Helicobacter cetorum sp. nov., a urease-positive Helicobacter species isolated from dolphins and whales

Abstract

A novel helicobacter with the proposed name Helicobacter cetorum, sp. nov. (type strain MIT 99-5656; GenBank accession number AF 292378), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins (Lagenorhynchus acutus) and from the feces of three captive cetaceans (a Pacific white-sided dolphin [Lagenorhynchus obliquidens]; an Atlantic bottlenose dolphin [Tursiops truncatus]; and a beluga whale [Delphinapterus leucas]). The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy. Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals. In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria. Helicobacter cetorum is a fusiform gram-negative bacterium with a single bipolar flagellum. The isolates grow under microaerobic conditions at 37 and 42 degrees C but not at 25 degrees C. H. cetorum is urease, catalase, and oxidase positive, and it is sensitive to cephalothin. The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant. By 16S rRNA sequencing it was determined that H. cetorum represented a distinct taxon that clusters most closely with H. pylori. Further studies are necessary to determine the role of H. cetorum in the development of gastric ulcers and gastritis of cetaceans. This is the first description and formal naming of a novel Helicobacter species from a marine mammal.

FIG. 1.

Transmission electron micrograph of novel cetacean microaerobic bacteria isolated from Atlantic white-sided dolphin. It is a fusiform Helicobacter sp., with the proposed name H. cetorum, with bipolar flagella. Bar, 0.5 μm (24).

FIG. 2.

Gel electrophoresis with ethidium bromide staining demonstrating 1,200-bp PCR target sequence with Helicobacter-specific primers (arrow). Lane 1, reagent control; lane 2, Helicobacter-positive control (H. hepaticus); lanes 3 and 4, MIT 99-5656 and MIT 99-5657 represent DNA from the main stomach of stranded Atlantic white-sided dolphins; lane 5, MIT 00-7128 DNA from beluga whale feces; lane 6, MIT 01-5903 DNA from Pacific white-sided dolphin feces; lane 7, MIT 01-6202 DNA from Atlantic bottlenose dolphin feces.

FIG. 3.

PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from cultures. Lanes 1 through 5 show results for DNA digested by the restriction enzymes HhaI and AluI as indicated. Lanes 1 and 2, MIT 99-5656 and MIT 99-5657 DNA from Atlantic white-sided dolphin stomach tissues; lanes 3 to 5, MIT 00-7128 (beluga whale), MIT 01-5903 (Pacific white-sided dolphin), and MIT 01-6201 (Atlantic bottlenose dolphin) DNA, respectively, obtained from fecal isolates.

FIG. 4.

Dendrogram depicting the phylogenetic location of the H. cetorum constructed on the basis of the 16S rRNA sequence similarity values. The sequences from the Atlantic white-sided dolphins (MIT 99-5656 and MIT 99-5657), beluga whale (MIT 00-7128), a Pacific white-sided dolphin (MIT 01-5903), and an Atlantic bottlenose dolphin (MIT 01-6202) are identified with arrows. The number in parentheses after the MIT accession number is the GenBank accession. The scale bar is equal to a 3% difference in nucleotide sequences, as determined by measuring the lengths of the horizontal lines connecting two species.

FIG. 5.

Photomicrograph of main stomach (antrum) tissue of a Pacific white-sided dolphin (MIT 01-5903). Mucosal erosion with mononuclear cell (primarily lymphocytic) infiltrates in the lamina propria, with some formation of pseudofollicles (H&E staining; magnification, ×25).

FIG. 6.

Photomicrograph of the main stomach (antrum) tissue of a Pacific white-sided dolphin (MIT 01-5903). Numerous ca. 2-μm by 5- to 8-μm curved and spiral argyrophilic bacteria are evident in the surface mucous layer (Steiner staining; magnification, ×250).