Parasitol Res DOI 10.1007/s00436-015-4835-y
SHORT COMMUNICATION
Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods Carlos Hermosilla 1 & Liliana M. R. Silva 1 & Sonja Kleinertz 2 & Rui Prieto 3 & Monica A. Silva 3,4 & Anja Taubert 1
Received: 9 October 2015 / Accepted: 12 November 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A number of parasitic diseases have gained importance as neozoan opportunistic infections in the marine environment. Here, we report on the gastrointestinal endoparasite fauna of three baleen whale species and one toothed whale: blue (Balaenoptera musculus), fin (Balaenoptera physalus), and sei whales (Balaenoptera borealis) and sperm whales (Physeter macrocephalus) from the Azores Islands, Portugal. In total, 17 individual whale fecal samples [n = 10 (B. physalus); n=4 (P. macrocephalus); n=2 (B. musculus); n=1 (B. borealis)] were collected from free-swimming animals as part of ongoing studies on behavioral ecology. Furthermore, skin biopsies were collected from sperm whales (n=5) using minimally invasive biopsy darting and tested for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA via PCR. Overall, more than ten taxa were detected in whale fecal samples. Within protozoan parasites, Entamoeba spp. occurred most frequently (64.7 %), followed by Giardia spp. (17.6 %) and Balantidium spp. (5.9 %). The most prevalent metazoan parasites were * Carlos Hermosilla
[email protected] 1
Institute of Parasitology, Biomedical Research Center Seltersberg, Justus Liebig University Giessen, Schubertstr. 81, 35392 Giessen, Germany
2
Aquaculture and Sea-Ranching, Faculty of Agricultural and Environmental Sciences, University of Rostock, 18059 Rostock, Germany
3
MARE—Marine and Environmental Sciences Centre and IMAR—Institute of Marine Research, University of the Azores, 9901-862 Horta, Portugal
4
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
Ascaridida indet. spp. (41.2 %), followed by trematodes (17.7 %), acanthocephalan spp., strongyles (11.8 %), Diphyllobotrium spp. (5.9 %), and spirurids (5.9 %). Helminths were mainly found in sperm whales, while enteric protozoan parasites were exclusively detected in baleen whales, which might be related to dietary differences. No T. gondii, N. caninum, or B. besnoiti DNA was detected in any skin sample. This is the first record on Giardia and Balantidium infections in large baleen whales. Keywords Balaenoptera musculus . B. borealis . B. physalus . Physeter macrocephalus . Parasites . Giardia . Balantidium
Introduction So far, no field technique is available to obtain blood samples from large whales without killing them (Hunt et al. 2013). Moreover, there are no facilities to accommodate whale species larger than ~8 m of length (e.g. larger than killer whales) which seriously hampers the application of most classical parasitological diagnostic methods. In addition, large whales are extremely difficult to sample in field since they spend most of their time in deep waters, coming to the surface only for brief instants. Consequently, the present knowledge on large whale diseases is still very limited when compared with most other terrestrial vertebrate taxa (Hunt et al. 2013). In fact, little is known about endo- and ecto-parasites of any free-swimming cetacean population (Kleinertz et al. 2014a; Kleinertz et al. 2014b; Raga et al. 1997). Up to date, most investigations on cetacean endo- and ectoparasites have been carried out on stranded, captive, or killed specimens, and the majority of studies on large whale
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parasitoses focused on macroscopic large-sized parasites. Analyses were generally performed post mortem by necropsy and only occasionally referred to the animal’s health or parasite pathogenicity (Andrade et al. 2001; Baylis 1932; Delyamure 1955; Geraci and St Aubin 1987; Gubanov 1951). An exception is a recently published study on freeswimming bottlenose dolphins (Tursiops aduncus) from the Red Sea, Egypt, which used non-invasive methods for their analyses by investigating fecal and vomitus samples (Kleinertz et al. 2014b). The present study aimed to identify the gastrointestinal parasite fauna of blue, fin, sei, and sperm whale populations within their natural habitats by analyzing fecal and skin biopsy samples collected from free-swimming individuals in the North Atlantic Ocean.
Material and methods Study area and sample collection Fecal and skin samples used for parasitological analyses of free-swimming whales were collected around the islands of Faial and Pico, Archipelago of the Azores (38° N 28° W), Portugal, during studies on whale migration and ecology. In total, 17 individual fecal samples were obtained in the spring and summer of 2014 from blue (n=2), fin (n=10), sei (n=1), and sperm (n=4) whales, respectively, (Fig. 1). In addition, skin samples from five sperm whales collected in 2002 and 2003 were also used to investigate the presence of parasites. Samples were obtained during focal-follows of individual whales to deploy satellite or radio transmitters and collect photo-identification data and biopsy samples. Whenever individual large whales defecated, floating feces (Fig. 1b) were collected at the water surface by using a fine nylon mesh net (pore size 400 μm) mounted on a telescoping pole (Fig. 1c). Fecal samples were immediately placed in vials containing 70 % ethanol for fixation. Upon arrival in the laboratory of the Institute of Marine Research (IMAR), University of the Azores, Portugal, the samples were stored at 4 °C until being transferred to the Institute of Parasitology (Justus Liebig University, Giessen, Germany) for parasitological analyses. Whale skin biopsies were collected from free-ranging sperm whales using a minimally invasive method according to Walton et al. (2008). Briefly, a crossbow (125-lb Barnett®) shooting stainless steel biopsy dart tips especially designed for cetaceans (Ceta-Dart®) were used for collecting skin and blubber samples from target whales (see Fig. 1d). Skin samples were stored in 90 % ethanol and kept at 4 °C until being transferred to the Institute of Parasitology (JLU Giessen, Germany). Photo-identification pictures taken from sampled individuals were used to check for duplicates, and only one sample per individual was used. Only adult animals were included in this parasitological survey.
All biological samples were obtained and handled under permits from the relevant authorities (Regional Directorate for Sea Affairs, Autonomous Region of the Azores). All field procedures followed the guidelines of the European Union on the protection of animals used for scientific purposes (Directive 2010/63/EU) and of the American Society of Mammalogists for the use of wild mammals in research (Gannon et al. 2007). Coprological analysis Coproscopical analyses were performed using the standard sodium acetate acetic acid formalin (SAF) technique as described elsewhere (Yang and Scholten 1977; Young et al. 1979). By this method, parasite eggs, cysts, sporocysts, and oocysts were diagnosed within fecal material of whales according to Kleinertz et al. (2014a, b). The parasitological identification of parasite eggs and cysts was based on morphological characteristics referring to other reports (Abril et al. 1991; Dailey 1985; Delyamure 1955; Eckert et al. 2008; Kleinertz et al. 2014b; Kumar et al. 1975; Mehlhorn and Peter 1983). Additionally, coproantigen-ELISAs (ProSpecT®, Oxoid) were performed for the detection of Cryptosporidium- and Giardia-antigens in fecal samples as described elsewhere (Kleinertz et al. 2014a, b). Molecular analyses of skin samples Skin biopsies (n = 5) were analyzed for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA by either qPCR (T. gondii) or conventional PCR (N. caninum, B. besnoiti). Genomic DNA was extracted from skin tissue using the QIAamp DNA Blood Tissue Mini Kit (QIAGEN) according to the mammalian tissue protocol. Briefly, 80 mg of skin tissue were lysed in ATL lysis buffer containing 20 mg/ml proteinase K (24 h, under permanent stirring conditions). The DNA was thereafter purified using an anion exchange column (QIAGEN) and eluted in 100 μl of distilled water. For the specific T. gondii qPCR, the following primers and probe sequences were used: the forward primer Tg529: 5′GAGAGTCGGAGAGGAAG-3′, the reverse primer: Tg529: 5′-GTCTCGTCTGGATCGCATTC-3′, and the probe: Tg529cp 5′-TCTTTCCGGCTTGGCTGCTTTTCC-3′. The qPCR was performed in a total volume of 20 μl, containing 5 μl of skin DNA sample, 400 nM of each primer, 250 nM probe, and 10 μl Hot Firepol Probe qPCR MasterMix Plus (Solis BioDyne), at 95 °C for 15 min; 40 cycles at 95 °C for 20 s and 60 °C for 1 min. Analyses of the qPCR data used the Ct method (ΔΔCt method) and reported as n-fold differences comparing to purified T. gondii tachyzoites DNA obtained from in vitro culture.
Parasitol Res Fig. 1 Feces and skin biopsy collection from free-swimming whales in the Azores archipelago, Portugal: a blue whale (Balaenoptera musculus), b floating blue whale feces (white arrows), c sperm whale feces (white arrows) collected via nylon net, and d collection of skin biopsy via a dart tip (white arrow) from a free-swimming sperm whale
A
B
C
D
Specific oligonucleotide sequences for the conventional N. caninum PCR were the following: the forward oligonucleotide Np6Plus: 5′-CTCGCCAGTCAACCTACGTCTTCT-3′ and the reverse oligonucleotide Np21Plus: 5′GGGTGTGCGTCCAATCCTGTAAC-3′. The PCR was performed in a total volume of 25 μl containing 5 μl skin DNA sample, 5 μl skin DNA sample (1:100), 1 μl Np6Plus oligonucleotides (10 μM), 1 μl Np21Plus oligonucleotides (10 μM), dNTPs 0.5 μl (10 mM), 0.5 μl Taqpolymerase (1 U/μl; PeqLab), and 14, 5 μl H2O. The following thermocyclin profiles were used: 95 °C for 5 min, 35 cycles at 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 30 s followed by a final extension step at 72 °C for 5 min and a final hold at 20 °C. PCR amplificates were visualized in GelRed- (Biotium Incorporation) stained 2 % agarose gels. For the B. besnoiti PCR, the following specific primers were used: the forward Bb primer: 5′TGACATTTAATAACAATCAACCCTT-3′ and the reverse Bb primer: 5′-GGTTTGTATTAACCAATCCGTGA-3′. The B. besnoiti PCR was performed in a total volume of 25 μl containing 5 μl skin DNA sample, 5 μl skin DNA sample (1:100), 1 μl forward Bb primer (10 μM), 1 μl reverse Bb primer (10 μM), dNTPs 0.5 μl (10 mM), 0.5 μl Taq-polymerase (1 U/μl; PeqLab), and 14, 5 μl H2O. The following PCR profiles were used: 95 °C for 15 min, 45 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s followed by a final extension step at 72 °C for 5 min and a final hold at 20 °C. PCR amplificates were visualized as described above.
Results and discussion Whale intestinal parasites Overall, more than ten taxa were detected in fecal samples of large whales revealing different protozoan (three) and metazoan (six) parasite species. Baleen whales were mainly infected with protozoans; blue whales were parasitized with one protozoan while sei and fin whales with three protozoans. Additionally, six different metazoan parasites were found in fin whales. In contrast, protozoan parasite infection was not detected in sperm whales and only shed eggs of metazoan parasite species were found. Protozoan parasites found in this study consisted of species belonging to three classes: the Entamoebidea, Trepomonadea, and Litostomatea. In total, parasitic stages of three enteric protozoan genera were exclusively found in baleen whales comprising cysts of Entamoeba, Giardia, and Balantidium. Entamoeba sp. infections occurred most frequently (64.7 %), followed by Giardia sp. (17.6 %) and Balantidium sp. (5.9 %) (Table 1). At the genus level, these parasitological findings included new host records for Entamoeba in blue, fin, and sei whales. In the case of Giardia, these findings included first new host records for sei and fin whales. Moreover, Balantidium were detected in fin whales, and, to the best of our knowledge, this is the first time this protozoan genus is reported for any cetacean species. The metazoan parasitizing large whales consisted of the classes Secernentea, Digenea, Cestoda, and Acanthocephalea. The nematode species consisted of
Parasitol Res Table 1 Prevalence (in percentage) of parasitic infections in blue (Balaenoptera musculus), sei (B. borealis), fin (B. physalus), and sperm whales (Physeter macrocephalus), technique, and sample origin Whales species
Groups
Blue-, fin-, sei-whale
Protozoa
Fin-, sei-whale Fin whale Fin-, sperm-whale
Nematoda
Fin-, sperm-whale
Parasites
(%)
Technique
Material
Entamoeba sp.
64.7
SAF
Feces
Giardia sp.
17.6
SAF/coproELISA
Feces
Balantidium sp. Ascaridida indet.
5.9 41.2
SAF SAF
Feces Feces
Strongyles indet.
11.8
SAF
Feces
Fin-whale Fin-, sperm-whale
Trematoda
Spirurids indet. Trematoda indet.
5.9 17.6
SAF SAF
Feces Feces
Fin whale Fin whale
Cestoda Acanthocephala
Diphyllobotrium sp. Acanthocephala indet.
5.9 11.8
SAF SAF
Feces Feces
specimens belonging to the families Ascarididae and Tetrameridae (Anderson 1992). Trematode species consisted of specimens belonging to the family Campulidae, the cestodes belonged to members of the family Diphyllobotriidae, and acanthocephalan species to the family Polymorphidae. Within these helminths and nematodes showed higher species richness (three species), followed by trematodes, cestodes, and acanthocephalans with only one species each. The most prevalent metazoan parasites found in baleen whales were Ascaridida indet. (41.2 %), followed by trematodes (17.6 %), strongyles (11.8 %), and acanthocephalans (11.8 %, most probably Bolbosoma). The lower prevalences were observed for Diphyllobotrium sp. and spirurids with prevalences of 5.9 %. A complete list of the parasites, prevalences, and diagnostic techniques used in the study is presented in Table 1. Illustrations of some parasitic stages found in baleen and sperm whale feces are depicted in Fig. 2. The metazoan parasite eggs were mainly found in the toothed sperm whales, which might be related to the diet of these apex predators which includes cephalopods but also bony and cartilaginous fishes (sharks and rays). All metazoan parasite species diagnosed in this study have already been described before for baleen and toothed whales of the same genera. Furthermore, no DNA of T. gondii, N. caninum, and B. besnoiti was detected in any sperm whale skin samples. The most prevalent parasites found in the present study were Entamoeba sp. The only report for an Entamoeba sp. infection that we found for a whale species was that by Raga et al. (2008) for a dead bowhead whale. In contrast to the marine ecosystem, Entamoeba sp. infections are frequently detected in several terrestrial vertebrate species including humans, apes, domestic livestock animals (Matsubayashi et al. 2015; Skappak et al. 2014; Stensvold et al. 2011), reptiles (De Cadiz et al. 2013; Eichinger 1997; Ojha et al. 2014; Vazquezdelara-Cisneros and Arroyo-Begovich 1984), and birds (Marietto-Goncalves et al. 2008). Besides several non/ low pathogenic species, such as Entamoeba coli,
Entamoeba hartmanni, Entamoeba suis, Entamoeba polecki, and Iodamoeba bütschlii (Matsubayashi et al. 2015), Entamoeba histolytica is well known as important zoonotic parasite and still represents a globally spread and leading parasitic cause of human death (Salles et al. 2007; Stanley 2003). Besides Entamoeba sp., the prevalence of Giardia sp. infections in large whales also proved rather high (17.6 %). Marine mammals are well known as final hosts of Giardia spp. (Kleinertz et al. 2014b; Mehlhorn and Piekarski 1998; Reboredo-Fernandez et al. 2014; Thompson et al. 2008). Keeping in mind that large whales may range closely to populated coastlines and that touristic attractions, such as ‘swimming-with-whales’ or ‘whale watching tours,’ are becoming more popular, whales may become infected by human excretions and/or vice versa. Giardia spp. infections can cause severe diarrhea in terrestrial mammals; nonetheless, very little is known on the pathogenesis of Giardia sp. within the marine ecosystems (Hughes-Hanks et al. 2005; Kleinertz et al. 2014b). Balantidium sp. cyst stages were also identified in large whales samples. Within the genus Balantidium, Balantidium coli is the only species of trichostome ciliates currently considered as pathogenic for mammals (Hassell et al. 2013; Ponce-Gordo et al. 2011). B. coli is a cosmopolitan zoonotic parasite of the large intestine infecting a wide range of terrestrial hosts including pigs, rodents, primates, and humans (Hassell et al. 2013; Schuster and Ramirez-Avila 2008). There is only one report on B. coli infections in Chilean sea lions (Otaria flavescens) (Hermosilla et al. 2013), but none in cetaceans, so far. Ascaridida nematodes were by far the most prevalent metazoan parasites in the current analysis, most probably corresponding to the genus Anisakis, since the closely related genera Contracaecum and Porrocaecum have rarely been described in large cetaceans (Delyamure 1955). Besides other cetaceans, large whales are known as definitive hosts of the zoonotic nematode Anisakis sp. (Cavallero et al. 2011), which
Parasitol Res Fig. 2 Protozoan and metazoan parasite stages from fecal samples of baleen and sperm whales: a Giardia sp. cyst, b Ascaridida indet. egg, c Entamoeba sp. cysts, d Trematoda indet. I egg (asterisk) and Ascaridida indet. egg (arrow), e Balantidium sp. cyst, and f Diphyllobotrium sp. egg
A
B *
C
D
E
F
has a worldwide distribution (Colon-Llavina et al. 2009; Delyamure 1955; Kleinertz et al. 2014b; Kleinertz et al. 2012; Klimpel et al. 2004; Mattiucci and Nascetti 2006; Nadler et al. 2005). Anisakis spp. reside freely within the stomach or firmly attached to the gastric mucosa (Geraci and St Aubin 1987). Mucosal damage by larvae (Young and Lowe 1969) or adults (McClelland 1980) may cause severe ulcers, perforation of the mucosa (Fiscus et al. 1962), and gastritis which may sometimes bear an allergic pathogenic component (Geraci and St Aubin 1987; Martinez-Aranguren et al. 2014; Prester 2015). Other nematode stages found in this study included strongyle- and spirurid-eggs. Metastrongylid lungworm species of cetaceans, such as Stenurus spp., and giant spirurids (up to 9 m in length, Anderson 1992), such as Crassicauda and Placentonema, could in principle shed these types of eggs
in whales. Crassicauda boopis, Crassicauda crassicauda, Crassicauda delamureana, and Placentonema gigantissima are found in the kidneys, renal veins, and intrarenal ureters of fin, blue, and sei whales in the North Atlantic Ocean (Baylis 1932; Lambertsen 1985; Skriabin 1966), while P. gigantissima additionally may occur in the uterus of sperm whales (Delyamure 1955; Gubanov 1951). So far, no data are available on the transmission mode of these parasites or the pathogenesis. Acanthocephalan-like eggs were also found in the whale feces but could not be identified at the genus level owing to a lack of adequate morphological characteristics. However, acanthocephalans have previously been described to parasitize the intestine of cetaceans (Arundel 1978; Baylis 1932; Golvan 1959). In principle, both genera, i.e., Bolbosoma and Corynosoma, were reported in marine mammals (Arundel
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1978; Baylis 1932; Golvan 1959), but in large whales mainly Bolbosoma spp. (Bolbosoma balaenae, Bolbosoma brevicolle, Bolbosoma nipponicum, Bolbosoma turbinella, and Bolbosoma capitatum in baleen whales and Bolbosoma physeteris in sperm whales) were detected so far (Baylis 1932; Delyamure 1955). Only one species of Corynosoma (Corynosoma curilensis) is known to reside in sperm whales (Delyamure 1955; Gubanov 1951). High parasitic burdens of acanthocephalans might be associated with hemorrhagic enteritis and severe peritonitis due to intestinal wall perforation (Geraci and St Aubin 1987). Furthermore, the genus Bolbosoma has been reported to bear zoonotic potential in Asia (Tada et al. 1983). Moreover, at least three morphologically different trematodes eggs were identified in this study. Unfortunately, due to the lack of literature on whale trematode egg identification based on morphological criteria, we were not able to determine these stages to genus level. Large whale trematodes include members of the genus Ogmogaster [Ogmogaster plicatus (Baylis 1932) and Ogmogaster antarcticus (Delyamure 1955)], which are mainly found in the stomach and small intestine. In the pancreas and hepatobiliary system of large whales, members of the genera Zalophotrema and Lecithodesmus [Zalophotrema curilensis, Lecithodesmus goliath, and Lecithodesmus spinosus (Delyamure 1955)] induce pathological alterations, such as parenchymal tissue necrosis, hepatitis/pancreatitis, and hyperplasia of bile ducts (Delyamure 1955; Fleischman and Squire 1970; Geraci and St Aubin 1987; Gubanov 1951; Woodard et al. 1969). Other digeneans with major health implications are the members of the genus Nasitrema which reside the sinus nasalis and sinus frontalis as adult stages (Kleinertz et al. 2014b; Ridgway 1972). Severe pathological findings of cetacean nasitremosis include extensive cerebral necrosis and meningoencephalitis. Cerebral nasitremosis was suggested as a cause of mortality of small cetaceans and to be associated with strandings (Dailey and Walker 1978; Kleinertz et al. 2014b; Raga et al. 2008). As also common for other marine mammals, we diagnosed Diphyllobotrium sp. infections in large whales but at a low prevalence. Large whales are known as final hosts for different enteric pseudophyllidean cestodes, such as Diphyllobothrium, Hexagonoporus, Priapocephalus, Tetrabothrium, and Diplogonoporus (Baylis 1932; Delyamure 1955; Raga et al. 2008). Infections with adult pseudophyllideans generally are considered as innocuous (Arundel 1978). Nonetheless, high tapeworm burdens may obstruct the intestinal lumen or result in debilitation and even death of the host (Geraci and St Aubin 1987; Kleinertz et al. 2014b). Overall, the current data confirm that large whales are exposed to anthropogenic parasites within the marine environment, as previously demonstrated (Davidson et al. 2012; Doney et al. 2012; Kleinertz et al. 2014b; Pompa et al. 2011; Reboredo-Fernandez et al. 2014; Van Bressem et al. 2009),
emphasizing the relevance of constant surveillance of marine mammals to prevent parasite transmission to humans (Hughes-Hanks et al. 2005; Kleinertz et al. 2014b; Reboredo-Fernandez et al. 2014; Van Bressem et al. 2009). In particular, water-borne zoonotic protozoans, such as Giardia, Entamoeba, and Balantidium, might have been acquired by the whales in coastal waters contaminated by sewage, agricultural run-off, and medical waste as proposed elsewhere (Bogomolni et al. 2008; Hermosilla et al. 2013; Hughes-Hanks et al. 2005; Kleinertz et al. 2014b; ReboredoFernandez et al. 2014). Moreover, cyst-forming coccidian parasites, such as T. gondii, Sarcocystis sp., and N. caninum, which typically infect terrestrial animals, were identified as neozoan parasites within the marine environment (Akao 1970; Fujii et al. 2007), and have emerged as detrimental pathogens for several pinnipeds (Cabezón et al. 2011; Reichel et al. 2015), dolphins (Dubey et al. 2008), sea otters (Conrad et al. 2005; Miller et al. 2008), and whales (Mazzariol et al. 2012). New insights into these diseases in large whales and other marine vertebrates will contribute to a better understanding of human-related impacts on marine ecosystem health and to the development of proper conservation tools. Moreover, the here analyzed whale species are well-known to swim from tropical into polar waters, crossing vast oceanic areas, and thus suggesting their potential role in the spreading of anthropozoonotic parasites or other pathogens throughout the marine environment, including remote areas with little or absent human settlements. Acknowledgements We are deeply thankful to those who contributed to the whale images included in this work: Claudia Oliveira (Fig. 1a) and N. Liebsich (Fig. 1b, c), and Christine Henrich for her molecular work. We further acknowledge funds and support from the Portuguese Fundação para a Ciência e a Tecnologia (FCT), Fundo Regional da Ciência e Tecnologia (FRCT), through research projects TRACEPTDC/MAR/74071/2006 and MAPCET-M2.1.2/F/012/2011 [FEDER, the Competitiveness Factors Operational (COMPETE), QREN European Social Fund, and Proconvergencia Açores/EU Program]. We furthermore acknowledge funds provided by FCT to MARE and by the FRCT— Government of the Azores pluri-annual funding. RP is supported by a research grant from the Azores Regional Fund for Science and Technology (M3.1.5/F/115/2012). MAS is supported by FCT through a Program Investigator FCT fellowship (IF/00943/2013).
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