Helicobacter marmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs

Abstract

Prairie dogs (Cynomys ludovicianus) are used to study the aetiology and prevention of gallstones because of the similarities of prairie dog and human bile gallstone composition. Epidemiological and experimental studies have suggested a connection between infection with Helicobacter species and cholesterol cholelithiasis, cholecystis and gallbladder cancer. Ten of the 34 prairie dogs in this study had positive Helicobacter species identified by PCR using Helicobacter genus-specific primers. Ten of 34 prairie dogs had positive Campylobacter species identified in the intestine by PCR with Campylobacter genus-specific primers. Six Helicobacter sp. isolates and three Campylobacter sp. isolates were identified taxonomically by 16S rRNA gene analysis. The prairie dog helicobacters fell into three clusters adjacent to Helicobacter marmotae. On the basis of 16S rRNA gene sequence analysis, three strains in two adjacent clusters were included in the species H. marmotae. Three strains were only 97.1 % similar to the sequence of H. marmotae and can be considered a novel species with the provisional designation Helicobacter sp. Prairie Dog 3. The prairie dog campylobacters formed a single novel cluster and represent a novel Campylobacter sp. with the provisional designation Campylobacter sp. Prairie Dog. They branched with Campylobacter cuniculorum at 96.3 % similarity and had the greatest sequence similarity to Campylobacter helveticus at 97.1 % similarity. Whether H. marmotae or the novel Helicobacter sp. and Campylobacter sp. identified in prairie dogs play a role in cholesterol gallstones or hepatobiliary disease requires further studies.

Fig. 1.

PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by AluI (a) or HhaI (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588 and MIT 07-5165; lane 5, woodchuck faecal isolate MIT 02-6901; lanes 6 and 7, prairie dog isolates MIT 04-8589 and MIT 04-8584; lane 8, H. marmotae MIT 98-6070 isolated from woodchuck liver; M, 100 bp DNA ladder.

Fig. 2.

Neighbour-joining tree for prairie dog Helicobacter isolates based on comparison of 16S rRNA gene sequences. Bar, percentage difference in nucleotide sequence. GenBank accession numbers are shown in brackets and host species is indicated. Numbers in bold are isolates described in this study.

Fig. 3.

Neighbour-joining tree for prairie dog Campylobacter isolates based on comparison of 16S rRNA gene sequences. Bar, percentage difference in nucleotide sequence. GenBank accession numbers are shown in brackets and host species is indicated. Numbers in bold are isolates described in this study.

Fig. 4.

Hepatocellular carcinoma seen in the liver of prairie dog 08-9090, whose liver was positive for Helicobacter species by nested PCR. (1) Normal liver parenchyma (N) is expanded by a tumour (T). An arrow marks the zone of demarcation between normal liver parenchyma and the hepatocellular carcinoma. Bar, 500 µm. (2) A fine connective tissue stroma (arrow) delineates nests of well-differentiated, neoplastic hepatocytes. Lipid vacuoles (*) scattered throughout characterize the fatty changes within the tumour. Bar, 50 µm. (3) A zone of liquefactive necrosis within the tumour, with characteristic loss of cell detail and mixed, large amounts of neutrophilic cell debris (arrow). Fatty changes are again evident by the presence of lipid vacuoles (*). Bar, 50 µm. (4) A portal area within the non-tumorous region of the liver is expanded by proliferations of bile ducts (white arrows) and arterioles (black arrows). Increased numbers of lymphocytes also surround the bile ducts. Bar, 20 µm.