target densities in log10 CFU per gram are presented in circles); the control treatment (“0”) was not inoculated with campylobacters. CFUs were enumerated in the inoculated and control feces by using dilution plating. Concurrently, DNA was extracted from all fecal treatments and subjected to PCR with the primers shown in objective A, and amplicons were electrophoresed. (C) In the third objective, uninoculated bovine feces were collected from eight dairy cows (C1 to C8). Campylobacters were isolated and enumerated on four media maintained at three temperatures. Representative colonies were selected, established in pure cultures, and identified by using physiological characters, PCR with primers for specific taxa and, if required, extraction of DNA and sequencing of a portion of the 16S rRNA gene. During the course of this experiment, C. lanienae was frequently isolated on Karmali medium at 40°C. As a result, a nested primer set was developed for this taxon, and the C. lanienae primers were used in a multiplex PCR with primers for C. fetus. DNA was extracted from fecal samples obtained from the eight cows and subjected to PCR, and amplicons were electrophoresed. The experiments in the second and third objectives were conducted three times on separate occasions.

Article Snippet: The following reference strains were used to test the primers that we developed: Arcobacter butzleri (ATCC 49616 [American Type Culture Collection]), A. cryaerophilus (ATCC 49942), C. coli (HC 1111 [Health Canada] and ATCC 49941), C. concisus (ATCC 33237), C. fetus subsp. fetus (ATCC 25936), C. fetus subsp. venerealis (ATCC 19438), C. hyointestinalis subsp. hyointestinalis (ATCC 35217), C. hyointestinalis subsp. lawsonii (NCTC 12901 [National Collection of Type Cultures and Pathogenic Fungi]), C. jejuni (HC 1102, HC 1104, HC 1115, HC 1108, ATCC 29428, ATCC 49943, and ATCC 33291), C. lanienae (NCTC 13004), C. lari (ATCC 35221), and C. upsaliensis (LCDC 5424 [Laboratory Centre for Disease Control]).

Techniques: Control, Multiplex Assay, Nested PCR, Labeling, Isolation, Extraction, Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

doi: 10.1128/aem.69.6.3435-3447.2003

Figure Lengend Snippet: FIG. 3. Impact of C. jejuni L102 genomic DNA concentration on the expression of the internal control amplicon. Lane 1, 100-bp mo- lecular weight marker (the dark band was at 500 bp); lane 2, 101

Article Snippet: The following reference strains were used to test the primers that we developed: Arcobacter butzleri (ATCC 49616 [American Type Culture Collection]), A. cryaerophilus (ATCC 49942), C. coli (HC 1111 [Health Canada] and ATCC 49941), C. concisus (ATCC 33237), C. fetus subsp. fetus (ATCC 25936), C. fetus subsp. venerealis (ATCC 19438), C. hyointestinalis subsp. hyointestinalis (ATCC 35217), C. hyointestinalis subsp. lawsonii (NCTC 12901 [National Collection of Type Cultures and Pathogenic Fungi]), C. jejuni (HC 1102, HC 1104, HC 1115, HC 1108, ATCC 29428, ATCC 49943, and ATCC 33291), C. lanienae (NCTC 13004), C. lari (ATCC 35221), and C. upsaliensis (LCDC 5424 [Laboratory Centre for Disease Control]).

Techniques: Concentration Assay, Expressing, Control, Amplification, Marker

Journal: Applied and Environmental Microbiology

Article Title: Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

doi: 10.1128/aem.69.6.3435-3447.2003

Figure Lengend Snippet: FIG. 4. Multiplex PCR for detection of Campylobacter species in bovine feces. Lane 1, 100-bp molecular weight marker (the dark band was at 500 bp); lane 2, DNA from uninoculated feces amplified with the Campylobacter genus-specific primer set; lane 3, DNA from unin- oculated feces amplified with the Campylobacter genus-specific primer set (note the weak genus amplicon and the internal control amplicon at 465 bp); lane 4, DNA from uninoculated feces amplified with the C. jejuni-C. coli multiplex nested primer set (note the C. jejuni amplicon at 413 bp); lane 5, DNA from uninoculated feces amplified with the C. fetus-C. lanienae nested primer set (note the C. lanienae amplicon at 360 bp); lane 6, DNA from feces inoculated with C. coli ATCC 49941 at a density of 103 CFU g1 and amplified with the C. jejuni-C. coli multiplex nested primer set (note the C. coli amplicon at 330 bp); lane 7, DNA from feces inoculated with C. hyointestinalis subsp. hyointesti- nalis ATCC 35217 at a density of 102 CFU g1 and amplified with the C. hyointestinalis seminested primer set (note the C. hyointestinalis amplicon at 468 bp); lane 8, DNA from feces inoculated with C. fetus ATCC 25936 at a density of 102 CFU g1 and amplified with the C. fetus-C. lanienae nested primer set (note the C. fetus amplicon at 473 bp); lane 9, negative control for the internal control and Campylobacter genus-specific primers (no template added); lane 10, negative control for C. jejuni-C. coli multiplex primers; lane 11, negative control for C. fetus-C. lanienae multiplex primers; lane 12, negative control for C. hyointestinalis subsp. hyointestinalis primers.

Article Snippet: The following reference strains were used to test the primers that we developed: Arcobacter butzleri (ATCC 49616 [American Type Culture Collection]), A. cryaerophilus (ATCC 49942), C. coli (HC 1111 [Health Canada] and ATCC 49941), C. concisus (ATCC 33237), C. fetus subsp. fetus (ATCC 25936), C. fetus subsp. venerealis (ATCC 19438), C. hyointestinalis subsp. hyointestinalis (ATCC 35217), C. hyointestinalis subsp. lawsonii (NCTC 12901 [National Collection of Type Cultures and Pathogenic Fungi]), C. jejuni (HC 1102, HC 1104, HC 1115, HC 1108, ATCC 29428, ATCC 49943, and ATCC 33291), C. lanienae (NCTC 13004), C. lari (ATCC 35221), and C. upsaliensis (LCDC 5424 [Laboratory Centre for Disease Control]).

Techniques: Multiplex Assay, Molecular Weight, Marker, Amplification, Control, Negative Control

Journal: Applied and Environmental Microbiology

Article Title: Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

doi: 10.1128/aem.69.6.3435-3447.2003

Figure Lengend Snippet: FIG. 6. Dendrogram based on a majority-rule consensus tree ob- tained from analyzing the partial 16S rRNA gene data set with the NEIGHBOR program (neighbor-joining option) and showing DNA sequence relatedness for campylobacters. The outgroup used in the analysis was C. jejuni, and isolates indicated by “T” represent type specimens. The bar represents 0.01 nucleotide substitution per base, and numbers at selected nodes indicate support obtained by bootstrap analysis (1,000 replicates) for the internal branches within the resulting trees.

Article Snippet: The following reference strains were used to test the primers that we developed: Arcobacter butzleri (ATCC 49616 [American Type Culture Collection]), A. cryaerophilus (ATCC 49942), C. coli (HC 1111 [Health Canada] and ATCC 49941), C. concisus (ATCC 33237), C. fetus subsp. fetus (ATCC 25936), C. fetus subsp. venerealis (ATCC 19438), C. hyointestinalis subsp. hyointestinalis (ATCC 35217), C. hyointestinalis subsp. lawsonii (NCTC 12901 [National Collection of Type Cultures and Pathogenic Fungi]), C. jejuni (HC 1102, HC 1104, HC 1115, HC 1108, ATCC 29428, ATCC 49943, and ATCC 33291), C. lanienae (NCTC 13004), C. lari (ATCC 35221), and C. upsaliensis (LCDC 5424 [Laboratory Centre for Disease Control]).

Techniques: Sequencing

Journal: Microbiology and immunology

Article Title: Isolation and characterization of bacteriophages specific for Campylobacter jejuni.

doi: 10.1111/j.1348-0421.2009.00163.x

Figure Lengend Snippet: Fig. 1. Electron microphotographs of isolated Campylobacter- specific bacteriophages, CPS1–6 (a–f). Bacteriophages were exam- ined by EF-TEM with 0.2% uranyl acetate negative-staining. Each scale bar corresponds to 100 nm.

Article Snippet: Bacterial strains and growth media Six Campylobacter reference strains (C. jejuni NCTC 11168, 81–176, ATCC 33291, ATCC 43429, A74/c, and NCTC12662 [phage type 14, PT14]), 11 C. jejuni isolates from chickens (12), SM1 (a phage CPS2-resistant NCTC 12662 derivative isolated by sequential cultivation with CPS2), and C. coli isolates were subcultured on brain heart infusion agar ([BHI] Difco, Franklin Lakes, NJ, USA) for 18 hr at 42◦C under micro-aerobic conditions (6% O2, 7.1% CO2, 3.6% H2, 83.3% N2), which was generated by using the Anoxomat system (Mart Microbiology, Lichtenvoorde, The Netherlands), unless otherwise indicated.

Techniques: Isolation, Negative Staining

Journal: Microbiology and immunology

Article Title: Isolation and characterization of bacteriophages specific for Campylobacter jejuni.

doi: 10.1111/j.1348-0421.2009.00163.x

Figure Lengend Snippet: Fig. 4. Electron microscopy images of CPS2 attachment to Campylobacter jejuni strains. Each strain cultured for 1 hr was incubated with CPS2 in Tris-HCl buffer for 30 min at 42 ◦C. CPS2 could attach to NCTC 12662 (a) and ATCC 43429 (d), but not the spontaneous mutant of NCTC 12662 (SM1 [b]) or NCTC 11168 (c). The arrows indicate CPS2.

Article Snippet: Bacterial strains and growth media Six Campylobacter reference strains (C. jejuni NCTC 11168, 81–176, ATCC 33291, ATCC 43429, A74/c, and NCTC12662 [phage type 14, PT14]), 11 C. jejuni isolates from chickens (12), SM1 (a phage CPS2-resistant NCTC 12662 derivative isolated by sequential cultivation with CPS2), and C. coli isolates were subcultured on brain heart infusion agar ([BHI] Difco, Franklin Lakes, NJ, USA) for 18 hr at 42◦C under micro-aerobic conditions (6% O2, 7.1% CO2, 3.6% H2, 83.3% N2), which was generated by using the Anoxomat system (Mart Microbiology, Lichtenvoorde, The Netherlands), unless otherwise indicated.

Techniques: Electron Microscopy, Cell Culture, Incubation, Mutagenesis