Fecal shedding of Helicobacter spp. by co-housed Australian sea lions (Neophoca cinerea) and Australian fur seals (Arctocephalus pusillus doriferus)
Introduction
Helicobacter species are ubiquitous colonizers of the gastrointestinal system (Solnick and Schauer, 2001) and have been found to occur in both humans and a broad range of animal hosts including domestic cats and dogs (Jalava et al., 1998), captive cats (Eaton et al., 1993), cattle (De Groote et al., 1999), pigs and birds (Dewhirst et al., 1994), rodents (Lee et al., 1992), sheep (Dewhirst et al., 2000), primates (Bronsdon et al., 1991), and marine mammals (Harper et al., 2002, Harper et al., 2003). To date 24 species have been formally recognized and have often been associated with conditions of gastric and/or enterohepatic disease including gastroenteritis, gastric ulcers, hepatitis and cancer (for a review, see Fox and Lee, 1997, Solnick and Schauer, 2001). However, not all Helicobacter species are considered pathogenic and instead may form part of the host’s indigenous intestinal microflora (Simmons et al., 2000). The emergence of disease may also be due to host-dependent factors (Cover, 1997) or the infection of the host with species that do not usually colonize its gut (Solnick and Schauer, 2001).
The pinniped species Neophoca cinerea (Australian sea lions) and Arctocephalus pusillus doriferus (Australian fur seals) are found predominantly within the southern waters of Australia (Bryden et al., 1998, Gales et al., 1994) and are often featured in oceanariums. The maintenance of such animals often entails widespread medical problems since disease, particularly that of the gastrointestinal system, is common (Lauckner, 1985). With the detection of Helicobacter species as agents of disease in cetaceans (Harper et al., 2002) and, more recently, in wild harp seals (Harper et al., 2003), its occurrence in captive animals may add to the already pervasive medical problems associated with the husbandry of these animals (for a review see Higgins, 2000, Lauckner, 1985). However, its occurrence in the captive host has not been previously reported.
This paper reports on the occurrence of Helicobacter species within the feces of several captive Australian sea lions (N. cinerea) and Australian fur seals (A. pusillus doriferus) using 16S rRNA PCR analysis. The occurrence of Helicobacter species within the feces of each animal was examined over a 31-day period using PCR and sequence analyses. Of the animals investigated only one (“Duran”) was previously diagnosed with a suspected Helicobacter related infection, all other animals were asymptomatic.
Section snippets
Sample collection
Fecal samples were collected from the holding pens of five individual seals (one fur seal, four sea lions) over a 31-day period. At each sampling period, approximately 20 g of fecal material from each animal was obtained, placed in an individual sterile collection vial and stored immediately on ice. Samples were collected from individuals within 12 h of defecation. Experiments showed that the recovery of template DNA and its amplification was not affected over this time period (data not shown).
Preparation of samples for DNA extraction
PCR sensitivity
To establish the sensitivity of the PCR assay, cells of H. pylori (106 to 101) were added to PBS and fecal samples (Fig. 1). DNA extracts of these samples were subjected to PCR and products were observed in samples of PBS spiked with as little as 104 cells. Taking into account the fraction subjected to PCR (1/125th) this represents a limit of detection of approximately 102 cells. Similarly, in fecal samples, a relatively strong PCR signal was observed with a limit of detection of 102 cells. The
Discussion
This paper reports on the occurrence of Helicobacter species within the feces of captive Australian sea lions (N. cinerea) and Australian fur seals (A. pusillus doriferus). Depending upon the individual animal, the presence of Helicobacter (as indicated by the amount of PCR product) was either variable or consistent over the 31-day period and in some cases occurred below the limits of detection (102 cells g−1). We cannot ascribe this variability to variations in amplification or to a variation in
Acknowledgements
The authors thank UnderWater World (Mooloolaba, Australia) and its staff for their support and cooperation in the collection of samples, in particular Greg Elks, Kristy Rychdalvalsky, Chris Groot, Lucas Robinson, Brad McKenzie and Malcolm Westwood. We also thank Melissa Wos, Jeffrey Argo and Alexis Oxley for their contributions. This work was supported by the School of Biological, Cellular and Molecular Sciences, University of New England, Armidale and the National Marine Science Center, Coffs
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