unsheathed flagella, with no periplasmic fibres, and appeared coccoid after 10-12 days of incubation. Phylogenetic analyses based on 16S rRNA gene revealed that all strains recognized Campylobacter species (Zanoni, et al. 2009). Routine detection of Campylobacter species in clinical laboratories is on culture on selective media and subsequent phenotypic identification. Culture methods are based toward the detection of C. jejuni and C. coli. The antimicrobial agents incorporated into used selective media may inhibit growth of some Campylobacter species (Collins, et al. 2001 and Reddington, et al. 2011). The true incidence of Campylobacter species may be under estimated because of the limitations of routine culture methods because conventional methods are relatively slow (Engberg, et al. 2000). Presumptive results may be available after 2 days. However, definitive species-level identification based on phenotypic methods may require a further 3 to 4 days (O'Connor, et al. 2000). Phenotypic identification can be challenging because of the fastidious growth requirements and the distinguishing of biochemical characteristics by Campylobacter species (Goossens, & Butzler, 1992). Molecular methods on PCR amplification may provide an alternative to culture methods for the detection of Campylobacter in different specimens. The application of PCR-based assays applied to the detection of Campylobacter species in rabbit specimens (Maher, et al. 2003). Amplification of DNA targets including the Campylobacter flagellin gene, 16S rRNA and the 16S/23S rRNA intergenic spacer region (Touzet, et al. 2009).
Centrifugation at full speed for 1 min was done. The QIAamp mini spin column was placed in a clean 1.5 ml micro centrifuge tube. The QIAamp mini spin column was carefully opened and 100 μl buffer AE were added. The QIAamp mini spin column was Incubated at room temperature for 1 min, and then centrifuged at 8000 rpm for 1 min. 3-4. Preparation of duplex PCR Master: Mix for each of (C. coli glyA and C. jejuni hipO), (cdtB and cdtC) and (virB11 and flaA) genes according to GoTaq® Hot Start Green Master Mix. 3-5. Agarose gel electrophoreses (Iroala et al., 2012). Electrophoresis grade agarose (1.5 g) was prepared in 100 ml TBE buffer in a sterile flask, 0.5μg/ml Ethidium bromide was added and mixed thoroughly. Twenty μl of each PCR product samples, negative control and positive control were loaded to the gel. The gel was photographed by a gel documentation system and the data was analyzed. Results: Out of 230 rabbit samples screened for the presence of Campylobacters, 58 samples yielded characteristic Campylobacter colonies on CCDA plates after 48 hours of incubation (Table, 4). They had characteristic corkscrew motility observed by the phase contrast microscope. The biochemical tests for isolates were found positive for catalase, oxidase, and nitrate. None of the isolates revealed positive reaction for urease activity (Table, 5). Out of the 51 (22.17%) Campylobacter isolates, 27 (52.94%) were identified as C. jejuni (323bp) , 17 (33.33%) isolates as C.coli (126 bp),
high incidence in liver and intestinal samples 50% while in cloacal swabs was 35.71%. A putative virulence gene associated with adhesion of the pathogen to intestinal epithelial cells (Rosenquist et al., 2003). This gene is 100% conserved among C. jejuni and C. coli isolates of diverse sources; therefore, it was used to detect virulent isolates of both species (Datta et al., 2003). Cytolethal distending toxin (CDT) is widely distributed among Gram-negative bacteria (Guo et al., 2010) and is the best characterized of the toxins produced by Campylobacter spp. It has been described as an important virulence factor of this pathogen (Asakura et al., 2008). CDT holotoxin, composed of three subunits encoded by the cdtA, cdtB and cdtC genes, causes eukaryotic cells to arrest in the G2/M phase of the cell cycle, preventing them from entering mitosis and consequently leading to cell death (Zilbauer et al., 2008). C.jejuni cytolethal distending toxins showed at 555 bp for CdtC and at 495 bp for CdtB of the genus Campylobacter 555 bp and 495 bp amplicons were confirmed. C. lari strains were about the same size as the C. jejuni and C. coli products during the PCR step did not amplify this product. Thus, the assay could be used to discriminate between C. lari and the C. jejuni-C. coli isolates. The remaining seven Campylobacter strains were belonging to other Campylobacter species in PCR assays. However, reducing the primer concentration from 0.25 to 0.2 μm during the first PCR step was sometimes necessary to reduce the number of nonspecific amplicons (Woz´niak and Wieliczko, 2011). We concluded that C. jejuni and C. coli are highly prevalent in rabbit farms in Egypt. Control measures for exposure and contamination of the rabbit supply should be identified so that methods can be developed to
Table (2): Oligonucleotide primers sequences. Reference Length of amplified product Primer sequence (5'-3') Target gene Wang et al., 2002 323 bp ACTTCTTTATTGCTTGCTGC C. jejuni hipO GCCACAACAAGTAAAGAAGC 126 bp GTAAAACCAAAGCTTATCGTG C. coli glyA TCCAGCAATGTGTGCAATG Datta et al., 2003 855 bp FlaA 494 bp virB11 Bang et al., 2003 495 bp GTTAAAATCCCCTGCTATCAACCA CdtB 555 bp cdtC TTGCACATAACCAAAAGGAAG Table (3): Cycling conditions of the different primers during PCR Gene Primary denaturation Secondary denaturation Annealing Extension No. of cycles Final extension Reference HipO and glyA 94˚C 6 min. 95˚C 30 sec. 55˚C 30 sec. 72˚C 30 sec. 35 72˚C 10 min. Wang et al., 2002
+ + + + Nitrate reduction -- -- -- -- Urease -- -- -- + Hippurate hydrolysis D + + -- + + -- + + -- + + Growth at: 25◦C 37◦C 43◦C + + + + Growth at 1% glycine -- -- -- -- %3.5 NaCl + + + -- + D + -- H2S, lead acetate strip H2S, TSI R S R R S R S R Susceptibility to: Nalidixic acid Cephalothin Table 6: Detection of Campylobacter by multiplex PCR in rabbit samples. Other Campylobacter species. C. coli C. jejuni Positive Campylobacter spp. by PCR No. of examined samples Type of Samples
4 12.5% 12 37.5% 16 50% 32 24.62% 130 Cloacal swabs -- -- 4 100% 4 10% 40 samples 3 21.43% 5 35.71% 6 42.86% 14 35% 40 Intestinal samples -- -- 1 100% 1 10% 10 Water -- -- -- -- 10 ration 7 13.73% 17 33.33% 27 52.94% 51 (22.17%) 230 Total