Introduction

Due to the geographical isolation of the Antarctic region, the contact between the Antarctic fauna and bacterial pathogens is limited. However, the presence of humans has significantly increased as a result of tourism and scientific expeditions (Bonnedahl et al. 2005). Given the limited data available regarding endemic and exotic diseases, it is rather difficult to determine the true origin of current diseases (Leotta et al. 2006a; Nievas et al. 2007). Edwardsiella tarda is considered a common inhabitant of the normal intestinal flora in aquatic animals (White 1984); however, under certain circumstances, this bacterium can cause intestinal and extra-intestinal diseases and wound infections in reptiles, amphibians and terrestrial endotherms, including humans (Bockemuhl et al. 1971; Sakazaki and Tamura 1992; Janda and Abbott 1993; Baya et al. 1997). This bacterium is also considered a common opportunistic agent in sick or injured marine wildlife (Coles et al. 1978). In Antarctic and sub-Antarctic animals, two cases associated with E. tarda have been reported. The first case was in Rockhopper penguins (Eudyptes crestatus) with chronic enteritis (Cook and Tappe 1985), and the second case, in an Adelie penguin (Pygoscelis adeliae) with a subcutaneous clostridial infection (Nievas et al. 2007). In addition, only two reports describe E. tarda in Antarctic wildlife (Zunino et al. 1985). However, the prevalence of E. tarda in Antarctic wildlife has never been reported. The purpose of this work was to determine the prevalence of Edwardsiella tarda in Antarctic wildlife, including birds, mammals and fish.

Methods

Sampling was carried out in Potter Peninsula, located on King George Island, South Shetland Islands (62°15′S, 58°36′W), and in the Hope Bay area, located on the tip of the Antarctic Peninsula (63°24′S, 56°59′W). During the spring and summer seasons, these are breeding areas for some seabirds (Hahn et al. 1998; Leotta et al. 2006a), and occasionally for Antarctic sea mammals. One of the main fish found in these regions is Nothotenidae, which is one of the food sources of both mammals and birds. A total of 1,855 samples were collected from Antarctic animals in the summer of 2000 and 2002 (Potter Peninsula), and 2001 and 2003 (Hope Bay). Non-probabilistic sampling was carried out in animals that were separated from their colonies for mere convenience. Breeding areas for brown skuas (Stercorarius lonnbergi), south polar skuas (Stercorarius maccormicki), kelp gulls (Larus dominicanus), south giant petrels (Macronectes giganteus), Adelie penguins, gentoo penguins (Pygoscelis papua), chinstrap penguins (Pygoscelis antarctica), and greater sheathbills (Chionis albus) were identified. The population was calculated using a census carried out by Hahn et al. (1998) and Leotta et al. (2006a). A total of 1,622 seabirds were captured, clinically evaluated by experienced veterinarians, and sampled with cloacal swabs, which were conserved on Stuart transport media (Difco Laboratories Incorporated, Cambridge, UK) at 4°C. Fifty infertile eggs of Adelie penguins were collected from the nest after the incubation period, they were washed with distilled water and neutral detergent, dried and immersed into alcohol 70° for 1 h. Then they were dried and the content was streaked onto hektoen agar (Difco) and incubated at 37°C for 48 h. In addition, 161 Antarctic fur seals (Arctocephalus gazella) and Weddell seals (Leptonychotes weddelli) were observed but not captured, and immediately after defecation an aliquot of fresh faeces was collected in sterile bags (Nasco’s Whirl-pak, Network International Technologies, Buenos Aires, Argentina) and conserved at 4°C. Finally, a total of 22 fishes (Nothotenia coriiceps) were captured, sacrificed, necropsied, and an aliquot of intestinal contents was collected in sterile bags (Nasco’s Whirl-pak) and conserved at 4°C. All samples of birds, mammals and fish were processed between 2 and 12 h after being collected. Each sample was plated directly onto hektoen enteric agar (Difco), and incubated at 37°C for 48 h. The suspected blue-green colonies with black centres were subcultured onto trypticase soy agar (Difco) and incubated at 37°C for 24 h. Pure colonies were subsequently inoculated into brain heart infusion broth (Difco) with 30% of glycerol and stored at −20°C. All suspected E. tarda colonies were subjected to a complete phenotypic characterisation. The Gram stain of the isolates was determined, and the following biochemical tests were carried out according to the manufacturer’s directions: catalase (Waco Pure Chemical Industries, Osaka, Japan), oxidase (Laboratorios Britania, Buenos Aires, Argentina), β-galactosidase (Laboratorios Britania), indole (Laboratorios Britania), methyl red (Difco), Voges-Proskauer (Difco), citrate (Difco), lysine (Difco), urea (Difco), malonate (Difco), ornitine (Difco), sulfhydric acid (tri sugar iron, Difco), glucose (ICN Biomedicals, Aurora, Ohio, USA), lactose (ICN), dulcitol (ICN), salicine (ICN), raffinose (ICN), sorbitol (ICN), arabinose (ICN), maltose (ICN), xylose (ICN), trehalose (ICN), and sucrose (ICN), according to the method described by Koneman et al. (1999). A statistical analysis of data was performed using Statgraphics Centurion XV version 15.2.05 (StatPoint Inc., Herdon, Virginia, USA).

Results

The classic Edwardsiella tarda was isolated from 281 (15.1%) of 1,855 Antarctic wildlife samples obtained from southern giant petrels, brown skuas, greater sheathbills, Adelie penguins, gentoo penguins and chinstrap penguins from Potter Peninsula in 2000 and 2002. E. tarda could not be isolated in 54 faecal samples from Antarctic fur seals, 22 samples from fish and 32 samples from south polar skuas in 2002. E. tarda was isolated from brown skuas, kelp gulls, greater sheathbills, Adelie penguins and their eggs, chinstrap penguins and Weddell seals from Hope Bay in 2001 and 2003. However, in the summer of 2003, E. tarda could not be isolated from the cloacal samples from 22 gentoo penguins. In all the birds sampled in both places, no clinical signs of disease were observed. In 15 fishes, no pathological lesions were observed, but in the intestine from seven fishes, endoparasites were observed. Animal species, seasons, geographical regions, identification of Antarctic wildlife population sampled, and the prevalence of E. tarda are shown in Table 1. As a result of the non-probabilistic sampling method used, the positive rate observed amongst seasons, areas and species did not exhibit a normal distribution. There were no statistical differences amongst seasons and/or geographic areas of the positive samples obtained (P = 0.24, Kruskal Wallis test) (P = 0.07, Kolmogorov–Smirnov test). Due to the lack of positive results amongst the fish evaluated, only the differences amongst the mean of positive results in birds and mammals were analysed, where no statistical differences were observed (P = 0.93, Kolmogorov–Smirnov test).

Table 1 Antarctic wildlife species, geographical region, animal population, season, number of analyzed samples and prevalence of E. tarda
Full size table

Discussion

In this study we isolated E. tarda in 15.1% of Antarctic wildlife animals sampled in two places of Antarctica, Hope Bay and Potter Peninsula. We considered that E. tarda is a common bacterium in the faeces of Antarctic birds and mammals, because no significant differences were found with regard to the areas and seasons investigated. This is supported by the fact that there are no significant differences in the distribution of positive rates obtained within the species evaluated. However, E. tarda was described as an opportunistic bacterial pathogen and has been commonly isolated from different healthy, sick and moribund fish, birds and reptiles in several ecosystems around the world (White and Simpson 1973; Trust and Bartlett 1974; Baya et al. 1997). Antarctic birds and mammals eat fish, particularly Nothotenia coriiceps. We considered that the trophic chain could be one of the links in E. tarda transmission. However, none of the 22 N. coriiceps samples was positive for E. tarda. The negative results, though inconclusive, could be due to the small number of fish sampled.

Several enteropathogens such as Campylobacter lari, Campylobacter jejuni, Salmonella spp., and enteropathogenic Escherichia coli, were isolated from Antarctic animals (Oelke and Steiniger 1973; Bonnedahl et al. 2005; Leotta et al. 2006a, b). The known prevalence of E. tarda in Antarctic animals is an important precedent for the future control and surveillance of populations of mammals, birds and fishes, since this enteropathogen could cause diseases in these animals, which could be an indicator of the health of the ecosystem.

This is the first report of E. tarda isolation from southern giant petrels, skuas, kelp gulls, greater sheathbills, chinstrap penguins, eggs of Adelie penguins, and Weddell seals. However, further investigations are necessary in order to determine the role of E. tarda as a pathogen of Antarctic wildlife fauna.