Introduction
Protozoan parasites are widely distributed and the cause of a range of different diseases in animals, including humans. Examples of these organisms are blood-inhabiting parasites, which affect body condition and reproduction of the hosts (Merino et al. Reference Merino, Moreno, Sanz and Arriero2000, Sanz et al. Reference Sanz, Arriero, Moreno and Merino2001), or coccidia, which are responsible for loss of body weight and diarrhoea (Dorrenstein Reference Dorrenstein1997). Polar environments also contain these parasites. For instance, Giardia sp. is prevalent in faeces of both land and marine mammals in Arctic and Sub-Arctic areas (Roach et al. Reference Roach, Olson, Whitley and Wallis1993, Olson et al. Reference Olson, Roach, Stabler and Chan1997). Coccidia, such as Tyzzeria parvula or Toxoplasma gondii, are also present in Arctic wildlife such as barnacle geese (Branta leucopsis Bechstein) and Arctic foxes (Alopex lagopus Miller) (Prestrud et al. Reference Prestrud, Asbakk, Fuglei, Mork, Stien, Ropstad, Tryland, Gabrielsen, Lydersen, Kovacs, Loonen, Sagerup and Oksanen2007, Dolnik & Loonen Reference Dolnik and Loonen2007). In Antarctica, several protozoa have been reported. Some of them, such as blood parasites, are restricted to the sub-Antarctic region where suitable vectors are present (Merino et al. Reference Merino, Barbosa, Moreno and Potti1997, Jones & Shellam Reference Jones and Shellam1999, Barbosa & Palacios Reference Barbosa and Palacios2009). Others include Cryptosporidium sp. (Fredes et al. Reference Fredes, Raffo and Muñoz2007a, Reference Fredes, Diaz, Raffo and Muñoz2008), Eimeria pygosceli (Golemanski Reference Golemanski2003), Sarcocystis sp. (Ippen & Henne Reference Ippen and Henne1989) and Isospora sp. (Golemanski Reference Golemanski2003). In general, information about diseases and parasites, including protozoa in Antarctic birds, is scarce and fragmented (Barbosa & Palacios Reference Barbosa and Palacios2009). Although penguins are among the best studied bird species in Antarctica, protozoa are one of the least studied groups of parasites in these birds (Clarke & Kerry Reference Clarke and Kerry2000, Barbosa & Palacios Reference Barbosa and Palacios2009), and information is restricted to a few locations. A reasonable distributional map of these parasites is lacking and further information about the presence/absence of pathogens is therefore needed. Such information is crucial in evaluating whether their presence is due to a natural process or introduced by human activities.
For this paper, we studied the presence of the protozoan parasites Cryptosporidium sp., Giardia sp. and Toxoplasma gondii in three species of Antarctic penguins (chinstrap - Pygoscelis antarctica Forster, gentoo - Pygoscelis papua Forster and Adélie - Pygoscelis adeliae (Hombron & Jacquinot)) in different locations along the western part of the Antarctic Peninsula in order to assess the distribution of these parasites in the same hosts within a wide geographical area as a reference point for future studies.
Materials and methods
During January and February 2006 and 2007, we visited several breeding colonies of chinstrap, gentoo, and Adélie penguins along the west coast of the Antarctic Peninsula, covering a geographical range from 62°10′–67°46′S (Table I). Chinstrap penguins range from 56–65°S, gentoo penguin from 46–65°S and Adélie penguins from 54–77°S (Williams Reference Williams1995). Therefore, our study covers the intermediate part of the Adélie penguin range, and the southern part of the ranges of chinstrap and gentoo penguins.
Table I Sample size analysed for Cryptosporidium sp., Giardia sp. and Toxoplasma gondii in each penguin species in the different locations. Mean percentage of population analysed = 1.27% (range = 0.06–5.64; own data except for Stranger Point where data were taken from Carlini et al. Reference Carlini, Coria, Santos, Negrete, Juares and Daneri2009)
Adult penguins were captured at random on the beach in order to minimize disturbance in the breeding colonies (see Barbosa et al. Reference Barbosa, Merino, Benzal, Martinez and García-Fraile2007 for a similar approach). From each individual (see sample sizes in Table I) a blood sample was taken from a foot vein with a heparinised capillary tube immediately after capture. This sample was later centrifuged at 12 000 rpm for 10 min (relative centrifugal force = 14 811 g) to separate plasma from red blood cells. After centrifugation both fractions were frozen for subsequent analyses. Cloacal swabs and faeces were also collected. Faeces were stored at -20°C and cloacal swabs were preserved in a sodium chloride–Tris–EDTA buffer (NaCl 0.5M, Tris 0.05M, EDTA 0.05M).
The number of samples recovered and analysed from the different penguin species in the different locations (Fig. 1) is summarized in Table I. Following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997) 95% confidence intervals of prevalence were calculated. Cryptosporidium and Giardia were studied by means of PCR assay while Toxoplasma was studied by serological methods.
Fig. 1 Localities sampled. 1. King George Island (Stranger Point), 2. Livingston Island (Hannah Point), 3. Deception Island (Vapor Col), 4. Ronge Island (George Point), 5. Paradise Bay, 6. Yalour Island, 7. Avian Island.
Cryptosporidium and Giardia detection
Oocyst/cyst disruption and DNA purification from faecal samples and cloacal swabs was performed as described in McLauchlin et al. (Reference McLauchlin, Pedraza-Díaz, Amar-Hoetzeneder and Nichols1999) and comprised disruption with zirconia beads in the presence of guanidinium thiocyanate followed by purification with activated silica (Boom et al. Reference Boom, Sol, Salimans, Jansen, Wertheim van Dillen and van der Noordaa1990). For Cryptosporidium detection and characterization, a nested PCR procedure was performed for amplification of an 827–840 bp polymorphic fragment of the 18S rDNA (Xiao et al. Reference Xiao, Escalante, Yang, Sulaiman, Escalante, Montali, Fayer and Lal1999, Reference Xiao, Alderisio, Limor, Royer and Lal2000). PCR products were amplified in 50μl volumes containing 1x PCR buffer, 6mM MgCl2 (primary reaction) or 3mM MgCl2 (secondary reaction), 200 μM dNTPs, 10pmoles of each primer, 2.5 units of DNA polymerase (Biotools, Madrid, Spain) and 5 μl of template DNA (primary reaction) or 2 μl PCR product (secondary reaction). Templates were subjected to an initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 60 s, and a final extension at 72°C for 7 min. Primers used include 18SX1F: 5′-TTCTAGAGCTAATACATGCG-3′ and 18SX1R: 5′-CCCATTTCCTTCGAAACAGGA-3′ in the first step and 5 μl of the first step product and primers 18SX2F: 5′-GGAAGGGTTGTATTTATTAGATAAAG-3′ and 18SX2R: 5′-AAGGAGTAAGGAACAACCTCCA-3′ in the second step. Positive and negative controls were included in each batch of tests.
For Giardia detection and characterization, a nested procedure was performed to amplify a 511 bp fragment of the beta-giardin gene (Lalle et al. Reference Lalle, Pozio, Capelli, Bruschi, Crotti and Caccio2005). PCR products were amplified in 25 μl volumes containing 1x PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 10 pmoles of each primer, 1.5 units of DNA polymerase (Biotools, Madrid, Spain) and 5 μl of template DNA (primary reaction) or 2.5 μl PCR product (secondary reaction). In the first reaction, templates were subjected to an initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 7 min. For the secondary reaction, an initial denaturation at 94°C for 5 min was followed by 35 cycles of 94°C for 60 s, 50°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 7 min. Markers used were G7: 5′-AAGCCCGACGACCTCACCCGCAGTGC-3′ and G759: 5′-GAGGCCGCCCTGGATCTTCGAGACGAC-3′ in the first step and BG-For: 5′-GAACGAGATCGAGGTCCG-3′ and BG-Rev: 5′-CTCGACGAGCTTCGTGTT-3′ in the second one. Positive and negative controls were included for all PCRs. A 5 μl aliquot of the PCR products was examined following electrophoresis in 1% agarose/ethidium bromide gels.
Toxoplasma detection
For detection of antibodies against Toxoplasma gondii, a commercial kit was used (Toxo-Screen DA, BioMerieux®, France). This commercial kit is based on detection of specific IgG from sera by direct agglutination with sensitized antigen of Sabin Strain Formolated Toxoplasma taquizoites obtained from mice ascitic fluid. The procedure was followed according to the manufacturer’s instructions. For initial screening, 1:20 and 1:100 final dilutions of sera were mixed in round bottom well microplates with the antigen suspension and 2-mercaptoethanol to avoid non-specific agglutination. Both positive and negative controls included in the kit were added. Results were read after overnight incubation (18 hours approximately). Samples that showed a smooth mat across the bottom of the well indicating the agglutination of specific antibodies in the serum with the antigen sensitized particles were recorded as positive. The presence of a compact button at the bottom of wells formed by the settling of the non-agglutinated particles, which indicates lack of specific antibody in serum samples, was recorded as negative. The sensitivity and specificity of this commercial kit, reported by the manufacturer, is 96.22% and 98.80% respectively.
Results and discussion
We did not find any positive result in any of the samples studied either for Cryptosporidium, Giardia or Toxoplasma gondii. Upper 95% confidence intervals for infection are as follows: Cryptosporidium/Giardia - gentoo penguin (3.6%), Cryptosporidium/Giardia - chinstrap penguin (1.5%), Cryptosporidium/Giardia - Adélie penguin (8.2%), Toxoplasma gondii - gentoo penguin (4.8%), Toxoplasma gondii - chinstrap penguin (2.3%), Toxoplasma gondii - Adélie penguin (4.8%). According to these results and although it would seem reasonable to assume that pygoscelid penguins are free from these parasites in the studied locations, we are cautious and consider that the prevalence could be as high as the figures shown by the upper confidence intervals (see Bush et al. Reference Bush, Lafferty, Lotz and Shostak1997). In the case of Cryptosporidium and Giardia, detection of these organisms was done by PCR and no specific amplicons were observed which indicates that these parasites might be absent or there was an insufficient amount of the parasites’ DNA present. Other possible reasons for the lack of amplification are degradation of the DNA during storage, and/or copurification of inhibitors of the PCR. We believe that degradation of the DNA is unlikely since it has been previously reported that, at least for Cryptosporidium, DNA can be successfully isolated from whole faeces by the methods described here after storage at room temperature for six weeks or at 4°C for four years (McLauchlin et al. Reference McLauchlin, Pedraza-Díaz, Amar-Hoetzeneder and Nichols1999). Copurification of inhibitors in the DNA extraction is also unlikely since unspecific bands were observed in some samples after PCR. In addition, dilutions of positive Cryptosporidium and Giardia control DNAs seeded into a subset of extracts from penguins resulted in successful amplification of the targets.
There have been three previous surveys looking for Cryptosporidium in Antarctic penguins (Fredes et al. Reference Fredes, Raffo and Muñoz2007a, Reference Fredes, Madariaga, Raffo, Valencia, Herrera, Godoy and Alcaino2007b, Reference Fredes, Diaz, Raffo and Muñoz2008). The first one described negative results in 52 gentoo penguin samples from Paradise Bay (64°49′S 62°51′W) using the Ziehl-Neelsen method (Fredes et al. Reference Fredes, Madariaga, Raffo, Valencia, Herrera, Godoy and Alcaino2007b). However, the other two studies, using acid-fast stain, found some parasite structures compatible with Cryptosporidium oocysts in Adélie penguins (Fredes et al. Reference Fredes, Raffo and Muñoz2007a) and gentoo penguins (Fredes et al. Reference Fredes, Diaz, Raffo and Muñoz2008) both in Ardley Island, South Shetland Islands (62°13′S 58°54′W). At the moment, only penguin populations breeding in this island seem to be infected by Cryptosporidium, while extensive parts of the South Shetland Islands and Antarctic Peninsula seem to be free or at least with low prevalences of this parasite (Fredes et al. Reference Fredes, Raffo and Muñoz2007a and results from this study). However, a positive record has been found in one elephant seal (Mirounga leonina (L.)) in Avian Island (Pedraza-Diaz et al. unpublished). Cryptosporidium is a significant pathogen of humans and other animals (Fayer et al. Reference Fayer, Speer and Dubey1997) that is transmitted through contamination with human and domestic animal faecal material (Appelbee et al. Reference Appelbee, Thompson and Olson2005). Specifically Cryptosporidium together with Giardia have been detected in marine water samples from areas of treated sewage disposal (Johnson et al. Reference Johnson, Reynolds, Gerba, Pepper and Rose1995a). The only place where Cryptosporidium has been detected in Antarctica in penguins is very close to the Fildes Peninsula (King George Island) (Fredes et al. Reference Fredes, Raffo and Muñoz2007a, Reference Fredes, Diaz, Raffo and Muñoz2008). In this place extensive scientific, logistic and tourist activities are carried out (Pfeiffer Reference Pfeiffer2005), and sewage water disposal without treatment was carried out in former times (Tin & Roura Reference Tin and Roura2004) therefore increasing the probability of its presence. However, we have not found Cryptosporidium in other parts of King George Island, such as the penguin rockery in Stranger Point (62°15′S 58°37′W). This place is the ASPA (Antarctic Special Protected Area) No. 132 and is located 5 km from the nearest research station. Moreover, other places with scientific research stations in the locality such as Paradise Bay have not shown the presence of Cryptosporidium (Fredes et al. Reference Fredes, Madariaga, Raffo, Valencia, Herrera, Godoy and Alcaino2007b and the present study). Results from Fredes et al. (Reference Fredes, Madariaga, Raffo, Valencia, Herrera, Godoy and Alcaino2007b) should be taken with caution because they used direct microscopic identification of oocyst for detection, which gives a high probability of false negatives and is around 100 to 1000 times less efficient than the use of molecular methods (Atías Reference Atías1998, but see Ballweber et al. Reference Ballweber, Panuska, Huston, Vasilopulos, Pharr and Mackin2009 who stated that microscopic and PCR methods are equivalent for Giardia detection). In addition, the use of molecular methods allows the identification of the species or genotypes involved in infection and may contribute to understanding the routes of transmission. However, in some cases, inhibitory substances can affect PCR performance leading to false-negative PCR results, therefore reducing its efficacy (Johnson et al. Reference Johnson, Pieniazek, Griffin, Misener and Rose1995b). In our study we can rule out this possibility since unspecific bands were detected after PCR and dilutions of positive Cryptosporidium and Giardia control DNAs seeded into a subset of extracts from the studied species resulted in successful amplification of the targets (see above). In any case, our results using PCR assays seem to confirm the absence of Cryptosporidium in this location obtained previously by means of microscopic techniques.
Giardia has been found in polar environments, specifically in marine mammals in the Arctic and sub-Arctic area (Appelbee et al. Reference Appelbee, Thompson and Olson2005). As far as we know, this is the first time that the presence of Giardia has been studied in Antarctica and we have obtained negative results. Giardia and Cryptosporidium share many characteristics. Both parasitize similar hosts, have the same route of transmission and produce similar effects on the hosts (Appelbee et al. Reference Appelbee, Thompson and Olson2005). Therefore, it is not surprising that the same results were found for both protozoan parasites, in this case negative.
In our study an agglutination test was used for the detection of antibodies to T. gondii. To our knowledge this is the first time this procedure has been used in penguins although it has been widely used, either as kit or as a modified agglutination test (MAT), in humans and other mammals and in birds (Desmonts & Remington Reference Desmonts and Remington1980, Dubey & Desmonts Reference Dubey and Desmonts1987, Dubey Reference Dubey2002, Meunier et al. Reference Meunier, Jourda, Deville and Guillot2006, de Camps et al. Reference De Camps, Dubey and Saville2008, Dubey & Jones Reference Dubey and Jones2008, Waap et al. Reference Waap, Vilares, Rebelo, Gomes and Angelo2008, Zhu et al. Reference Zhu, Yin, Xiao, Jiang, Ankarlev, Lindh and Chen2008, Salant et al. Reference Salant, Landau and Baneth2009). It has been reported that amongst different serological tests available, the agglutination test is most useful because it is species independent (does not require species specific conjugates), sensitive, and specific (Desmonts & Remington Reference Desmonts and Remington1980, Dubey Reference Dubey2002, Negash et al. Reference Negash, Tilahun, Patton, Prévot and Dorchies2004), and no cross-reactivity of T. gondii with other possible infective organisms has been reported (Salant et al. Reference Salant, Landau and Baneth2009). Most authors have considered titres of 1:25 as positive, although as low as 1:2 or 1:5 have also been reported (Dubey & Jones Reference Dubey and Jones2008), therefore screenings were done at 1:20 and 1:100 final dilutions. We did not detect the presence of antibodies to T. gondii in any of the samples analysed. However, Toxoplasma has been found in different groups of birds (Dubey Reference Dubey2002) including penguins in captivity (Ratcliffe & Worth Reference Ratcliffe and Worth1951, Mason et al. Reference Mason, Hartley and Dubey1991).
To our knowledge the study presented here constitutes the first investigation on the presence of Toxoplasma in Antarctica, although it was found previously in the Iles Kerguelen in the sub-Antarctic region (Afonso et al. Reference Afonso, Thulllez, Pontier and Gilot-Fromont2007) and in the Arctic (Prestrud et al. Reference Prestrud, Asbakk, Fuglei, Mork, Stien, Ropstad, Tryland, Gabrielsen, Lydersen, Kovacs, Loonen, Sagerup and Oksanen2007). As domestic cats and other felids are the only definitive hosts for Toxoplasma (Frenckel et al. Reference Frenckel, Dubey and Miller1970), the presence of this parasite in Antarctica is not expected a priori. However, a wide range of birds serve as intermediate hosts, being infected by direct ingestion of oocysts and also by vertical transmission (Dubey & Beattie Reference Dubey and Beattie1988). Therefore, the introduction of Toxoplasma in the Antarctic ecosystem by migrating birds could be a risk, although when it happens, the probability of transmission to penguins would be very low. However, scavenger species like the skuas or giant petrels are probably at a higher risk. On the other hand, Toxoplasma transmission by marine invertebrates have been suspected (Prestrud et al. Reference Prestrud, Asbakk, Fuglei, Mork, Stien, Ropstad, Tryland, Gabrielsen, Lydersen, Kovacs, Loonen, Sagerup and Oksanen2007) and transmission from human activities in the Arctic has not been discarded (Prestrud et al. Reference Prestrud, Asbakk, Fuglei, Mork, Stien, Ropstad, Tryland, Gabrielsen, Lydersen, Kovacs, Loonen, Sagerup and Oksanen2007), posing a risk for a similar situation in Antarctica.
We report negative findings, although calculating the upper 95% confidence intervals prevalence could be between 1.53% and 8.22%. Therefore, it would be important to implement long-term monitoring programs to confirm the presence/absence of this kind of organism in different places with different infection susceptibility according to human presence or environmental factors (Barbosa & Palacios Reference Barbosa and Palacios2009). Searching in the future for parasites and diseases in places where they seem to be absent nowadays will allow us to detect new introductions and their causes.
Acknowledgments
This study was funded by the Spanish Ministry of Science projects CGL2004-01348 and POL2006-05175 and by the European Regional Development Fund. MJP was supported by a PhD grant from the Spanish Ministry of Science and Innovation (BES-2005-8465). We thank the Spanish Antarctic Base “Gabriel de Castilla”, the Argentinean Polar Base “Teniente Jubany”, the Spanish polar ship Las Palmas and the Maritime Logistic Unit (CSIC) for logistic support and transport. We are greatly indebted to the avian and mammal groups of the Argentinean Antarctic Institute for their help in the fieldwork. Permissions to work in the study area and for penguin manipulation were given by the Spanish Polar Committee. The suggestions of two anonymous referees improved an earlier version of this paper. This is a contribution to the International Polar Year project 172 BIRDHEALTH and to PINGUCLIM project.
