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Original article
First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli) Frédéric Schramm a,∗,1 , Michel Gauthier-Clerc b,c,1 , Jean-Charles Fournier d , Karen D. McCoy e , Cathy Barthel a , Danièle Postic f , Yves Handrich d,g , Yvon Le Maho d,g , Benoît Jaulhac a,∗∗ a
Université de Strasbourg, EA 7290, Faculté de Médecine, Strasbourg, France Centre de Recherche de la Tour du Valat, Le Sambuc, Arles, France c Université de Franche-Comté, Département Chrono-Environnement, UMR UFC/CNRS 6249 USC INRA, Besanc¸on, France d Université de Strasbourg, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France e MIVEGEC (Maladies infectieuses et vecteurs: écologie, génétique, évolution et contrôle), UMR 5290 CNRS/IRD, UR 244 IRD UM1 UM2, Montpellier, France f Institut Pasteur, Laboratoire des Spirochètes, Paris, France g CNRS, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France b
a r t i c l e
i n f o
Article history: Received 26 December 2013 Received in revised form 15 July 2014 Accepted 16 July 2014 Available online xxx Keywords: King penguins Borrelia burgdorferi Lyme disease Spirochetemia Crozet Archipelago Epidemiology
a b s t r a c t The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes. © 2014 Elsevier GmbH. All rights reserved.
Introduction Lyme borreliosis, caused by spirochetes of the Borrelia burgdorferi sensu lato (Bbsl) group and transmitted by Ixodes spp. ticks, is the most common human tick-borne disease of the Northern hemisphere (Stanek et al., 2012). In their obligate enzootic life cycle, these spirochetes are transmitted by Ixodes ticks through a terrestrial cycle involving a variety of vertebrate hosts, including mammals, reptiles (Kurtenbach et al., 2006; Gern, 2009) and birds
∗ Corresponding author at: Institut de Bactériologie, EA 7490, Faculté de Médecine, Université de Strasbourg, 3 rue Koeberlé, 67000 Strasbourg, France. Tel.: +33 3 69 55 14 61; fax: +33 3 69 55 16 98. ∗∗ Corresponding author. E-mail address: [email protected] (F. Schramm). 1 These authors contributed equally to the paper.
(Comstedt et al., 2006; Dubska et al., 2009; Hamer et al., 2011). Birds are thought to play an important role in the dissemination and global circulation of Borrelia (Humair, 2002; Ogden et al., 2008; Comstedt et al., 2011). This is particularly the case for seabirds via a marine cycle which involves B. garinii and ticks of the I. uriae complex (Olsen et al., 1993, 1995; Gylfe et al., 2001; Duneau et al., 2008; Lobato et al., 2011). I. uriae parasitises an extensive range of seabirds species (Dietrich et al., 2011), and has been shown to be infected with Bbsl in the circumpolar regions of both the Northern (Olsen et al., 1993, 1995; Duneau et al., 2008; Comstedt et al., 2009; Gómez-Díaz et al., 2010, 2011) and the Southern Hemisphere, including the Crozet Archipelago (Olsen et al., 1995). The French sub-Antarctic Crozet Archipelago lies in the Southern Indian Ocean (45◦ 68 –46◦ 26 S, 50◦ 14 –52◦ 15 E), some 2500 km southeast of South Africa and 2400 km north of the coast of Antarctica (Fig. 1A). This archipelago is an important breeding area for seabirds, with an estimated 25 million breeding birds
http://dx.doi.org/10.1016/j.ttbdis.2014.07.013 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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Fig. 1. (A) Location of Crozet Archipelago in the Southern Indian Ocean. (B) Location of the study site (Baie du Marin) in Possession Island (Crozet Archipelago).
(Frenot et al., 2001) of 37 species (Shirihai and Jarrett, 2008). The breeding population of king penguins (Aptenodytes patagonicus halli) alone consists of about one million pairs (Guinet et al., 1995) and thus may represent a significant host for I. uriae in this part of the world (Gauthier-Clerc et al., 1998, 1999; Frenot et al., 2001; McCoy et al., 2005). In a previous study, we showed that seroprevalence for anti-Bbsl antibodies was 14% in tick-infested adult
king penguins (Gauthier-Clerc et al., 1999). However, to our knowledge, spirochetes have never been detected in seabirds of Southern Hemisphere, which means that there is until now no direct evidence of the involvement of these seabirds in the epizootiology of Bbsl in this region. The aim of the present study was therefore to investigate the potential role of king penguins as a reservoir for Bbsl and, in this case, to precise the genomic species of Borrelia involved.
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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Materials and methods Site and field collection This study was approved by the Ethical Committee of the French Polar Institute. The study was conducted in a large king penguin colony located at Baie du Marin on the eastern coast of Possession Island (46◦ 25 S, 51◦ 45 E), Crozet Archipelago (Fig. 1B) where approximately 25,000 pairs of king penguin breed. I. uriae is the only tick species recorded on the king penguins examined in this colony (Gauthier-Clerc et al., 1999). A total of 50 tick-infested adult king penguins, and 10 other tick-free adults were randomly caught while departing to sea after an incubation shift. The presence of ticks was checked as previously described by searching the head and neck of each bird (Mangin et al., 2003). Blood samples were collected by a brachial vein puncture without anaesthesia. The sampled birds were then coloured with picric acid on the leg to avoid recapturing the same individual. Serology, PCR and Borrelia species typing For Borrelia serology, 4–6 mL of blood samples were collected in dry tubes (Vacuette, Greiner, France) and left for 2 h to clot. Tubes were then centrifuged at 4000 × g for 15 min. Following centrifugation, sera were frozen at −80 ◦ C until use. As previously described (Gauthier-Clerc et al., 1999), Lyme disease serodiagnosis on penguins was performed using a commercially-available passive hemagglutination-inhibition technique (Lymag, Diagast France). The kit had been previously reported to be appropriate for serology for numerous animal species (Doby et al., 1992), and was the only commercial kit available for birds. Antibody titres of 1/200 or above were considered as positive, according to the manufacturer’s recommendations. For the PCR detection of Borrelia in plasma, 4 mL of blood samples were collected in sodium citrate tubes (Vacutainer CPT, Becton-Dickinson, France) and, after the 2 h period, were centrifuged for 20 min at 1500 × g. After centrifugation, tubes were again directly frozen at −80 ◦ C until analysis. DNA extraction, amplification targeting the fla gene, detection, and southern blot hybridisation, as well as a control of Taq DNA polymerase inhibitors were carried out as previously described (Jaulhac et al., 1996). PCRpositive DNA extracts were then amplified using the rrf (5S) – rrl (23s) intergenic spacer as a second Borrelia target and species identifications were made by PCR-RFLP (Postic et al., 1994), using negative and positive controls (including DNA of the following reference strains: B. burgdorferi sensu stricto B31, B. afzelii VS461, B. valaisiana VS116, and B. garinii 20047). Results Among the tested animals, 5 penguins (8%) had a positive serology against Bbsl: four individuals were positive among the 50 tick-infested penguins, and one individual was positive among the 10 tick-free penguins. Borrelia DNA of the fla gene was detected by PCR in the blood of two of the 50 tick-infested penguins and was not detected in the 10 tick-free penguins (Table 1). As a confirmation test, PCR targeting the rrf (5S) – rrl (23s) intergenic spacer was also positive. After cleavage by MseI, PCR-RFLP patterns of the two
3
PCR-positive samples identically matched the RFLP pattern produced by the B. garinii positive control (with an initial amplicon size of 253 pb and MseI restriction fragments of 108, 98 and 50 bp). It is interesting to note that none of the two spirochetemic penguins was positive by Borrelia serology. Discussion Among the 18 genomic species currently described in the Bbsl complex, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. bavariensis and B. spielmanii are clearly known to be pathogenic for humans, whereas B. bissettii, B. valaisiana and B. lusitaniae are only occasionally detected in patients (Stanek and Reiter, 2011; Stanek et al., 2012). In the marine ecosystem of Bbsl, B. garinii appears to be the main Bbsl species infecting I. uriae, although atypical genospecies such as B. lusitaniae, B. afzelii and B. burgdorferi sensu stricto may also be present (Dietrich et al., 2008; Duneau et al., 2008). Several studies in the literature have reported the detection (by PCR or culture) of Bbsl in I. uriae collected from various seabirds colonies (Olsen et al., 1993, 1995; Smith et al., 2006; Larsson et al., 2007; Duneau et al., 2008; Comstedt et al., 2009; Gómez-Díaz et al., 2010, 2011), and on the presence of anti-Bbsl antibodies in blood samples collected from seabirds of both hemispheres (Gauthier-Clerc et al., 1999; Gylfe et al., 1999; Staszewski et al., 2008; Lobato et al., 2011). However, direct reports on Bbsl detection in seabirds are scarce (Gylfe et al., 1999) and to our knowledge, our study is the first one reporting direct Bbsl detection from seabirds in the Southern Hemisphere. We previously described the presence of anti-Bbsl antibodies in king penguin sera sampled in the Crozet Archipelago (GauthierClerc et al., 1999), and the present study confirms the moderate seroprevalence of Bbsl in this seabird species (∼8%). However, the results of hemagglutination assay should be considered with caution as the presence of serum antibodies indicates exposure, but does not imply the presence of clinical disease. The duration of these antibodies in the blood of birds is also unknown and this test may therefore underestimate seabird exposure rates. Finally a positive test provides little information about the antigenic characteristics of the infecting organisms and the Borrelia species responsible for the infection. Evidence by DNA amplification of Bbsl spirochetemia in king penguins fills a part of this void, indicating that the infecting spirochete was able to disseminate within the body of king penguins, and strongly supports a Bbsl reservoir status for this seabird species. As I. uriae ticks tend to show restricted host use within colonies (Gómez-Díaz et al., 2010) and king penguins do not frequently change colonies (Weimerskirch et al., 1992), our results suggest that Bbsl is maintained locally and, thus, Bbsl enzootic foci are present in the Southern Hemisphere. RLFP results also suggest that Bbsl DNA detected in king penguins is related to B. garinii, matching an earlier study that found B. garinii in I. uriae from the Crozet Islands (Olsen et al., 1995). However, further studies including a larger number of individuals and the sequencing of multi-locus PCR products are needed to confirm this hypothesis and to analyse the genetic variability of Bbsl in king penguins. In our study, no spirochetemic king penguins were seropositive for Bbsl and the percentage of individuals with active (and detectable) infections was very small. This could be due to the
Table 1 Prevalence of Borrelia burgdorferi sensu lato spirochetemia and seropositivity in adult king penguins tested. Tick-infested penguins
PCR (−) PCR (+)
Tick-free penguins
Serology (−)
Serology (+)
Serology (−)
Serology (+)
44 (88%) 2 (4%)
4 (8%) 0
9 0
1 0
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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fact that king penguins are spirochetemic for very short periods after infection, as has been experimentally demonstrated for some bird species (Bishop et al., 1994; Richter et al., 2000), or that blood samples are not the most appropriate specimens for sensitive Bbsl detection in king penguins. Further studies are now needed to better understand the epizootiology of the marine Bbsl cycle in the Crozet Archipelago, notably by studying the other enzootic seabird species, including those infested by the other tick species (I. kerguelenensis) recorded on these islands. Conflicts of interest There are no conflicts of interest. Acknowledgements We thank Nathalie Boulanger for fruitful discussion. We thank the Institut Polaire Franc¸ais-Paul-Emile Victor for financial and logistical support (Programme 137). This work was partly supported by the project Zones Ateliers of the Programme Environnement Vie et Société of the CNRS (France). References Bishop, K.L., Khan, M.I., Nielsen, S.W., 1994. Experimental infection of northern bobwhite quail with Borrelia burgdorferi. J. Wildl. Dis. 30, 506–513. Comstedt, P., Bergström, S., Olsen, B., Garpmo, U., Marjavaara, L., Mejlon, H., Barbour, A.G., Bunikis, J., 2006. Migratory passerine birds as reservoirs of Lyme borreliosis in Europe. Emerg. Infect. Dis. 12, 1087–1095. Comstedt, P., Asokliene, L., Eliasson, I., Olsen, B., Wallensten, A., Bunikis, J., Bergström, S., 2009. Complex population structure of Lyme borreliosis group spirochete Borrelia garinii in subarctic Eurasia. PLoS ONE 4, e5841. Comstedt, P., Jakobsson, T., Bergström, S., 2011. Global ecology and epidemiology of Borrelia garinii spirochetes. Infect. Ecol. Epidemiol. 1, 1–10. Dietrich, M., Gómez-Díaz, E., Boulinier, T., McCoy, K.D., 2008. Local distribution and genetic structure of tick-borne pathogens: an example involving the marine cycle of Lyme disease. In: Bertrand, M., Kreiter, S., McCoy, K.D., Migeon, A., Navajas, M., Tixier, M.S., Vial, L. (Eds.), Integrative Acarology. Proceedings of the 6th European Congress. European Association of Acarologists, pp. 33–42. Dietrich, M., Gómez-Díaz, E., McCoy, K.D., 2011. Worldwide distribution and diversity of seabird ticks: implications for the ecology and epidemiology of tick-borne pathogens. Vector Borne Zoonotic Dis. 11, 453–470. Doby, J.M., Betremieux, C., Degeilh, B., 1992. Interest of using antigens prepared from different Borrelia burgdorferi strains for the diagnosis of Lyme borreliosis in animals. Rev. Méd. Vét. 143, 617–622. Dubska, L., Literak, I., Kocianova, E., Taragelova, V., Sychra, O., 2009. Differential role of passerine birds in distribution of Borrelia spirochetes, based on data from ticks collected from birds during the postbreeding migration period in Central Europe. Appl. Environ. Microbiol. 75, 596–602. Duneau, D., Boulinier, T., Gómez-Díaz, E., Petersen, A., Tveraa, T., Barrett, R.T., McCoy, K.D., 2008. Prevalence and diversity of Lyme borreliosis bacteria in marine birds. Infect. Genet. Evol. 8, 352–359. Frenot, Y., de Oliveira, E., Gauthier-Clerc, M., Deunff, J., Bellido, A., Vernon, P., 2001. Life cycle of the tick Ixodes uriae in penguin colonies: relationships with host breeding activity. Int. J. Parasitol. 31, 1040–1047. Gauthier-Clerc, M., Clerquin, Y., Handrich, Y., 1998. Hyperinfestation by ticks Ixodes uriae: a possible cause of death in adult king penguins, a long lived seabird. Colon. Waterbirds 21, 229–233. Gauthier-Clerc, M., Jaulhac, B., Frenot, Y., Bachelard, C., Monteil, H., Le Maho, Y., Handrich, Y., 1999. Prevalence of Borrelia burgdorferi (the Lyme disease agent) antibodies in king penguin Aptenodytes patagonicus in Crozet Archipelago. Polar Biol. 22, 141–143. Gern, L., 2009. Life cycle of Borrelia burgdorferi sensu lato and transmission to humans. In: Lipsker, D., Jaulhac, B. (Eds.), Current Problems in Dermatology. Karger, Basel, pp. 18–30.
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Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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Original article
First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli) Frédéric Schramm a,∗,1 , Michel Gauthier-Clerc b,c,1 , Jean-Charles Fournier d , Karen D. McCoy e , Cathy Barthel a , Danièle Postic f , Yves Handrich d,g , Yvon Le Maho d,g , Benoît Jaulhac a,∗∗ a
Université de Strasbourg, EA 7290, Faculté de Médecine, Strasbourg, France Centre de Recherche de la Tour du Valat, Le Sambuc, Arles, France c Université de Franche-Comté, Département Chrono-Environnement, UMR UFC/CNRS 6249 USC INRA, Besanc¸on, France d Université de Strasbourg, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France e MIVEGEC (Maladies infectieuses et vecteurs: écologie, génétique, évolution et contrôle), UMR 5290 CNRS/IRD, UR 244 IRD UM1 UM2, Montpellier, France f Institut Pasteur, Laboratoire des Spirochètes, Paris, France g CNRS, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France b
a r t i c l e
i n f o
Article history: Received 26 December 2013 Received in revised form 15 July 2014 Accepted 16 July 2014 Available online xxx Keywords: King penguins Borrelia burgdorferi Lyme disease Spirochetemia Crozet Archipelago Epidemiology
a b s t r a c t The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes. © 2014 Elsevier GmbH. All rights reserved.
Introduction Lyme borreliosis, caused by spirochetes of the Borrelia burgdorferi sensu lato (Bbsl) group and transmitted by Ixodes spp. ticks, is the most common human tick-borne disease of the Northern hemisphere (Stanek et al., 2012). In their obligate enzootic life cycle, these spirochetes are transmitted by Ixodes ticks through a terrestrial cycle involving a variety of vertebrate hosts, including mammals, reptiles (Kurtenbach et al., 2006; Gern, 2009) and birds
∗ Corresponding author at: Institut de Bactériologie, EA 7490, Faculté de Médecine, Université de Strasbourg, 3 rue Koeberlé, 67000 Strasbourg, France. Tel.: +33 3 69 55 14 61; fax: +33 3 69 55 16 98. ∗∗ Corresponding author. E-mail address: [email protected] (F. Schramm). 1 These authors contributed equally to the paper.
(Comstedt et al., 2006; Dubska et al., 2009; Hamer et al., 2011). Birds are thought to play an important role in the dissemination and global circulation of Borrelia (Humair, 2002; Ogden et al., 2008; Comstedt et al., 2011). This is particularly the case for seabirds via a marine cycle which involves B. garinii and ticks of the I. uriae complex (Olsen et al., 1993, 1995; Gylfe et al., 2001; Duneau et al., 2008; Lobato et al., 2011). I. uriae parasitises an extensive range of seabirds species (Dietrich et al., 2011), and has been shown to be infected with Bbsl in the circumpolar regions of both the Northern (Olsen et al., 1993, 1995; Duneau et al., 2008; Comstedt et al., 2009; Gómez-Díaz et al., 2010, 2011) and the Southern Hemisphere, including the Crozet Archipelago (Olsen et al., 1995). The French sub-Antarctic Crozet Archipelago lies in the Southern Indian Ocean (45◦ 68 –46◦ 26 S, 50◦ 14 –52◦ 15 E), some 2500 km southeast of South Africa and 2400 km north of the coast of Antarctica (Fig. 1A). This archipelago is an important breeding area for seabirds, with an estimated 25 million breeding birds
http://dx.doi.org/10.1016/j.ttbdis.2014.07.013 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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Fig. 1. (A) Location of Crozet Archipelago in the Southern Indian Ocean. (B) Location of the study site (Baie du Marin) in Possession Island (Crozet Archipelago).
(Frenot et al., 2001) of 37 species (Shirihai and Jarrett, 2008). The breeding population of king penguins (Aptenodytes patagonicus halli) alone consists of about one million pairs (Guinet et al., 1995) and thus may represent a significant host for I. uriae in this part of the world (Gauthier-Clerc et al., 1998, 1999; Frenot et al., 2001; McCoy et al., 2005). In a previous study, we showed that seroprevalence for anti-Bbsl antibodies was 14% in tick-infested adult
king penguins (Gauthier-Clerc et al., 1999). However, to our knowledge, spirochetes have never been detected in seabirds of Southern Hemisphere, which means that there is until now no direct evidence of the involvement of these seabirds in the epizootiology of Bbsl in this region. The aim of the present study was therefore to investigate the potential role of king penguins as a reservoir for Bbsl and, in this case, to precise the genomic species of Borrelia involved.
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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Materials and methods Site and field collection This study was approved by the Ethical Committee of the French Polar Institute. The study was conducted in a large king penguin colony located at Baie du Marin on the eastern coast of Possession Island (46◦ 25 S, 51◦ 45 E), Crozet Archipelago (Fig. 1B) where approximately 25,000 pairs of king penguin breed. I. uriae is the only tick species recorded on the king penguins examined in this colony (Gauthier-Clerc et al., 1999). A total of 50 tick-infested adult king penguins, and 10 other tick-free adults were randomly caught while departing to sea after an incubation shift. The presence of ticks was checked as previously described by searching the head and neck of each bird (Mangin et al., 2003). Blood samples were collected by a brachial vein puncture without anaesthesia. The sampled birds were then coloured with picric acid on the leg to avoid recapturing the same individual. Serology, PCR and Borrelia species typing For Borrelia serology, 4–6 mL of blood samples were collected in dry tubes (Vacuette, Greiner, France) and left for 2 h to clot. Tubes were then centrifuged at 4000 × g for 15 min. Following centrifugation, sera were frozen at −80 ◦ C until use. As previously described (Gauthier-Clerc et al., 1999), Lyme disease serodiagnosis on penguins was performed using a commercially-available passive hemagglutination-inhibition technique (Lymag, Diagast France). The kit had been previously reported to be appropriate for serology for numerous animal species (Doby et al., 1992), and was the only commercial kit available for birds. Antibody titres of 1/200 or above were considered as positive, according to the manufacturer’s recommendations. For the PCR detection of Borrelia in plasma, 4 mL of blood samples were collected in sodium citrate tubes (Vacutainer CPT, Becton-Dickinson, France) and, after the 2 h period, were centrifuged for 20 min at 1500 × g. After centrifugation, tubes were again directly frozen at −80 ◦ C until analysis. DNA extraction, amplification targeting the fla gene, detection, and southern blot hybridisation, as well as a control of Taq DNA polymerase inhibitors were carried out as previously described (Jaulhac et al., 1996). PCRpositive DNA extracts were then amplified using the rrf (5S) – rrl (23s) intergenic spacer as a second Borrelia target and species identifications were made by PCR-RFLP (Postic et al., 1994), using negative and positive controls (including DNA of the following reference strains: B. burgdorferi sensu stricto B31, B. afzelii VS461, B. valaisiana VS116, and B. garinii 20047). Results Among the tested animals, 5 penguins (8%) had a positive serology against Bbsl: four individuals were positive among the 50 tick-infested penguins, and one individual was positive among the 10 tick-free penguins. Borrelia DNA of the fla gene was detected by PCR in the blood of two of the 50 tick-infested penguins and was not detected in the 10 tick-free penguins (Table 1). As a confirmation test, PCR targeting the rrf (5S) – rrl (23s) intergenic spacer was also positive. After cleavage by MseI, PCR-RFLP patterns of the two
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PCR-positive samples identically matched the RFLP pattern produced by the B. garinii positive control (with an initial amplicon size of 253 pb and MseI restriction fragments of 108, 98 and 50 bp). It is interesting to note that none of the two spirochetemic penguins was positive by Borrelia serology. Discussion Among the 18 genomic species currently described in the Bbsl complex, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. bavariensis and B. spielmanii are clearly known to be pathogenic for humans, whereas B. bissettii, B. valaisiana and B. lusitaniae are only occasionally detected in patients (Stanek and Reiter, 2011; Stanek et al., 2012). In the marine ecosystem of Bbsl, B. garinii appears to be the main Bbsl species infecting I. uriae, although atypical genospecies such as B. lusitaniae, B. afzelii and B. burgdorferi sensu stricto may also be present (Dietrich et al., 2008; Duneau et al., 2008). Several studies in the literature have reported the detection (by PCR or culture) of Bbsl in I. uriae collected from various seabirds colonies (Olsen et al., 1993, 1995; Smith et al., 2006; Larsson et al., 2007; Duneau et al., 2008; Comstedt et al., 2009; Gómez-Díaz et al., 2010, 2011), and on the presence of anti-Bbsl antibodies in blood samples collected from seabirds of both hemispheres (Gauthier-Clerc et al., 1999; Gylfe et al., 1999; Staszewski et al., 2008; Lobato et al., 2011). However, direct reports on Bbsl detection in seabirds are scarce (Gylfe et al., 1999) and to our knowledge, our study is the first one reporting direct Bbsl detection from seabirds in the Southern Hemisphere. We previously described the presence of anti-Bbsl antibodies in king penguin sera sampled in the Crozet Archipelago (GauthierClerc et al., 1999), and the present study confirms the moderate seroprevalence of Bbsl in this seabird species (∼8%). However, the results of hemagglutination assay should be considered with caution as the presence of serum antibodies indicates exposure, but does not imply the presence of clinical disease. The duration of these antibodies in the blood of birds is also unknown and this test may therefore underestimate seabird exposure rates. Finally a positive test provides little information about the antigenic characteristics of the infecting organisms and the Borrelia species responsible for the infection. Evidence by DNA amplification of Bbsl spirochetemia in king penguins fills a part of this void, indicating that the infecting spirochete was able to disseminate within the body of king penguins, and strongly supports a Bbsl reservoir status for this seabird species. As I. uriae ticks tend to show restricted host use within colonies (Gómez-Díaz et al., 2010) and king penguins do not frequently change colonies (Weimerskirch et al., 1992), our results suggest that Bbsl is maintained locally and, thus, Bbsl enzootic foci are present in the Southern Hemisphere. RLFP results also suggest that Bbsl DNA detected in king penguins is related to B. garinii, matching an earlier study that found B. garinii in I. uriae from the Crozet Islands (Olsen et al., 1995). However, further studies including a larger number of individuals and the sequencing of multi-locus PCR products are needed to confirm this hypothesis and to analyse the genetic variability of Bbsl in king penguins. In our study, no spirochetemic king penguins were seropositive for Bbsl and the percentage of individuals with active (and detectable) infections was very small. This could be due to the
Table 1 Prevalence of Borrelia burgdorferi sensu lato spirochetemia and seropositivity in adult king penguins tested. Tick-infested penguins
PCR (−) PCR (+)
Tick-free penguins
Serology (−)
Serology (+)
Serology (−)
Serology (+)
44 (88%) 2 (4%)
4 (8%) 0
9 0
1 0
Please cite this article in press as: Schramm, F., et al., First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli). Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.07.013
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