salmonella presence - an indicator of direct and indirect human impact ...

SALMONELLA PRESENCE - AN INDICATOR OF DIRECT AND INDIRECT HUMAN IMPACT ON GENTOO IN ANTARCTICA K. Dimitrov1, R. Metcheva2, A. Kenarova3 1 Department of Experimental Zoology, Institute of Zoology, BAS, 1 Tzar Osvoboditel, 1000 Sofia, Bulgaria 2 Department Ecology of Terrestrial Animals, Institute of Zoology, BAS, 1 Tzar Osvoboditel, 1000 Sofia, Bulgaria 3 Department of Ecology and EP, Faculty of Biology, Sofia University “St. Kl. Ochridski”, 8 Dragan Tzankov, 1164 Sofia, Bulgaria Correspondence to: Krastio Dimitrov E-mail: [email protected]

ABSTRACT Antarctica remains one of the last areas, where direct human impact is limited. Compared to the rest of the world Antarctic environment could be considered as unpolluted. This explains the significant scientific interest to the effects of touristic activities, and especially during the last decade due to the increased number of tourists visiting diverse parts of the continent. The role of human activity for Salmonella presence in penguins remains unclear. The aim of the present study was to contribute to the knowledge to what extend Salmonella presence in penguins interstitial fauna might be used as an indicator of direct or indirect human impact on the Antarctic ecosystem, by investigating three Gentoo colonies, subjected to different levels of human impact. 16 individual fecal samples were taken per each Gentoo colony. The potentially pathogenic Salmonella typhimurium/enteridis occurred in both studied populations at King George Island at the rates 37,5% and 18,8% respectively, or at rates 85,7% and 60,0% of the positive for Salmonella sp. samples respectively. Unlike both populations at Admiralty bay, King George Island, all samples taken from the Caleta Argentina population at Livingston Island were positive neither for both pathogenic species nor for Salmonell sp. Keywords: Antarctica, human impact, Salmonella, SS and TSI agar

Introduction Antarctica remains one of the last areas, where direct human impact is limited. Compared to the rest of the world Antarctic environment could be considered as unpolluted. This explains the significant scientific interest to the effects of tourist activities, and especially during the last decade due to the increased number of tourists visiting diverse parts of the continent. Antarctic cruises may cause significant damages to the ecosystem, as the visiting objects are placed on the coastal zones, which are used by Antarctic animals during their daily activities. Further, Antarctic tourism is practiced mainly in summer (November-February) during ice melting, which coincides with the breeding periods of seals and sea birds (11), and according to Hall and Wouters (8) this substantially increases the environmental risks (Fig.1, 2) This applies to a full extend to Subantarctc islands, where the specialized island biota is especially susceptible to external

XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

impacts, and to human activity in particular (2, 7). Current knowledge is insufficient to enable motivated predictions of the possible impacts of human induced changes of abiotic and biotic factors, and of induced microorganisms in particular, on Antarctic animals and plants (9). During the first half of the 20th century the main human activities in Antarctica (whaling, seal hunting, and use of dogs during expeditions (10) created conditions for inducing exotic pathogens which were common for the northern hemisphere. Nova days due to the requirements of the Antarctic treaty these activities were ceased, and replaced by the operation of the Antarctic bases and tourism (4, 5). The likelihood for development of enteric infections increases substantially during summer, when human activity rises and coincides in time and space with the activities of see animals in coastal zones.

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Gentoo colonies, subjected to different levels of human impact.

Materials and methods

Fig 1.Criuse ship landing site for tourist. King George Island appears to be the most heavily visited research station in Antarctica

Three breeding colonies of Gentoo penguins in Antarctic peninsula were studied during summer 2006/2007 – at Livingston Island (Caleta Argentina), and at King George Island (Peter Lenie station and Rakussa point, Admiralty Bay). The Caleta Argentina colony is not visited by any tourists, and just occasionally is sampled by scientists. The Rakussa point colony is part of originally designated as a sites of special scientific interest (SSSI No 34) and Antarctic special protected area (ASPA 151) after a proposal by Poland and it is placed ca. 1 000 m far from a colony of P. adelie , near which tourists are permitted to gain access (14). The Peter Lenie station colony is placed ca. 5 000 m apart of the Rakussa point colony and is only visited by scientists (Fig.3). Research has now shown that the colonies of Adelie penguin (15 151 nests) and Gentoo Penguin (2 287 nests) are the largest on the King George Island (Menagement Plan for SSSI, 2005)

Fig 2. Tourists are brought ashore by inflatable boats (Zodiac) in parties of a 10 to 15, and they are accompanied and closely supervised by guides.

There were a few investigations dealing with the question if Salmonella is part of the normal interstitial fauna of penguins. First data about non-pathogenic Salmonella strains in penguins were provided by Oelke and Stainiger (12). Cockburn (3) found that Salmonella typhimurium acts as a pathogen in penguins in captivity. Baker (1) found S. typhimurium in Gentoo penguins (Pygoscelis papua) at Aukland Island (Subantarctica). Olsen et al. (13) discussed the infection role of the pathogenic strain S. enteridis (type 4) in Gentoo penguins at Bird Islan (South Georgia), which appears in 80% of the clinic isolations in the Western hemisphere. The role of human activity for Salmonella presence in penguins remains unclear. Even more Olsen et al. (1996) speculated that Salmonella infections in Antarctic birds might be introduced by other bird species, which distribution areas cover more northern parts of the world as well. The aim of the present study was to contribute to the knowledge to what extend Salmonella presence in penguins interstitial fauna might be used as an indicator of direct or indirect human impact on the Antarctic ecosystem, by investigating three BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE

Fig 3. Gentoo breeding colony near Peter Lenie station

Enrichment and selective procedure were used to isolate all members of genus Salmonella from fecal samples. 16 individual fecal samples were taken per each Gentoo colony. Pre-enrichment and selective enrichment of samples were done stepwise on Lactose broth and Selenite-Cystine broth at 370C for 24 hrs for each procedure. 10 µl from inoculated and incubated Selenite-Cystine broth was streaked on Salmonella/Shigella (SS) agar plates to obtained isolated colonies and incubated at 370C for 24 hrs. SS agar is a highly selective and differential medium used for the isolation of Salmonella and some Shigella species (6). Triple Sugar Iron (TSI) agar slants were inoculated with suspicious colonies from SS agar (colorless with or without black centers) and cultivated at 370C for 24 – 48 hrs. Positive cultures have alkaline (red) slants and acid (yellow) butts, with or without

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H2S production (blackened agar). The use of TSI agar allowed the identification of several Salmonella species, among which S. typhimurium and S. enteridis were considered as pathogenic in penguins (1, 3, 13).

Results and Discussion Salmonella sp. was present in the intestinal flora of the two sampled Gentoo populations in Admiralty Bay, King George Island. It was determined in 43.8% of the samples from Peter Lenie station population and in 31.3% of the samples from

Rakussa point population (Table 1). The potentially pathogenic Salmonella typhimurium/enteridis occurred in both populations at the rates 37.5% and 18.8% respectively, or at rates 85.7% and 60.0% of the positive for Salmonella sp. samples respectively. Unlike both populations at Admiralty bay, King George Island, all samples taken from the Caleta Argentina population at Livingston Island were positive neither for both pathogenic species nor for Salmonell sp.

TABLE 1. Salmonella presence in the intestinal flora in three Gentoo penguins (Pygoscelis papua) colonies subjected to different levels of human impact. Number of Number of Number of Salmonella Name of Gentoo population sampled Salmonella typhimurium/enteridis penguins positive samples positive samples Livingston Island “Caleta 16 0 0 Argentina” King George Island “Peter Lenie 16 7 6 station” King George Island “Rakussa 16 5 3 point” Both studied populations at King George Island were subjected to limited direct human impact, their nesting sites being visited only by few well trained scientists. It has to be pointed out that the two populations were located approximately 1 000 and 5 000 m far from a large breeding colony of Adelie penguins (P.adelie). The last has been a major attraction point for decades for the tourist visiting Admiralty bay. Olsen et al. (13) explained similar observations with an infection pathway going through human or other bird populations, infected previously by humans. The present results did not deny any of both possibilities. It was shown that both populations at Admiralty bay carried Salmonella at relatively higher rates compared to previous investigations in penguins (1, 12, 13). Taking into account the disadvantages of microbial cultivation methods it was hardly to state that both studied Gentoo populations at King George Island were highly infected with the potentially pathogenic Salmonella typhimurium/enteridis. On the other hand the negative for Salmonella samples taken from the visited by scientists only once per few years Gentoo population at Caleta Argentina, Livingston Island, together with the relative microbial sterility of feather samples of the same colony (Roumiana XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

Metcheva, person communication), showed that direct or indirect human impact played a role for the microbial diversity of Gentoo body.

Conclusion Salmonella presence in penguin intestinal flora could by successfully used as indication of direct or indirect human impact on Antarctic birds. In order to resolve the general question how pathogenic and nonpathogenic Salmonella species disperse among Sub Antarctic penguin populations long term surveillance of particular colonies should be started and fast and species specific diagnostic methods for Salmonella determination should be applied. Acknowledgements This article was supported by Ministry of Education and Science of Bulgaria by grant number NSF B1615 to Dr. Roumiana Mecheva. The authors would like to express their gratitude to the Bulgarian Antarctic Institute and to the Polish Antarctic Institute, which provided excellent work conditions for field works and preliminary processing of samples at their Antarctic stations on Livingston and King George Islands respectively. The authors are thankful also to eng. Iordan

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Jankov, MSc, for his help in the field works at Livingston Island.

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