http://orcid.org/0000-0001-8920-6061
Figures
Abstract
A lack of knowledge of naturally occurring pathogens is limiting our ability to use the Antarctic to study the impact human-mediated introduction of infectious microorganisms have on this relatively uncontaminated environment. As no large-scale coordinated effort to remedy this lack of knowledge has taken place, we rely on smaller targeted efforts to both study present microorganisms and monitor the environment for introductions. In one such effort, we isolated Campylobacter species from fecal samples collected from wild birds in the Antarctic Peninsula and the sub-Antarctic island of South Georgia. Indeed, in South Georgia, we found Campylobacter lari and the closely related Campylobacter peloridis, but also distantly related human-associated multilocus sequence types of Campylobacter jejuni. In contrast, in the Antarctic Peninsula, we found C. lari and two closely related species, Campylobacter subantarcticus and Campylobacter volucris, but no signs of human introduction. In fact, our finding of human-associated sequence types of C. jejuni in South Georgia, but not in the Antarctic Peninsula, suggests that efforts to limit the spread of infectious microorganisms to the Antarctic have so far been successful in preventing the introduction of C. jejuni. However, we do not know how it came to South Georgia and whether the same mode of introduction could spread it from there to the Antarctic Peninsula.
Citation: Johansson H, Ellström P, Artursson K, Berg C, Bonnedahl J, Hansson I, et al. (2018) Characterization of Campylobacter spp. isolated from wild birds in the Antarctic and Sub-Antarctic. PLoS ONE 13(11): e0206502. https://doi.org/10.1371/journal.pone.0206502
Editor: Patrick Jon Biggs, Massey University, NEW ZEALAND
Received: February 1, 2018; Accepted: October 15, 2018; Published: November 9, 2018
Copyright: © 2018 Johansson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was funded by the Chilean Antarctic Institute (INACH number T-12-13) and the Swedish Research Council Formas (2014-829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jonas Waldenström currently serves as an academic editor for PLOS ONE. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction
The Antarctic is among the most isolated places on Earth. By virtue of inhabiting such a remote location, Antarctic animals were long thought to be protected from disease introduction from other regions. However, recent studies have reported the presence of human and animal pathogens previously believed to be absent from the region [1, 2], including Salmonella enterica serovar Enteriditis phage type 4 [3–5] and influenza A viruses [6]. In addition to finding pathogens with presumed non-Antarctic origin in Antarctic wildlife, it has been shown that penguins kept in captivity are susceptible to a range of infectious diseases not observed in the Antarctic (see [2], and references therein). Sustained transmission of some of these pathogens are unlikely, due to the absence of suitable vectors in the Antarctic. Others may only be limited by geographical barriers. The breakdown of such barriers due to human activity may therefore pose a threat to the Antarctic ecosystem.
There has been no causal evidence of human-mediated pathogen introduction to the Antarctic [7]. However, due to a lack of knowledge concerning naturally occurring pathogens in the region, it is difficult to determine whether a detected pathogen has been introduced by humans or not. Furthermore, any study of disease in the Antarctic faces several challenges, including the environment, which poses a major hurdle to longitudinal monitoring of individuals and populations, and limited access to sufficient laboratory infrastructure, which makes the study of fastidious microorganism difficult. Nevertheless, overcoming these obstacles and furthering our understanding of disease in the region is a priority for both conservation efforts and our ability to use the Antarctic to study human impact on a relatively uncontaminated environment [7–9].
In the present study, we focused on Campylobacter, a genus of bacteria that are often found in the gut microbiota of both wild and domestic animals, especially in avian species [10]. This genus includes Campylobacter jejuni, one of the leading causes of bacterial gastroenteritis in humans (e.g. [11–13]). At least five species of Campylobacter have been found in the Antarctic and the surrounding sub-Antarctic: Campylobacter insulaenigrae [14], Campylobacter jejuni [15, 16], Campylobacter lari [14, 17, 18], Campylobacter subantarcticus [19] and Campylobacter volucris [18]. In addition, at least one unidentified C. lari-like bacterium has been reported [20]. So far, three isolates of C. jejuni ST-45 from Macaroni penguins (Eudyptes chrysolophus) on Bird Island, South Georgia, constitutes the only detection plausibly associated with human activity [15, 16]. Therefore, the aim of our study was twofold: i) to look for potentially introduced Campylobacter, i.e. human-associated strains of primarily C. jejuni, and ii) to further increase our knowledge of Campylobacter spp. in the Antarctic and sub-Antarctic, particularly in light of recent characterizations of novel C. lari-like Campylobacter species [19, 21, 22].
Materials and methods
Ethics statement
Samples were collected in accordance with the Wildlife and Protected Areas (WPA) Ordinance enacted by the Government of South Georgia and the South Sandwich Islands, and the Protocol on Environmental Protection to the Antarctic Treaty. Permission to collect samples were granted by the Government of South Georgia and the South Sandwich Islands (WPA/2012/034), the Swedish Polar Research Secretariat (2012-169) and the Chilean Antarctic Institute (INACH 654/2014, 23/2015, 46/2016). Ethical consideration of sample methodology was approved by the Swedish animal ethics committee (Linköpings djurförsöksetiska nämnd, permits 112-11, 2-15).
Sampling
Fieldwork was conducted during the austral summer in the Antarctic and Sub-Antarctic in four years. In November 2012, we sampled birds at three locations in South Georgia: Stromness (-54.16°, -36.71°), Grytviken (-54.27°, -36.51°) and Gold Harbor (-54.63°, -35.93°); and six locations in the Antarctic Peninsula: Danco Harbor (-64.73°, 62.59°), Deception Island (-62.98°, -60.65°), Orne Harbor (-64.62°, -62.53°), Paradise Harbor (-64.82°, -62.87°), Petermann Island (-65.17°, -64.14°) and Yankee Harbor (-62.53°, -59.77°). In January and February 2014, we sampled birds at five locations in the Antarctic Peninsula: Ardley Island (-62.21°, -58.93°), base Gabriel González Videla (-64.82°, -62.85°), Cape Legoupil (-63.32°, -57.90°), Kopaitik Island (-63.32°, -57.85°) and Neko Harbor (-64.84°, -62.53°). In January and February 2015, we sampled birds at three locations in the Antarctic Peninsula: Cape Shirreff (-62.46°, -60.79°), Kopaitik Island and Narebski Point (-62.24°, -58.78°). In January 2016, we sampled birds at four locations in the Antarctic Peninsula: Ardley Island, Cape Legoupil, Kopaitik Island and Rakusa Point (-62.16°, 58.46°).
In total, 2,278 samples were collected. Samples were predominantly collected from brush-tailed penguins (Pygoscelis spp.): Adélie penguins (Pygoscelis adeliae; n = 134), chinstrap penguins (Pygoscelis antarctica; n = 960) and gentoo penguins (Pygoscelis papua; n = 828). In addition, samples were collected from giant petrels (Macronectes spp.; n = 43), kelp gulls (Larus dominicanus; n = 151), king penguins (Aptenodytes patagonicus; n = 27), skuas (Stercorarius spp.; n = 46) and snowy sheathbills (Chionis albus; n = 89).
Sampling strategy is one factor that can affect prevalence estimates. Bearing this in mind, samples were obtained from birds captured with hand nets or from fresh feces directly from the nest when possible; when not, fecal samples were obtained from the spots on the ground where the birds had been seen standing still for a while, either alone or in single-species groups. In the latter case—which was particularly common for king penguins, kelp gulls, skuas and snowy sheathbills—care was taken to avoid droppings involving material from more than one bird. Consequently, the risk of one sample containing bacteria from several birds was limited, although occasional contamination cannot be ruled out.
Sampling methodology was similar in all years, and consisted of either fecal samples or cloacal swabs. Collected samples were kept in Amies charcoal medium (Copan Diagnostics, Inc. Murrieta, CA, USA) at +4°C. In 2012, the samples were kept refrigerated in Amies medium for about three weeks until they reached the Swedish National Veterinary Institute (SVA) where they were cultured immediately. In 2014, 2015 and 2016, the samples were kept in Amies charcoal medium for less than 24 h and then either cultured in a field-based laboratory (2015) or frozen to -70°C in lysogeny broth (LB) with 5% glycerol and transported in an unbroken freeze chain to Linnaeus University, Sweden (2014 and 2016). In the latter cases, the time from sampling to culturing was no longer than 3 months.
Isolation and identification
All samples were enriched in Bolton broth (X135, Lab M, Lancashire, England; or CM0983, Oxoid, Basingstoke, England) supplemented with CVTN selective supplement (X132, Lab M) or modified Bolton broth selective supplement (SR0208, Oxoid,) and incubated at 37 ± 1°C for 48 ±4 h. Samples were plated on mCCDA (modified charcoal cefoperazone deoxycholate agar, SR0155, Oxoid) and incubated at 41.5 ± 0.5°C for 48 ± 4 h. Samples showing presumed Campylobacter growth were re-cultured on conventional blood agar and incubated at 41.5 ± 0.5°C for 48 ± 4 h. All incubations were performed in a microaerobic environment generated using CampyGen sachets (CN0025, Oxoid).
Isolates from 2012 were identified to species using phenotypic tests [23], PCR [24], and MALDI-TOF mass spectrometry [25]. Five of the isolates could not be unambiguously identified to species using MALDI-TOF. One of these isolates could not be analyzed further, but the remaining four were identified to species level by whole-genome sequencing and subsequent 16S rRNA gene analysis. Briefly, sequencing libraries were prepared using the Nextera XT kit (Illumina, San Diego, CA, USA) and 250 bp paired-end sequencing was performed on a MiSeq sequencer (Illumina). A partial (1,313 bp) 16S rRNA sequence that was shared between all Campylobacter spp. 16S rRNA gene sequences available in GenBank at the time (November, 2013) was identified and used as a reference sequence. For each isolate, the partial 16S rRNA gene sequence was determined by mapping the reads to the reference sequence using the crossmatch function of Consed [26]. The sequences were subsequently aligned with all Campylobacter spp. 16S rRNA gene sequences available in GenBank at the time (November, 2013), and a phylogenetic analysis was performed using MrBayes [27]. The four isolates (74507, 74514, 74521 and 74521) grouped with the C. peloridis reference sequence (GenBank accession number: AM922331) (see S1 Fig).
Isolates from 2014, 2015 and 2016 were identified to species following the atpA determination scheme developed by Miller et al. [28], supplemented with additional atpA reference sequences from C. blaseri 17S00004-5T (GenBank accession number: MG958595), C. ornithocola WBE38T (KX467979), C. pinnipediorum RM17260T (CP012546), C. hepaticus HV10T (LUKK01000000), C. iguaniorum 1485ET (CP009043), C. geochelonis RC20T (FIZP01000001), C. corcagiensis CIT 045T (JFAP00000000). Briefly, the atpA gene was amplified and sequenced using a primer pair capable of targeting all known species of Campylobacter at the time of the schemes development (March, 2014). The sequences were subsequently aligned with the reference sequences using MAFFT v. v7.313 [29], and a phylogenetic analysis was performed using RAxML v. 8.2.9 [30]. All species formed monophyletic clades with the exception of C. lari which was paraphyletic with respect to C. subantarcticus (see S2 Fig). However, as there was strong support for the C. subantarcticus delimitation, samples falling within the larger C. lari-C. subantarcticus clade was treated as C. subantarcticus if they fell within the C. subantarcticus-clade and otherwise as C. lari.
All C. jejuni strains and a subset of the C. lari strains were typed using multilocus sequence typing (MLST) and the PubMLST databases (http://pubmlst.org/campylobacter/) as previously described [31–33].
Results
We isolated Campylobacter in samples from the majority of the sampling locations and from almost all of the sampled species (Table 1, with detailed information in S1 Table). Campylobacter colonization was modest in penguins, nowhere exceeding 8.5%. The colonization was similarly modest in giant petrels (14.0%) and kelp gulls (13.9%), although locally it reached as high as 30.6% in kelp gulls. The colonization was markedly higher in skuas (50%) and sheathbills (48.3%) and in some locations reached 100% for these species. However, sample sizes were generally small for the non-penguin species.
Isolates recovered from the Antarctic Peninsula were identified as C. lari (75 isolates) or one of two closely related species: C. subantarcticus (25 isolates) and C. volucris (3 isolates). In addition, three isolates were identified as C. lari-like. C. lari was found in chinstrap and gentoo penguins, as well as kelp gulls, skuas and snowy sheathbills, whereas C. subantarcticus was only found in chinstrap penguins and a snowy sheathbill and C. volucris only in gentoo penguins (Table 2, with detailed information in S1 Table).
Numbers indicate samples for which species were determined by atpA sequencing; numbers in parentheses indicate additional samples for which species were determined by phenotypic tests, PCR and MALDI-TOF, but not by atpA sequencing. In the latter case, the methods used do not distinguish between C. lari and C. subantarcticus or C. volucris; these samples should therefore be considered positive for C. lari-like bacteria.
Isolates recovered from South Georgia were identified as C. jejuni (18 isolates) or either C. peloridis (8 isolates) or C. lari-like bacteria (9 isolates). There were large overlaps between host species, with giant petrels and skuas carrying both C. jejuni and C. lari-like bacteria, and snowy sheathbills carrying C. jejuni, C. peloridis and C. lari-like bacteria (Table 2).
All but two of the 18 C. jejuni isolates recovered belonged to known MLST sequence types (ST-45, ST-227 and ST-883) (Table 3). Sequence types ST-45 and ST-883 were found in multiple locations and in samples from multiple host species. Sequence type ST-227 was only found in kelp gulls in Grytviken. The remaining two isolates belonged to a novel sequence type. Both isolates were from giant petrels in Stromness (Table 3).
New STs are shown in bold.
Of the 24 C. lari isolates chosen for MLST analysis, 20 could be assigned to one of 17 novel sequence types (Table 4). Of the remaining four, the tkt locus could not be amplified and thus no sequence type assigned.
New STs are shown in bold.
Discussion
In the worst-case scenario, the introduction of novel pathogens to an ecosystem may prelude an ecological catastrophe [34]. Nevertheless, in the absence of mass mortality, the establishment of a novel pathogen may impact reproductive investment and success, which in turn may reduce the population size, disrupt the food web and increase the risk of species extinction [35, 36]. Appropriately, the threat of such introductions to the Antarctic has been recognized [7, 37]. However, whether the current measures put in place to mitigate the threat are sufficient, especially in the face of the predicted increase in human presence, has been called into question [9, 38, 39].
We isolated Campylobacter spp. from apparently healthy birds, as was done in previous studies [18, 40]. While the absence of overt signs of disease suggests commensal colonization rather than infection, clinical signs are rarely observed even in birds that mount an immune response to infection [41–43], and mild symptoms or opportunistic infections cannot be ruled out. Even if this is taken into account, it seems unlikely that the introduction of Campylobacter spp. would have a substantial adverse impact on the Antarctic ecosystem. They may, however, be used as indicators for microbial pollution, signaling areas where care must be taken lest we cause outbreaks of more virulent pathogens.
While the chosen culturing method generates the microaerobic atmosphere required for growth of most of the Campylobacter species previously observed in the Antarctic and sub-Antarctic, it does not generate hydrogen or formate. This excludes several species—C. concisus, C. curvus, C. rectus, C. mucosalis, C. showae, C. gracilis—that require hydrogen or formate as electron donors for microaerobic growth [10]. In addition, little is known about how different species of Campylobacter respond to prolonged storage in Amies medium or lysogeny broth. Barring these limitations, our findings corroborate earlier work suggesting that wild birds in the Antarctic are predominantly colonized by C. lari and closely related species [17–20]. Due to the limited number of studies of C. lari in wild birds, it is difficult to draw conclusions as to whether the isolated strains are indigenous or if the Antarctic acts as a sink, repeatedly reseeded from an outside source. Some evidence favoring the former is provided by the MLST of the 24 C. lari isolates yielding 17 novel sequence types, but without a clearer picture of C. lari host association outside of the Antarctic this remains largely speculative.
Notably, to our knowledge, this is only the second time that C. subantarcticus has been isolated in the wild. C. subantarcticus—initially described during a polyphasic taxonomic study of C. lari-like isolates from Bird Island, South Georgia [19]—responds well to isolation with routine protocols used in studies of other Campylobacter species. That it is largely absent in the literature suggests that it may be geographically restricted to the Antarctic and sub-Antarctic, restricted to the host species that occur in the region, or both. However, Campylobacter species other than C. jejuni and C. coli have generally received little attention and the apparent absence of C. subantarcticus in other regions and in non-Antarctic species may be the result of such oversight.
While we found no evidence of introduction of human-associated strains of Campylobacter to the Antarctic Peninsula, we did isolate such human-associated strains in South Georgia. Two of the three known sequence types recovered—ST-227 and ST-883—belong to clonal complexes frequently isolated from humans and domestic animals [44–46], but rarely from wild birds [47, 48]. The third of the three known sequence types recovered—ST-45—has frequently been isolated from humans and domestic animals [44–46], but unlike the other two is also common in wild birds [47, 49, 50].
There are several routes by which human-associated C. jejuni may have found its way to South Georgia. Some of the potential routes are historical and associated with the whaling era (1904–1965); alongside direct transmission from humans, these include the introduction of other known hosts for Campylobacter, including chickens, geese, pigeons, ducks, pigs and sheep [51]. Other potential routes may be more recent and include transmission from tourists or personnel, and yet another potential route is through transmission from remote areas by migrating birds. While the re-isolation of C. jejuni ST-45—the same sequence type isolated in 1998 on Bird Island, South Georgia, by Broman et al. [15]—may reflect persistent circulation of C. jejuni following a single introduction event, the presence of two additional human-associated sequence types suggests repeated introduction, but offers no further clues on the route of introduction.
In contrast to South Georgia, C. jejuni has never been found in the Antarctic, despite considerable monitoring effort [17, 18, 20]. The reason for this discrepancy remains unclear. Since the abandonment of the whaling stations in the 1960s, South Georgia houses no permanent residents, and personnel and tourist numbers are similar to comparable regions on the Peninsula [52, 53]. Furthermore, even though South Georgia is not encompassed by the Antarctic treaty regulations, similar management guidelines to limit the human impact are in place [52].
Thus, the presence of several human-associated MLST sequence types of C. jejuni in South Georgia is worrying because we do not know how they found their way there. At the same time, it is encouraging that we did not find C. jejuni south of the 60°S latitude—within the Antarctic Treaty Area and the pristine Antarctic—which suggests that current measures to reduce the risk of pathogen introduction may be paying off.
Supporting information
S1 Fig. Species identification of Campylobacter strains based on partial (1,313 bp) 16S rRNA gene sequences.
https://doi.org/10.1371/journal.pone.0206502.s001
(PDF)
S2 Fig. Species identification of Campylobacter strains based on atpA gene sequences.
Reference sequences are indicated by species names. Bootstrap values shown at nodes represent support in >95% (black), >85% (grey) and >75% (white) of 1,000 replicates, respectively.
https://doi.org/10.1371/journal.pone.0206502.s002
(EPS)
S1 Table. Inferred Campylobacter species, host species, year, region, location, sample type and method of Campylobacter species determination for all samples.
https://doi.org/10.1371/journal.pone.0206502.s003
(HTML)
Acknowledgments
In carrying out the expeditions, we enjoyed the support of the Chilean Antarctic Institute, the Swedish Polar Research Secretariat, Quark Expeditions and the authorities of South Georgia and the South Sandwich Islands.
We thank Michele Thompson and María Fernanda González-Moraga, whose help during the fieldwork was indispensable. We also thank Birgitta Hellqvist and Mattias Myrenås, the captains and crews of the Ocean Diamond, Aquiles and Lautaro, as well as the officers, staff and personnel at the Antarctic bases Arctowski, Bernardo O’Higgins, Eduardo Frei, Escudero and Gabriel González Videla.
Our work was improved by the much appreciated input of two anonymous reviewers.
This study was funded by the Chilean Antarctic Institute (INACH number T-12-13) and the Swedish Research Council Formas (2014-829). The study made use of the Campylobacter Multi Locus Sequence Typing website (https://pubmlst.org/campylobacter/) sited at the University of Oxford [33], the development of which was funded by the Wellcome Trust.
References
- 1.
Woods R, Jones HI, Watts J, Miller GD, Shellam GR. 2. In: Kerry KR, Riddle M, editors. Diseases of Antarctic seabirds. Berlin, Germany: Springer-Verlag; 2009. p. 35–55.
- 2. Grimaldi WW, Seddon PJ, Lyver PO’B, Nakagawa S, Tompkins DM. Infectious diseases of Antarctic penguins: current status and future threats. Polar Biology. 2015;38(5):591–606.
- 3. Olsen B, Bergström S, McCafferty DJ, Sellin M, Wiström J. Salmonella enteritidis in Antarctica: zoonosis in man or humanosis in penguins? The Lancet. 1996;348(9037):1319–1320.
- 4. Palmgren H, McCafferty D, Aspan A, Broman T, Sellin M, Wollin R, et al. Salmonella in sub-Antarctica: low heterogeneity in Salmonella serotypes in South Georgian seals and birds. Epidemiology and Infection. 2000;125(2):257–262. pmid:11117947
- 5. Iveson JB, Shellam GR, Bradshaw SD, Smith DW, Mackenzie JS, Mofflin RG. Salmonella infections in Antarctic fauna and island populations of wildlife exposed to human activities in coastal areas of Australia. Epidemiology and Infection. 2009;137(6):858–870. pmid:18789175
- 6. Hurt AC, Su YCF, Aban M, Peck H, Lau H, Baas C, et al. Evidence for the introduction, reassortment, and persistence of diverse influenza A viruses in Antarctica. Journal of Virology. 2016;90(21).
- 7.
Kerry KR, Riddle M. 1. In: Kerry KR, Riddle M, editors. Health of Antarctic wildlife: an introduction. Berlin, Germany: Springer-Verlag; 2009. p. 1–10.
- 8. S.C.A.R. Annex to S.C.A.R. bulletin no. 3: scientific investigations recommended by S.C.A.R. Polar Record. 1959;9:596–603.
- 9.
Woehler EJ, Ainley D, Jabour J. 2. In: Tin T, Liggett D, Maher PT, Lamers M, editors. Human Impacts to Antarctic Wildlife: Predictions and Speculations for 2060. Dordrecht: Springer Netherlands; 2014. p. 27–60.
- 10. Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global Epidemiology of Campylobacter Infection. Clinical Microbiology Reviews. 2015;28(3):687–720. pmid:26062576
- 11. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne illness acquired in the United States—major pathogens. Emerging infectious diseases. 2011;17(1).
- 12. Platts-Mills JA, Kosek M. Update on the burden of Campylobacter in developing countries. Current opinion in infectious diseases. 2014;27(5). pmid:25023741
- 13. European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA Journal. 2016;14(12).
- 14. García-Peña FJ, Pérez-Boto D, Jiménez C, San Miguel E, Echeita A, Rengifo-Herrera C, et al. Isolation and Characterization of Campylobacter spp. from Antarctic Fur Seals (Arctocephalus gazella) at Deception Island, Antarctica. Applied and Environmental Microbiology. 2010;76(17):6013–6016. pmid:20639356
- 15. Broman T, Bergström S, On SLW, Palmgren H, McCafferty DJ, Sellin M, et al. Isolation and Characterization of Campylobacter jejuni subsp. jejuni from Macaroni Penguins (Eudyptes chrysolophus) in the Subantarctic Region. Applied and Environmental Microbiology. 2000;66(1):449–452. pmid:10618265
- 16. Griekspoor P, Olsen B, Waldenström J. Campylobacter jejuni in penguins, Antarctica. Emerging infectious diseases. 2009;15(5):847–848. pmid:19402996
- 17. Leotta G, Vigo G, Giacoboni G. Isolation of Campylobacter lari from seabirds in Hope Bay, Antarctica. Polish Polar Research. 2006;27(4):303–308.
- 18. García-Peña FJ, Llorente MT, Serrano T, Ruano MJ, Belliure J, Benzal J, et al. Isolation of Campylobacter spp. from Three Species of Antarctic Penguins in Different Geographic Locations. EcoHealth. 2017;14(1):78–87. pmid:28091764
- 19. Debruyne L, Broman T, Bergström S, Olsen B, On SLW, Vandamme P. Campylobacter subantarcticus sp. nov., isolated from birds in the sub-Antarctic region. International Journal of Systematic and Evolutionary Microbiology. 2010;60(4):815–819. pmid:19661523
- 20. Bonnedahl J, Broman T, Waldenström J, Palmgren H, Niskanen T, Olsen B. In search of human-associated bacterial pathogens in Antarctic wildlife: report from six penguin colonies regularly visited by tourists. Ambio. 2005;34(6):430–432. pmid:16201212
- 21. Debruyne L, On SLW, De Brandt E, Vandamme P. Novel Campylobacter lari-like bacteria from humans and molluscs: description of Campylobacter peloridis sp. nov., Campylobacter lari subsp. concheus subsp. nov. and Campylobacter lari subsp. lari subsp. nov. International Journal of Systematic and Evolutionary Microbiology. 2009;59(5):1126–1132. pmid:19406805
- 22. Debruyne L, Broman T, Bergström S, Olsen B, On SLW, Vandamme P. Campylobacter volucris sp. nov., isolated from black-headed gulls (Larus ridibundus). International Journal of Systematic and Evolutionary Microbiology. 2010;60(8):1870–1875. pmid:19767353
- 23.
Nachamkin I. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Campylobacter and Arcobacter. 6th ed. Washington, D.C., USA: ASM Press; 1995. p. 113–117.
- 24. Wang G, Clark CG, Taylor TM, Pucknell C, Barton C, Price L, et al. Colony Multiplex PCR Assay for Identification and Differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. Journal of Clinical Microbiology. 2002;40(12):4744–4747. pmid:12454184
- 25. Mandrell RE, Harden LA, Bates A, Miller WG, Haddon WF, Fagerquist CK. Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Applied and Environmental Microbiology. 2005;71(10):6292–6307. pmid:16204551
- 26. Gordon D, Green P. Consed: a graphical editor for next-generation sequencing. Bioinformatics. 2013;29(22):2936–2937. pmid:23995391
- 27. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–1574. pmid:12912839
- 28. Miller WG, Yee E, Jolley KA, Chapman MH. Use of an improved atpA amplification and sequencing method to identify members of the Campylobacteraceae and Helicobacteraceae. Letters in Applied Microbiology. 2014;58(6):582–590. pmid:24517729
- 29. Kazutaka K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution. 2013;30(4):772–780.
- 30. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–1313. pmid:24451623
- 31. Dingle KE, Colles FM, Wareing DRA, Ure R, Fox AJ, Bolton FE, et al. Multilocus Sequence Typing System for Campylobacter jejuni. Journal of Clinical Microbiology. 2001;39(1):14–23. pmid:11136741
- 32. Miller WG, On SLW, Wang G, Fontanoz S, Lastovica AJ, Mandrell RE. Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus. Journal of Clinical Microbiology. 2005;43(5):2315–2329. pmid:15872261
- 33. Jolley KA, Maiden MCJ. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 2010;11(1). pmid:21143983
- 34. Daszak P, Cunningham AA, Hyatt AD. Emerging infectious diseases of wildlife–threats to biodiversity and human health. Science. 2000;287(5452):443–449. pmid:10642539
- 35. Tompkins DM, Begon M. Parasites Can Regulate Wildlife Populations. Parasitology Today. 1999;15(8):311–313. pmid:10407375
- 36. Smith KF, Acevedo-Whitehouse K, Pedersen AB. The role of infectious diseases in biological conservation. Animal Conservation. 2009;12(1).
- 37.
Final Report of the Third Antarctic Treaty Consultative Meeting (Article IX, paragraph 4); 1964.
- 38. Convey P, Hughes KA, Tin T. Continental governance and environmental management mechanisms under the Antarctic Treaty System: sufficient for the biodiversity challenges of this century? Biodiversity. 2012;13(3–4):234–248.
- 39. Walton D. Keeping the aliens out. Antarctic Science. 2012;24(4):321.
- 40. González-Acuña D, Hernández J, Moreno L, Herrmann B, Palma R, Latorre A, et al. Health evaluation of wild gentoo penguins (Pygoscelis papua) in the Antarctic Peninsula. Polar Biology. 2013;36(12):1749–1760.
- 41. Cawthraw S, Ayling R, Nuijten P, Wassenaar T, Newell DG. Isotype, Specificity, and Kinetics of Systemic and Mucosal Antibodies to Campylobacter jejuni Antigens, Including Flagellin, during Experimental Oral Infections of Chickens. Avian Diseases. 1994;38(2):341–349. pmid:7526839
- 42. Waldenström J, Axelsson-Olsson D, Olsen B, Hasselquist D, Griekspoor P, Jansson L, et al. Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment. PLOS ONE. 2010;5(2).
- 43. Humphrey S, Chaloner G, Kemmett K, Davidson N, Williams N, Kipar A, et al. Campylobacter jejuni Is Not Merely a Commensal in Commercial Broiler Chickens and Affects Bird Welfare. mBio. 2014;5(4):e01364–14. pmid:24987092
- 44. E DK, Colles FM, Ure R, Wagenaar JA, Duim B, Bolton FJ, et al. Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation. Emerging infectious diseases. 2002;8(9):949–55.
- 45. Manning G, Dowson CG, Bagnall MC, Ahmed IH, West M, Newell DG. Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni. Applied and Environmental Microbiology. 2003;69(11):6370–6379. pmid:14602588
- 46. Colles FM, Jones K, Harding RM, Maiden MCJ. Genetic Diversity of Campylobacter jejuni isolates from farm animals and the farm environment. Applied and Environmental Microbiology. 2003;69(12):7409–7413. pmid:14660392
- 47. Griekspoor P, Colles FM, McCarthy ND, Hansbro PM, Ashhurst-Smith C, Olsen B, et al. Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds. Molecular Ecology. 2013;22(5):1463–1472. pmid:23356487
- 48.
Waldenström J, Griekspoor P. In: Sheppard SK, editor. Ecology and host associations of Campylobacter in wild birds. Norfolk: Caister Academic Press; 2014. p. 265–284.
- 49. Colles FM, McCarthy ND, Howe JC, Devereux CL, Gosler AG, Maiden MCJ. Dynamics of Campylobacter colonization of a natural host, Sturnus vulgaris (European Starling). Environmental Microbiology. 2009;11(1):258–267. pmid:18826435
- 50. French NP, Midwinter A, Holland B, Collins-Emerson J, Pattison R, Colles F, et al. Molecular epidemiology of Campylobacter jejuni isolates from wild-bird fecal material in children’s playgrounds. Applied and Environmental Microbiology. 2009;75(3):779–783. pmid:19047378
- 51.
Tønnessen JN, Johnsen AO. The history of modern whaling. Berkeley and Los Angeles, CA, USA: University of California Press; 1982.
- 52.
Government of South Georgia and the South Sandwich Islands. Biodiversity action plan for South Georgia and the South Sandwich Islands 2016–2020; 2016.
- 53.
International Association of Antarctica Tour Operators (IAATO). 2015–2016 Number of Tourists per Site per Vessel—Peninsula Sites; 2016. Available from: https://iaato.org/sv/tourism-statistics.