PCR and Gel Electrophoresis

DNA was extracted from ≈25 mg cecum, colon, jejunum, and liver tissue with a commercial tissue kit (DNeasy Tissue Kit, Qiagen, Venlo, the Netherlands). A PCR assay amplifying a 447-bp fragment of the 16S rRNA gene of H. pullorum was then used for detection purposes (1). From each sample, 2 μL template was added to 8 μL PCR mixture containing 0.03 U/μL Taq polymerase Platinum (Invitrogen Life Technologies, Merelbeke, Belgium), 10× PCR Buffer (Invitrogen Life Technologies), 3 mmol MgCl₂ (Invitrogen, Life Technologies), 40 μmol/L each of deoxynucleoside triphosphate (Invitrogen Life Technologies), a final primer concentration of 0.5 μmol/L, and sterile distilled water. The conditions used for the amplifications were the following: an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 90 s, elongation at 72°C for 90 s, and a final elongation at 72°C for 5 min.

Five microliters of the PCR products of each sample were mixed with 3 μL of sample buffer 5× (50% glycerol, 1 mmol cresol red) and were subjected to electrophoresis through an agarose gel containing 1.5% Multi Purpose agarose (Boehringer, Mannheim, Germany) and 50 ng ethidium bromide in per milliliter 1× Tris-acetate ethylenediaminetetraacetic acid buffer (Amresco, Solon, OH, USA), pH 8. As molecular size marker, the Gene Ruler 100-bp DNA ladder plus (MBI Fermentas, St. Leon-Rot, Germany) was used. Electrophoresis was implemented at a constant voltage of 170 V in 0.5× Tris-acetate ethylenediaminetetraacetic buffer for 75 min. The gels were visualized by using the Image Master VDS (Pharmacia Biotech, Puurs, Belgium).

Isolation of H. pullorum

Recovery of H. pullorum isolates was attempted on all positive samples in the PCR analysis described above. The samples (200 mg) for isolation of H. pullorum were placed in a 1.5-mL tube with 400 μL of a mixture of 7.5 g glucose, 25 mL brain heart infusion broth (Oxoid, Basingstoke, England), and 75 mL sterile inactivated horse serum, and then homogenized. The various isolates were inoculated on brain heart infusion agar that was supplemented with 10% horse blood, amphotericin B 20 μg/mL (Fungizone, Bristol-Myers Squibb, Epernon, France), and Vitox (Oxoid) (blood agar). A modified filter technique of Steele and McDermott (13) was then used. Briefly, a sterile cellulose acetate membrane filter (0.45 μm) was applied with a sterile pair of tweezers directly onto the surface of the agar. When the filter was totally absorbed on the agar, ≈300 μL of the mixture was placed in the middle of the filter. After at least 1 hour of incubation at 37°C and 5% CO₂, the filter was removed with a sterile pair of tweezers and the filtrate was streaked on the agar with a loop. Incubation was conducted in microaerobic conditions (5% H₂, 5% CO₂, 5% O₂, and 85% N₂) at 37°C for a minimum of 3 days. Very small, gray-white, hemolytic colonies were selected and purified on a blood agar plate. The colonial form and phenotypic characteristics (gram-negative, slightly curved rod, catalase and oxidase positive, and indoxyl acetate negative) of the isolates were used for presumptive identification. Confirmation was based on PCR and sequencing of a 447-bp fragment of the 16S ribosomal RNA gene, as described below.

Analysis of Nucleotide Sequences

The PCR product of the retrieved H. pullorum isolates was purified with the Qiaquick purification kit (Qiagen) and sequenced by using the same primers applied in the assay with the BigDye Terminator cycle sequencing kit (Applied Biosystems, Lennik, Belgium). Sequencing products were run on the ABI prism 3100 Genetic Analyzer (Applied Biosystems) by using 50-cm capillaries filled with Performance-Optimized-Polymer 6. The electrophoregrams were exported and converted to the Kodon software package (Applied Maths, Sint-Martems-Latem, Belgium). Sequences were compared to published H. pullorum 16S rRNA sequences obtained from GenBank (accession nos. AY631956, L36143, and L36144) by using BLAST software (available from http://www.ncbi.nlm.nih.gov/blast/).

Restriction Endonuclease Digestion and Ligation of Adaptors for AFLP

DNA of H. pullorum isolates was extracted by using a commercial tissue kit (DNeasy Tissue Kit, Qiagen). An aliquot containing 200 ng DNA, determined by optic density (260/280 nm) measurement by using the Spectra Fluor (TECAN, Grödig, Salzburg, Austria), was digested for 2 h at 37°C with BglII (10U/μL) and Csp6I (10U/μL) (MBI Fermentas) in TAC-buffer as described by Vos et al. (14). Five microliters of DNA digest was used in a ligation reaction containing 130 μg/mL BglII adaptor-oligonucleotide and 13 μg/mL Csp6I adaptor-oligonucleotide (Invitrogen) (14), 10× T4 DNA ligase buffer, T4 DNA ligase (1 U/μL) (Amersham Pharmacia), and TAC-buffer in a final volume of 20 μL. After incubation for 2 h at 25°C, the 20 μL ligation reaction was diluted 25 times.

Direct Selective PCR Amplification of Diluted Ligation

Five microliters of the diluted ligation reaction were applied in the PCR assay. The primers used in this assay were BGL2F-0, 5´-GAG TAC ACT GTC GAT CT-3´ (FAM labeled, 5´-end) and CSP6I-A, 5´-GAG CTC TCC AGT ACT ACA-3´ (15). The PCR conditions were as follows: an initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and elongation at 72°C for 90 s; and a final elongation at 72°C for 10 min.

Capillary Electrophoresis

PCR products were run on the ABI prism 3100 Genetic Analyzer (Applied Biosystems) by using the Fragile X Rox-1000 size standard and 50-cm capillaries filled with Performance-Optimized-Polymer 6. Electropherograms were analyzed with Genemapper U 3.5 Software (Applied Biosystems).

Numerical Analyses of AFLP Profiles

The program BioNumerics version 2.5 (Applied Maths) was used to perform numerical analyses of AFLP profiles. Strain relationships were inferred by use of the Pearson product-moment correlation coefficient and unweighted pair-group with mathematical average (UPGMA) clustering and depicted in a dendrogram (16).