Coinfection of enteric Helicobacter spp. and Campylobacter spp. in cats

Abstract

During a 6-year period, 64 of 227 commercially reared cats had microaerobic bacteria isolated from their feces. All the isolates were initially identified as Campylobacter-like organisms based on biochemical and phenotypic characteristics. DNA extractions from 51 of these isolates were subjected to PCR using primers specific for Helicobacter spp. and Campylobacter spp. Of the isolates, 92% (47 of 51 isolates) were positive for Campylobacter spp., 41% (21 of 51 isolates) were positive for Helicobacter spp., 33% (17 of 51 isolates) were positive for both genera, 59% (30 of 51 isolates) were positive only for Campylobacter spp., and 8% (4 of 51) were positive only for Helicobacter spp. Sixteen of the 47 Campylobacter-positive cultures were positive for more than one Campylobacter spp. Based on a species-specific PCR assay, 83% of the isolates were identified as Campylobacter helveticus, 47% of the isolates were identified as Campylobacter upsaliensis, and 6% of the isolates were classified as Campylobacter jejuni. The 1.2-kb PCR products of the 16S rRNA genes of 19 Helicobacter species isolates were subjected to restriction fragment length polymorphism (RFLP) analysis. Of the five different RFLP patterns obtained, two clustered with Helicobacter ("Flexispira") taxon 8, one clustered with Helicobacter bilis, one clustered with Helicobacter canis, and the remaining pattern was closely related to a novel Helicobacter sp. strain isolated from a woodchuck. The sequence data for the 16S rRNA genes of 10 Helicobacter spp. validated the RFLP-based identification of these isolates. This study demonstrated that biochemical and phenotypic characteristics of microaerobic organisms in cat feces were insufficient to characterize mixed Helicobacter and Campylobacter infections. Molecular structure-based diagnostics using genus- and species-specific PCR, RFLP analysis, and 16S rRNA sequence analysis enabled the identification of multiple microaerobic species in individual animals. The clinical relevance of enteric Helicobacter and Campylobacter coinfection in cats will require further studies.

FIG. 1

Cocolonization of cats by Campylobacter spp. and Helicobacter spp. (A) Campylobacter genus-specific primers were used to amplify DNA extracted from cat fecal isolates. Lane MW, 100-bp DNA ladder; lane 1, reagent control; lane 2, Campylobacter-positive control; lanes 3 to 19, isolates from cat feces. (B) Helicobacter genus-specific primers were used to amplify DNA extracted from cat fecal isolates. Lane MW, 1-kb DNA ladder; lane 2, Helicobacter-positive control; lane 3, reagent control; lanes 3 to 19, isolates from cat feces.

FIG. 2

Results of genus-specific PCR. (A) Primers specific for C. helveticus amplified 16S rRNA PCR products from 12 of 17 cat fecal isolates. Lane MW, 1-kb DNA ladder; lane 1, reagent control; lane 2, positive control; lanes 3 to 19, isolates from cat feces. (B) Primers specific for C. upsaliensis amplified 16S rRNA products from 8 of 17 isolates from cat feces. Lane MW, 1-kb DNA ladder; lane 1, reagent control; lane 2, positive control; lanes 3 to 19, isolates from cat feces. (C) Primers specific for C. jejuni amplified hippuricase gene PCR products from 3 of 17 cat isolates. Lane MW, 1-kb DNA ladder; lane 1, reagent control; lane 2, positive control; lanes 3 to 19, isolates from cat feces.

FIG. 3

Products (1.2 kb) of PCR using Helicobacter genus-specific primers were digested by AluI and analyzed by electrophoresis on 6% Visigel matrix. Five patterns were observed. Lane MW, 100-bp DNA ladder; lane 1, Helicobacter (“Flexispira”) taxon 8 (ATCC 49317); lane 2, MIT 98-90; lane 3, MIT 95-1850-65; lane 4, MIT 95-513-27; lane 5, H. bilis ATCC 51630; lane 6, MIT 95-234-6; lane 7, H. canis cat isolate (11); lane 8, MIT 95-1114-42; lane 9, MIT 95-1114-46; lane 10, MIT 94-55-4; lane 11, Helicobacter sp. woodchuck isolate (19); lane 12, MIT 95-513-29; lane 13, MIT 94-2635-37; lane 14, MIT 98-1705-4.

FIG. 4

Neighbor-joining phylogenetic tree for Helicobacter spp. isolated from feces of cats and reference Campylobacter and Helicobacter species. The scale bar represents phylogenetic distance as estimated using the Jukes Cantor correction. Distances can be determined by adding the lengths of all of the horizontal lines connecting any two species. GenBank accession numbers appear in brackets. The 1,200-bp partial sequences (marked as such) for which accession numbers are not provided were not deposited in GenBank and are available from the corresponding author.