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Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from São Paulo Zoo, Brazil
Veterinary Parasitology 173 (2010) 123–127
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from São Paulo Zoo, Brazil Marina Galvão Bueno a , Rodrigo Pinho Gomez Lopez a , Regiane Maria Tironi de Menezes c , Maria de Jesus Costa-Nascimento b , Giselle Fernandes Maciel de Castro Lima b , Radamés Abrantes de Sousa Araújo b , Fernanda Junqueira Vaz Guida a , Karin Kirchgatter b,∗ a
Fundac¸ão Parque Zoológico de São Paulo, São Paulo, Brazil Núcleo de Estudos em Malária, Superintendência de Controle de Endemias/Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil c Laboratório de Entomologia Médica, Superintendência de Controle de Endemias, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 27 May 2010 Accepted 21 June 2010 Keywords: Plasmodium relictum Spheniscus magellanicus Brazil Penguins Diagnosis
a b s t r a c t This study reports avian malaria caused by Plasmodium relictum in Magellanic Penguins (Spheniscus magellanicus) from São Paulo Zoo. The disease was highly infective among the birds and was clinically characterized by its acute course and high mortality. The penguins of São Paulo Zoo were housed for at least 2 years without malaria; however, they had always been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were freed at night for a short period. São Paulo Zoo is located in one of the last forest remnants of the city, an area of original Atlantic forest. In the winter, the space destined for Zoo birds is shared with migratory species. Hence the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes of the genera Aedes and Culex, common vectors in the Atlantic forest. In this study, one Culex (Cux.) sp. was found, infected with P. relictum. There are diverse problems in housing distinct species of animals in captivity, principally when occupying the same enclosure, since it facilitates the transmission of diseases with indirect cycles, as is the case of Plasmodium spp., because certain species that cause discrete infections in some bird species can become a serious danger for others, especially penguins, which do not possess natural resistance. Thus, serious implications exist for periodically testing and administrating malaria therapy in captive penguins potentially exposed to mosquitoes during the night period, as well as other captive birds from São Paulo Zoo. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The Magellanic Penguin, Spheniscus magellanicus, is a South American penguin that breeds in coastal Argentina, Chile and the Falkland Islands. The birds, especially the young, move between the Argentinean and Brazilian coast
∗ Corresponding author. Tel.: +55 11 3081 8039; fax: +55 11 3081 8039. E-mail address: [email protected] (K. Kirchgatter). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.06.026
(Stokes and Boersma, 1999). Along this route, many of these birds are exposed to adverse environmental conditions and some of them are found on Brazilian beaches with hypothermia, wounds, oil on the skin and dehydration and are forwarded for rehabilitation (García-Borboroglu et al., 2006). During the period that the penguins are in captivity, diseases that cause death, such as aspergillosis, pododermatitis and malaria, affect many of them. Malaria in penguins is caused by Plasmodium relictum and P. elongatum. It is the most important cause of mortality in outdoor
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zoological penguin exhibits, causing over 50% mortality in untreated juvenile and adult penguins when first exposed to the parasite (Cranfield et al., 1991). Malaria in penguins was first reported in 1926 in a King Penguin at the London Zoo (Scott, 1927) and the author identified the parasite as P. praecox. Rodhain (1937) described accounts of P. relictum infections in the Blackfoot Penguin, S. demersus, in the Antwerp Zoological Gardens. Since then, P. relictum has been described in many zoos around the world, mainly in African (S. demersus) (Stoskopf and Beier, 1979; Cranfield et al., 1994; Graczyk et al., 1994; McConkey et al., 1996; Lombard et al., 1999; Brossy et al., 1999) and Humboldt Penguins (S. humboldti) (Bak et al., 1984). The first record of P. relictum in captive Magellanic Penguins occurred in 1986 at the Blank Park Zoo in Des Moines, Iowa, USA (Fix et al., 1988). Here we report avian malaria caused by P. relictum in Magellanic Penguins (S. magellanicus) from São Paulo Zoo and the objective of this paper was to investigate the nature of this disease, in order to answer some of the questions relating to mosquito fauna and parasite transmission. 2. Cases history São Paulo Zoo had five Spheniscus magellanicus. Three females (numbers 26153, 26157 and 26159) were obtained from the Tamar Project of Ubatuba, northern coast of São Paulo State, in December 2000. Two females (numbers 29092 and 29093) came from Guarujá Aquarium, southern coast of São Paulo State, in September 2005. In February 2007, penguin 26153 died suddenly and was diagnosed with enteritis. All the thin blood smears collected were negative for malaria. Some days later, penguin 29092 presented dyspnea, apathy, bilateral ocular congestion and constant protrusion of the third eyelid. Symptomatic treatment was administered with inhalation, injectable aminophylline, hydrocortisone and oxygen therapy. After intensive treatment, with no response, euthanasia was performed due to severe and irreversible respiratory distress. Plasmodium sp. (recently identified as P. relictum by PCR and sequencing) was verified in thin blood smears collected during necropsy. The other three penguins (26157, 29093 and 26159) were also verified as positive for malaria and were treated orally with chloroquine diphosphate (10 mg/kg) at 0, 6, 12, 18 and 24 h. The treatment was completed with chloroquine diphosphate (5 mg/kg) and primaquine phosphate (1 mg/kg), orally, and repeated every 24 h for 3 days. At the end of the treatment, penguin 29093 presented the same clinical signals and convulsions and euthanasia was performed. Its blood was Plasmodium positive by PCR. One week after the end of treatment, a new blood sample was collected from the two survivors (26157 and 26159) and PCR was negative. 3. Materials and methods 3.1. Penguin sampling and thin blood smears Cardiac blood was collected from the three penguins that died (26153, 29092 and 29093) during necropsy examination for the preparation of thin blood smears and/or freezing for DNA extraction. Next, a clinical examination of
all live birds was conducted, while under physical restraint. Venous blood was collected from the median metatarsal vein for hemogram, biochemistry investigation, thin blood smears and DNA extraction. Thin blood smears were fixed by methanol, stained by Giemsa and examined under light microscopy (1000×) in 200 microscopic fields, searching for parasites. 3.2. Mosquito sampling and handling Mosquitoes were collected in March 2007 at one site within the perimeter of the penguins’ habitat and two sites within the perimeter of the Cape Barren Geese’s habitat (Cereopsis novaehollandiae), São Paulo Zoo, Brazil (for the geographical distribution of the mosquito collection sites in São Paulo Zoo, see Supplemental file 1). Initially, the mosquitoes were collected using a Nasci aspirator (Nasci, 1981) from 4 to 5 pm, in the lake in front of the Cape Barren Geese’s habitat. Then from 5 to 8 pm, a Shannon trap (Shannon, 1939) was used in the same place. During this period, four UV-baited Mini CDC light traps (Sudia and Chamberlain, 1962) were set up, two within the penguins’ habitat perimeter and two within the Cape Barren Geese’s habitat perimeter. Adult mosquitoes were killed with chloroform steam and transported to the laboratory, where they were identified according to species on chill tables with a stereomicroscope using descriptive keys appropriate for the collection sites (Forattini, 2002). All mosquitoes with fresh or visible blood remnants were individually placed in 1.5 ml microcentrifuge tubes, sealed with parafilm, labeled according to species and capture site and stored at −20 ◦ C. 3.3. Penguin genomic DNA (gDNA) extraction, PCR amplification of Plasmodium cytb fragment and Plasmodium SSU fragment, cloning, sequencing and data analysis of sequences After thawing, the samples were centrifuged and 100–150 l of red blood pellet were used for the DNA extraction procedure. First, initial lyses were performed with 1% saponin. Next, the pellets were washed twice in ultrapure water and submitted to the extraction protocol with the GFXTM Genomic Blood DNA Purification Kit (Amersham Biosciences, GE Healthcare), following the manufacturer’s instructions. The genomic DNA was eluted in 100 l of ultrapure water and stored at −20 ◦ C. A fragment of ∼1.1 kb (approximately 92% of the gene) from the mitochondrial cytochrome b gene (cytb) was amplified using a nested PCR, taking standard precautions to prevent cross-contamination of samples. The PCR reactions were conducted as previously described (Perkins and Schall, 2002) using primers DW2 and DW4 and 5 l of genomic DNA in the first reaction and 1 l aliquot of this product was used as a template for a nested reaction with primers DW1 and DW6. All these primers are specific to malarial parasites and do not amplify host DNA or that of other Apicomplexa. The PCR products of ∼1 kb were purified from agarose gels by PureLinkTM Quick Gel Extraction Kit (Invitrogen), cloned in pTZ57R/T plasmid (Fermentas) and sequenced by Big Dye Terminator v3.0 Cycle Sequenc-
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Fig. 1. Photomicrographs of parasites visualized from thin blood smears obtained from penguins 26157 (A), 29092 (B) and 29093 (C). Characteristic of P. relictum, the gametocytes appear round and take up a large portion of the cell, pushing the red blood cell nucleus to one side.
ing Kit in ABI Genetic Analyzer (ABI, USA), using plasmid oligonucleotides (M13 forward and M13 reverse) and internal primers DW3 and DW8 (Perkins and Schall, 2002). Sequences obtained were aligned with sequences from the GenBank using BLASTN (Basic Local Alignment Search Tool) available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi (Altschul et al., 1997). PCR amplification of Plasmodium SSU fragments was performed according to a nested genus-specific protocol (Dos Santos et al., 2009), which uses oligonucleotides in conserved sequences in the small subunit (SSU) ribosomal RNA of all Plasmodium organisms. PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide. The PCR fragment was purified from agarose gels by PureLinkTM Quick Gel Extraction Kit (Invitrogen), cloned in pGEM-T Easy Vector (Promega) and sequenced by Big Dye Terminator v3.0 Cycle Sequencing Kit in ABI Genetic Analyzer (ABI, USA), using PCR oligonucleotides (rPLU3 and rPLU4). The sequences obtained were aligned with sequences from the GenBank using BLASTN available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
3.5. gDNA extraction from blood-fed mosquitoes and blood meal identification
3.4. Mosquito gDNA extraction and PCR amplification of Plasmodium SSU fragment
This study reports avian malaria caused by P. relictum in Magellanic Penguins (S. magellanicus) from São Paulo Zoo. To our knowledge, this is the first report of this parasite in Brazil. The disease was highly infective among the birds and clinically characterized by its acute course and high mortality. Thin blood smears obtained from four penguins (26157, 26159, 29092 and 29093) were positive for
DNA from each mosquito was extracted as previously described (Cerutti et al., 2007). The Plasmodium SSU fragment was amplified, purified, cloned and analysed as described above for penguin blood samples.
Genomic DNA of blood-fed mosquitoes was obtained using PureLinkTM Genomic DNA Purification Kit (Invitrogen). PCR was used to amplify host DNA from the mosquito blood meal using primers L14841 and H15149 (Kocher et al., 1989) or B1 and B6 (Pereira and Baker, 2004), designed to amplify, respectively, fragments with ∼300 bp and ∼1 kb of the mitochondrial cytb gene from a wide array of animals, including mammals, birds, amphibians, reptiles and fishes. Amplified fragments were purified from gels and sequenced directly using the corresponding flanking primers. The sequences were identified by comparison with the GenBank DNA sequence database (National Center for Biotechnology Information available online: www.ncbi.nlm.nih.gov/blast/Blast.cgi). Positive identification and host species assignment were determined when exact or nearly exact matches (>95%) were obtained. 4. Results and discussion
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malaria (with 0.4, 0.4, 6.8 and 11% of parasitemia, respectively). Parasites visualized from penguins 26157, 29092 and 29093 are shown in Fig. 1. Young trophozoites are observed as round. Old trophozoites are about the size of the erythrocyte nucleus, beginning to displace the latter. Mature schizonts are large, exceeding the size of the host cell nucleus and also commonly displace the erythrocyte nucleus. A clear vacuole is frequently present and the nuclei are observed in the periphery. Gametocytes appear round and take up a large portion of the cell, pushing the red blood cell nucleus to one side. The hemogram and biochemistry investigation results were mostly within normal range. Differences were observed in the hematocrit (37 ± 5.3%) and hemoglobin (12 ± 2.4 g/dl) values, which were below the reference range (39–55.4% and 12.3–18.7 g/dl, respectively), and total protein (8.3 ± 0.8 g/dl) values, which were above the reference range (4.8–6.8 g/dl). There were also differences in heterophil (13,780 ± 6380/mm3 ) numbers (reference range 2515–9215/mm3 ). Reference values were obtained from ISIS 2002 (www.isis.org). The PCR reactions were performed with 8 samples obtained from 5 penguins (26153 after death, 26157 before and after treatment, 26159 before and after treatment, 29092 after death and 29093 before and after death). It was possible to amplify a fragment of ∼1.1 kb from the cytb gene in samples 26159, 29092 after death and 29093 after death. The other five samples presented negative results. The PCR products from samples 29092 and 29093 were purified from agarose gels, cloned and sequenced. The sequences obtained were aligned with sequences from the GenBank using BLASTN. The 29092 sequence (GenBank HM031936) presented 98% identity with P. relictum obtained from Zenaida macroura in Nebraska, USA (GenBank AY733089), while the 29093 sequence (GenBank HM031937) presented 99% identity with P. relictum obtained from Spheniscus demersus (GenBank AY733088) from Baltimore Zoo, Maryland, USA. Since the parasite detected in the penguins was confirmed as P. relictum by the cytb sequences and considering that the SSU marker used here (rPLU3/rPLU4) is on a different gene region than that used by other authors and available in GenBank (Jarvi et al., 2002), we decided to amplify, clone and sequence this fragment from two penguins (29092 and 29093). Five different sequences were obtained (three in sample 29092 and two in sample 29093) and were deposited in the GenBank (GenBank HM242418–HM242422). P. relictum described here was highly similar to parasites identified in another species of penguin and in a species of dove, both detected in the USA, which is consistent with the fact that this kind of Plasmodium has been reported from a very broad host (over 300 bird ¯ species) and geographical range (Valkiunas, 2005). In Brazil, two studies were performed to detect avian malaria in birds from fragments of Atlantic Forest in Minas Gerais and in these works, 39.6% and 36% of birds were verified as positive for Plasmodium spp. (Ribeiro et al., 2005; Belo et al., 2009). Therefore the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes
Table 1 Distribution of species or groups of female mosquitoes collected with Shannon trap, Centers for Disease Control (CDC) miniature light traps and Nasci aspirator in São Paulo Zoo. Shannon Mini trap CDC trap Anopheles (Nys.) evansae 06 Coquillettidia (Rhy.) chrysonotum/albifera 02 Culex (Cux.) habilitator/pseudojanthinosoma 06 Culex (Cux.) restuans/declarator Culex (Cux.) spp. 03 Culex (Cux.) Coronator group 18 Culex (Mel.) Melanoconion section 67 (02a ) Culex (Cux.) ameliae Mansonia (Man.) indubitans 41 Aedes (Och.) crinifer 01 Aedes (Och.) scapularis 07 Aedes (Sth.) albopictus 11 (03a ) Uranotaenia (Ura.) lowi Total 162 (05a ) a
Nasci aspirator
04
07 (04a )
04 01
06
06 01 02 03 01 21
14 (04a )
With blood meal.
of the genera Aedes and Culex and rarely by Anopheles (Bruce-Chwatt, 1985), which are all common vectors in the Neotropics, particularly in the Atlantic forest or urban areas of Brazil (Forattini, 2002). In this study, a total of 208 adult mosquitoes (188 females and 20 males) without visible blood meal were collected. The species or groups of females obtained in each capture method are shown in Table 1. PCR amplification was performed for these 188 females. Only one individual was positive for Plasmodium SSU fragment. This specimen, collected from a Nasci aspirator, was found damaged and thus identified as Culex (Cux.) sp. The amplified fragment was cloned and sequenced and two sequences were obtained (GenBank HM242416 and HM242417) that presented identities of 97% and 98% with sequences obtained from penguin 29092 (HM242420 and HM242419, respectively), confirming the mosquito infection with P. relictum (Table 2, Supplemental file 2). Nine mosquitoes with apparent blood meals were collected (Table 1): 2 Culex (Melanoconion) Melanoconion section; 3 Aedes albopictus using Shannon trap; and 4 Anopheles evansae using a Nasci aspirator. Using this methodology for the first time in Brazil, blood meal sources were successfully identified by DNA sequencing of the cytb fragment in 5 out of 9 mosquitoes (two Cx. (Mel.) Melanoconion section and three Ae. albopictus collected using the Shannon trap). One species was identified as avian (Nycticorax nycticorax) and one species as mammalian (Hippopotamus amphibius) hosts for Culex mosquitoes. N. nycticorax, popularly known as Socó, is an opportunistic bird that does not belong to the Zoo, but lives in the neighborhood and feeds on the leftover portions of food of the captive animals, mainly marine lions and penguins. All the blood meals identified from Ae. albopictus were human-derived. For all the An. evansae collected with apparent blood meals (44.4% of the total number of mosquitoes examined), it was not possible identify the blood meal source, since visible amplification products were not obtained. Using other methodologies, some authors were unable to identify the blood meal source in 37.5% of An. evansae tested (Forattini et al., 1987). The blood meal begins to be digested soon after ingestion and as a
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consequence, the DNA is degraded; thus, the longer the post-ingestion time, the lower the possibility of DNA detection (Oshaghi et al., 2006). Moreover, it is possible that the females had fed on sugar substances and not blood. Mosquitoes of both sexes seek out energy substances, generally carbohydrates, such as the nectar of flowers, dew and fruits, to fulfill functions that involve expending energy, including flight and dispersion (Foster, 1995). Although the study of avian malaria transmission by mosquitoes was not entirely conclusive, since the blood meal sources identified did not match with the penguins, the study showed that Culex mosquitoes could be the vectors of avian malaria in the park given the fact that the blood of one species of bird was successfully identified in these mosquitoes. Penguins from São Paulo Zoo were maintained in this location for at least 2 years without presenting malaria; however, they always had been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were allowed to move freely at night for a short period. São Paulo Zoo is located in one of the last forest remnants of the city, an area of 824.529 m2 of original Atlantic forest that contains springs of the historical stream of the Ipiranga, which rise in the State Park and whose waters form three lakes. In the winter period and also in the beginning of the summer, the space destined for Zoo birds is shared by other migratory species, as well as opportunistic species that live freely in the area of the park. In light of the evidence presented here, captive penguins must be managed to avoid contact with mosquitoes at night. These birds must also be periodically tested for malaria and should receive malaria therapy in cases where it is impossible to avoid contact with the mosquitoes. Acknowledgments The authors are grateful to Robson de Almeida Zampaulo and Caroline Perez Girardelli for the collection and identification of the mosquitoes. They would also like to thank Almir Robson Ferreira (Instituto de Medicina Tropical de São Paulo) for drawing the map in Supplemental File 1 and the staff of the São Paulo Zoological Park Foundation (Fundac¸ão Parque Zoológico de São Paulo) for their support during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetpar. 2010.06.026. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bak, U.B., Park, J.C., Lim, Y.J., 1984. An outbreak of malaria in penguins at the Farm-land Zoo. Kisaengchunghak Chapchi 22, 267–272. Belo, N.O., Passos, L.F., Júnior, L.M., Goulart, C.E., Sherlock, T.M., Braga, E.M., 2009. Avian malaria in captive psittacine birds: detection by microscopy and 18S rRNA gene amplification. Prev. Vet. Med. 88, 220–224.
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Brossy, J.J., Plös, A.L., Blackbeard, J.M., Kline, A., 1999. Diseases acquired by captive penguins; what happens when they are released into the wild? Marine Ornithol. 27, 185–186. Bruce-Chwatt, L.J., 1985. Essential Malariology, 2nd ed. William Heinemann Medical Books, London. Cerutti, C., Boulos, M., Coutinho, A.F., Hatab, M.C., Falqueto, A., Rezende, H.R., Duarte, A.M., Collins, W., Malafronte, R.S., 2007. Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil. Malar. J. 6, 33. Cranfield, M.R., Beall, F.B., Skjoldager, M.T., Ialeggio, D.M., 1991. Avian malaria. Spheniscus Penguin Newslett. 4, 5–7. Cranfield, M.R., Graczyk, T.K., Beall, F.B., Ialeggio, D.M., Shaw, M.L., Skjoldager, M.L., 1994. Subclinical avian malaria infections in African black-footed penguins (Spheniscus demersus) and induction of parasite recrudescence. J. Wildl. Dis. 30, 372–376. Dos Santos, L.C., Curotto, S.M., de Moraes, W., Cubas, Z.S., CostaNascimento, M.J., Filho, I.R., Biondo, A.W., Kirchgatter, K., 2009. Detection of Plasmodium sp. in capybara. Vet. Parasitol. 163, 148–151. Fix, A.S., Waterhouse, C., Greiner, E.C., Stoskopf, M.K., 1988. Plasmodium relictum as a cause of avian malaria in wild-caught Magellanic penguins (Spheniscus magellanicus). J. Wildl. Dis. 24, 610–619. Forattini, O.P., Gomes, A.C., Natal, D., Kakitani, I., Marucci, D., 1987. Food preferences of Culicidae mosquitoes in the Ribeira valley, São Paulo, Brazil. Rev. Saude Publica 21, 171–187. Forattini, O.P., 2002. Culicidologia Médica, vol. 2: Identificac¸ão, Biologia, Epidemiologia. Edusp, São Paulo, 860 pp. Foster, W.A., 1995. Mosquito sugar feeding and reproductive energetics. Annu. Rev. Entomol. 40, 443–474. García-Borboroglu, P., Boersma, P.D., Ruoppolo, V., Reyes, L., Rebstock, G.A., Griot, K., Heredia, S.R., Adornes, A.C., Silva, R.P., 2006. Chronic oil pollution harms Magellanic penguins in the outhwest Atlantic. Mar. Pollut. Bull. 52, 193–198. Graczyk, T.K., Shaw, M.L., Cranfield, M.R., Beall, F.B., 1994. Hematologic characteristics of avian malaria cases in African black-footed penguins (Spheniscus demersus) during the first outdoor exposure season. J. Parasitol. 80, 302–308. Jarvi, S.I., Schultz, J.J., Atkinson, C.T., 2002. PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines. J. Parasitol. 88, 153–158. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Pääbo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. U.S.A. 86, 6196–6200. Lombard, E., Brossy, J.J., Blackbeard, J., 1999. Malaria in an African penguin. Br. J. Haematol. 106, 577. McConkey, G.A., Li, J., Rogers, M.J., Seeley, D.C., Graczyk, T.K., Cranfield, M.R., McCutchan, T.F., 1996. Parasite diversity in an endemic region for avian malaria and identification of a parasite causing penguin mortality. J. Eukaryot. Microbiol. 43, 393–399. Nasci, R.S., 1981. A lightweight battery-powered aspirator for collecting resting mosquitoes in the field. Mosq. News 41, 808–811. Oshaghi, M.A., Chavshin, A.R., Vatandoost, H., Yaaghoobi, F., Mohtarami, F., Noorjah, N., 2006. Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes. Exp. Parasitol. 112, 232–236. Pereira, S.L., Baker, A.J., 2004. Vicariant speciation of curassows (Aves, Cracidae): a hypothesis based on mitochondrial DNA phylogeny. Auk 121, 682–694. Perkins, S.L., Schall, J.J., 2002. A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences. J. Parasitol. 88, 972–978. Ribeiro, S.F., Sebaio, F., Branquinho, F.C., Marini, M.A., Vago, A.R., Braga, E.M., 2005. Avian malaria in Brazilian passerine birds: parasitism detected by nested PCR using DNA from stained blood smears. Parasitology 130, 261–267. Rodhain, J., 1937. Une infection à Plasmodium chez Spheniscus demersus (Manchot du cap). Ann. Parasitol. Hum. Comp. 15, 235–238. Scott, H.H., 1927. Report on the deaths occurring in the Society’s gardens during the year 1926. Proc. Zool. Soc. Lond. 1927, 173–198. Shannon, R.C., 1939. Methods for collecting and feeding mosquitoes in jungle yellow fever studies. Am. J. Trop. Med. s1–19, 131–140. Stokes, D.L., Boersma, P.D., 1999. Where breeding Magellanic Penguins Spheniscus Magellanicus Forage: satellite telemetry results and their implications for penguin conservation. Marine Ornithol. 27, 59–65. Stoskopf, M.K., Beier, J., 1979. Avian malaria in African black-footed penguins. J. Am. Vet. Med. Assoc. 175, 944–947. Sudia, W.D., Chamberlain, R.W., 1962. Battery-operated light trap, an improved model. Mosq. News 22, 126–129. ¯ Valkiunas, G., 2005. Avian Malaria Parasites and Other Haemosporidia. CRC Press, Boca Raton, FL, USA.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from São Paulo Zoo, Brazil Marina Galvão Bueno a , Rodrigo Pinho Gomez Lopez a , Regiane Maria Tironi de Menezes c , Maria de Jesus Costa-Nascimento b , Giselle Fernandes Maciel de Castro Lima b , Radamés Abrantes de Sousa Araújo b , Fernanda Junqueira Vaz Guida a , Karin Kirchgatter b,∗ a
Fundac¸ão Parque Zoológico de São Paulo, São Paulo, Brazil Núcleo de Estudos em Malária, Superintendência de Controle de Endemias/Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil c Laboratório de Entomologia Médica, Superintendência de Controle de Endemias, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 27 May 2010 Accepted 21 June 2010 Keywords: Plasmodium relictum Spheniscus magellanicus Brazil Penguins Diagnosis
a b s t r a c t This study reports avian malaria caused by Plasmodium relictum in Magellanic Penguins (Spheniscus magellanicus) from São Paulo Zoo. The disease was highly infective among the birds and was clinically characterized by its acute course and high mortality. The penguins of São Paulo Zoo were housed for at least 2 years without malaria; however, they had always been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were freed at night for a short period. São Paulo Zoo is located in one of the last forest remnants of the city, an area of original Atlantic forest. In the winter, the space destined for Zoo birds is shared with migratory species. Hence the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes of the genera Aedes and Culex, common vectors in the Atlantic forest. In this study, one Culex (Cux.) sp. was found, infected with P. relictum. There are diverse problems in housing distinct species of animals in captivity, principally when occupying the same enclosure, since it facilitates the transmission of diseases with indirect cycles, as is the case of Plasmodium spp., because certain species that cause discrete infections in some bird species can become a serious danger for others, especially penguins, which do not possess natural resistance. Thus, serious implications exist for periodically testing and administrating malaria therapy in captive penguins potentially exposed to mosquitoes during the night period, as well as other captive birds from São Paulo Zoo. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The Magellanic Penguin, Spheniscus magellanicus, is a South American penguin that breeds in coastal Argentina, Chile and the Falkland Islands. The birds, especially the young, move between the Argentinean and Brazilian coast
∗ Corresponding author. Tel.: +55 11 3081 8039; fax: +55 11 3081 8039. E-mail address: [email protected] (K. Kirchgatter). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.06.026
(Stokes and Boersma, 1999). Along this route, many of these birds are exposed to adverse environmental conditions and some of them are found on Brazilian beaches with hypothermia, wounds, oil on the skin and dehydration and are forwarded for rehabilitation (García-Borboroglu et al., 2006). During the period that the penguins are in captivity, diseases that cause death, such as aspergillosis, pododermatitis and malaria, affect many of them. Malaria in penguins is caused by Plasmodium relictum and P. elongatum. It is the most important cause of mortality in outdoor
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zoological penguin exhibits, causing over 50% mortality in untreated juvenile and adult penguins when first exposed to the parasite (Cranfield et al., 1991). Malaria in penguins was first reported in 1926 in a King Penguin at the London Zoo (Scott, 1927) and the author identified the parasite as P. praecox. Rodhain (1937) described accounts of P. relictum infections in the Blackfoot Penguin, S. demersus, in the Antwerp Zoological Gardens. Since then, P. relictum has been described in many zoos around the world, mainly in African (S. demersus) (Stoskopf and Beier, 1979; Cranfield et al., 1994; Graczyk et al., 1994; McConkey et al., 1996; Lombard et al., 1999; Brossy et al., 1999) and Humboldt Penguins (S. humboldti) (Bak et al., 1984). The first record of P. relictum in captive Magellanic Penguins occurred in 1986 at the Blank Park Zoo in Des Moines, Iowa, USA (Fix et al., 1988). Here we report avian malaria caused by P. relictum in Magellanic Penguins (S. magellanicus) from São Paulo Zoo and the objective of this paper was to investigate the nature of this disease, in order to answer some of the questions relating to mosquito fauna and parasite transmission. 2. Cases history São Paulo Zoo had five Spheniscus magellanicus. Three females (numbers 26153, 26157 and 26159) were obtained from the Tamar Project of Ubatuba, northern coast of São Paulo State, in December 2000. Two females (numbers 29092 and 29093) came from Guarujá Aquarium, southern coast of São Paulo State, in September 2005. In February 2007, penguin 26153 died suddenly and was diagnosed with enteritis. All the thin blood smears collected were negative for malaria. Some days later, penguin 29092 presented dyspnea, apathy, bilateral ocular congestion and constant protrusion of the third eyelid. Symptomatic treatment was administered with inhalation, injectable aminophylline, hydrocortisone and oxygen therapy. After intensive treatment, with no response, euthanasia was performed due to severe and irreversible respiratory distress. Plasmodium sp. (recently identified as P. relictum by PCR and sequencing) was verified in thin blood smears collected during necropsy. The other three penguins (26157, 29093 and 26159) were also verified as positive for malaria and were treated orally with chloroquine diphosphate (10 mg/kg) at 0, 6, 12, 18 and 24 h. The treatment was completed with chloroquine diphosphate (5 mg/kg) and primaquine phosphate (1 mg/kg), orally, and repeated every 24 h for 3 days. At the end of the treatment, penguin 29093 presented the same clinical signals and convulsions and euthanasia was performed. Its blood was Plasmodium positive by PCR. One week after the end of treatment, a new blood sample was collected from the two survivors (26157 and 26159) and PCR was negative. 3. Materials and methods 3.1. Penguin sampling and thin blood smears Cardiac blood was collected from the three penguins that died (26153, 29092 and 29093) during necropsy examination for the preparation of thin blood smears and/or freezing for DNA extraction. Next, a clinical examination of
all live birds was conducted, while under physical restraint. Venous blood was collected from the median metatarsal vein for hemogram, biochemistry investigation, thin blood smears and DNA extraction. Thin blood smears were fixed by methanol, stained by Giemsa and examined under light microscopy (1000×) in 200 microscopic fields, searching for parasites. 3.2. Mosquito sampling and handling Mosquitoes were collected in March 2007 at one site within the perimeter of the penguins’ habitat and two sites within the perimeter of the Cape Barren Geese’s habitat (Cereopsis novaehollandiae), São Paulo Zoo, Brazil (for the geographical distribution of the mosquito collection sites in São Paulo Zoo, see Supplemental file 1). Initially, the mosquitoes were collected using a Nasci aspirator (Nasci, 1981) from 4 to 5 pm, in the lake in front of the Cape Barren Geese’s habitat. Then from 5 to 8 pm, a Shannon trap (Shannon, 1939) was used in the same place. During this period, four UV-baited Mini CDC light traps (Sudia and Chamberlain, 1962) were set up, two within the penguins’ habitat perimeter and two within the Cape Barren Geese’s habitat perimeter. Adult mosquitoes were killed with chloroform steam and transported to the laboratory, where they were identified according to species on chill tables with a stereomicroscope using descriptive keys appropriate for the collection sites (Forattini, 2002). All mosquitoes with fresh or visible blood remnants were individually placed in 1.5 ml microcentrifuge tubes, sealed with parafilm, labeled according to species and capture site and stored at −20 ◦ C. 3.3. Penguin genomic DNA (gDNA) extraction, PCR amplification of Plasmodium cytb fragment and Plasmodium SSU fragment, cloning, sequencing and data analysis of sequences After thawing, the samples were centrifuged and 100–150 l of red blood pellet were used for the DNA extraction procedure. First, initial lyses were performed with 1% saponin. Next, the pellets were washed twice in ultrapure water and submitted to the extraction protocol with the GFXTM Genomic Blood DNA Purification Kit (Amersham Biosciences, GE Healthcare), following the manufacturer’s instructions. The genomic DNA was eluted in 100 l of ultrapure water and stored at −20 ◦ C. A fragment of ∼1.1 kb (approximately 92% of the gene) from the mitochondrial cytochrome b gene (cytb) was amplified using a nested PCR, taking standard precautions to prevent cross-contamination of samples. The PCR reactions were conducted as previously described (Perkins and Schall, 2002) using primers DW2 and DW4 and 5 l of genomic DNA in the first reaction and 1 l aliquot of this product was used as a template for a nested reaction with primers DW1 and DW6. All these primers are specific to malarial parasites and do not amplify host DNA or that of other Apicomplexa. The PCR products of ∼1 kb were purified from agarose gels by PureLinkTM Quick Gel Extraction Kit (Invitrogen), cloned in pTZ57R/T plasmid (Fermentas) and sequenced by Big Dye Terminator v3.0 Cycle Sequenc-
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Fig. 1. Photomicrographs of parasites visualized from thin blood smears obtained from penguins 26157 (A), 29092 (B) and 29093 (C). Characteristic of P. relictum, the gametocytes appear round and take up a large portion of the cell, pushing the red blood cell nucleus to one side.
ing Kit in ABI Genetic Analyzer (ABI, USA), using plasmid oligonucleotides (M13 forward and M13 reverse) and internal primers DW3 and DW8 (Perkins and Schall, 2002). Sequences obtained were aligned with sequences from the GenBank using BLASTN (Basic Local Alignment Search Tool) available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi (Altschul et al., 1997). PCR amplification of Plasmodium SSU fragments was performed according to a nested genus-specific protocol (Dos Santos et al., 2009), which uses oligonucleotides in conserved sequences in the small subunit (SSU) ribosomal RNA of all Plasmodium organisms. PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide. The PCR fragment was purified from agarose gels by PureLinkTM Quick Gel Extraction Kit (Invitrogen), cloned in pGEM-T Easy Vector (Promega) and sequenced by Big Dye Terminator v3.0 Cycle Sequencing Kit in ABI Genetic Analyzer (ABI, USA), using PCR oligonucleotides (rPLU3 and rPLU4). The sequences obtained were aligned with sequences from the GenBank using BLASTN available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
3.5. gDNA extraction from blood-fed mosquitoes and blood meal identification
3.4. Mosquito gDNA extraction and PCR amplification of Plasmodium SSU fragment
This study reports avian malaria caused by P. relictum in Magellanic Penguins (S. magellanicus) from São Paulo Zoo. To our knowledge, this is the first report of this parasite in Brazil. The disease was highly infective among the birds and clinically characterized by its acute course and high mortality. Thin blood smears obtained from four penguins (26157, 26159, 29092 and 29093) were positive for
DNA from each mosquito was extracted as previously described (Cerutti et al., 2007). The Plasmodium SSU fragment was amplified, purified, cloned and analysed as described above for penguin blood samples.
Genomic DNA of blood-fed mosquitoes was obtained using PureLinkTM Genomic DNA Purification Kit (Invitrogen). PCR was used to amplify host DNA from the mosquito blood meal using primers L14841 and H15149 (Kocher et al., 1989) or B1 and B6 (Pereira and Baker, 2004), designed to amplify, respectively, fragments with ∼300 bp and ∼1 kb of the mitochondrial cytb gene from a wide array of animals, including mammals, birds, amphibians, reptiles and fishes. Amplified fragments were purified from gels and sequenced directly using the corresponding flanking primers. The sequences were identified by comparison with the GenBank DNA sequence database (National Center for Biotechnology Information available online: www.ncbi.nlm.nih.gov/blast/Blast.cgi). Positive identification and host species assignment were determined when exact or nearly exact matches (>95%) were obtained. 4. Results and discussion
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malaria (with 0.4, 0.4, 6.8 and 11% of parasitemia, respectively). Parasites visualized from penguins 26157, 29092 and 29093 are shown in Fig. 1. Young trophozoites are observed as round. Old trophozoites are about the size of the erythrocyte nucleus, beginning to displace the latter. Mature schizonts are large, exceeding the size of the host cell nucleus and also commonly displace the erythrocyte nucleus. A clear vacuole is frequently present and the nuclei are observed in the periphery. Gametocytes appear round and take up a large portion of the cell, pushing the red blood cell nucleus to one side. The hemogram and biochemistry investigation results were mostly within normal range. Differences were observed in the hematocrit (37 ± 5.3%) and hemoglobin (12 ± 2.4 g/dl) values, which were below the reference range (39–55.4% and 12.3–18.7 g/dl, respectively), and total protein (8.3 ± 0.8 g/dl) values, which were above the reference range (4.8–6.8 g/dl). There were also differences in heterophil (13,780 ± 6380/mm3 ) numbers (reference range 2515–9215/mm3 ). Reference values were obtained from ISIS 2002 (www.isis.org). The PCR reactions were performed with 8 samples obtained from 5 penguins (26153 after death, 26157 before and after treatment, 26159 before and after treatment, 29092 after death and 29093 before and after death). It was possible to amplify a fragment of ∼1.1 kb from the cytb gene in samples 26159, 29092 after death and 29093 after death. The other five samples presented negative results. The PCR products from samples 29092 and 29093 were purified from agarose gels, cloned and sequenced. The sequences obtained were aligned with sequences from the GenBank using BLASTN. The 29092 sequence (GenBank HM031936) presented 98% identity with P. relictum obtained from Zenaida macroura in Nebraska, USA (GenBank AY733089), while the 29093 sequence (GenBank HM031937) presented 99% identity with P. relictum obtained from Spheniscus demersus (GenBank AY733088) from Baltimore Zoo, Maryland, USA. Since the parasite detected in the penguins was confirmed as P. relictum by the cytb sequences and considering that the SSU marker used here (rPLU3/rPLU4) is on a different gene region than that used by other authors and available in GenBank (Jarvi et al., 2002), we decided to amplify, clone and sequence this fragment from two penguins (29092 and 29093). Five different sequences were obtained (three in sample 29092 and two in sample 29093) and were deposited in the GenBank (GenBank HM242418–HM242422). P. relictum described here was highly similar to parasites identified in another species of penguin and in a species of dove, both detected in the USA, which is consistent with the fact that this kind of Plasmodium has been reported from a very broad host (over 300 bird ¯ species) and geographical range (Valkiunas, 2005). In Brazil, two studies were performed to detect avian malaria in birds from fragments of Atlantic Forest in Minas Gerais and in these works, 39.6% and 36% of birds were verified as positive for Plasmodium spp. (Ribeiro et al., 2005; Belo et al., 2009). Therefore the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes
Table 1 Distribution of species or groups of female mosquitoes collected with Shannon trap, Centers for Disease Control (CDC) miniature light traps and Nasci aspirator in São Paulo Zoo. Shannon Mini trap CDC trap Anopheles (Nys.) evansae 06 Coquillettidia (Rhy.) chrysonotum/albifera 02 Culex (Cux.) habilitator/pseudojanthinosoma 06 Culex (Cux.) restuans/declarator Culex (Cux.) spp. 03 Culex (Cux.) Coronator group 18 Culex (Mel.) Melanoconion section 67 (02a ) Culex (Cux.) ameliae Mansonia (Man.) indubitans 41 Aedes (Och.) crinifer 01 Aedes (Och.) scapularis 07 Aedes (Sth.) albopictus 11 (03a ) Uranotaenia (Ura.) lowi Total 162 (05a ) a
Nasci aspirator
04
07 (04a )
04 01
06
06 01 02 03 01 21
14 (04a )
With blood meal.
of the genera Aedes and Culex and rarely by Anopheles (Bruce-Chwatt, 1985), which are all common vectors in the Neotropics, particularly in the Atlantic forest or urban areas of Brazil (Forattini, 2002). In this study, a total of 208 adult mosquitoes (188 females and 20 males) without visible blood meal were collected. The species or groups of females obtained in each capture method are shown in Table 1. PCR amplification was performed for these 188 females. Only one individual was positive for Plasmodium SSU fragment. This specimen, collected from a Nasci aspirator, was found damaged and thus identified as Culex (Cux.) sp. The amplified fragment was cloned and sequenced and two sequences were obtained (GenBank HM242416 and HM242417) that presented identities of 97% and 98% with sequences obtained from penguin 29092 (HM242420 and HM242419, respectively), confirming the mosquito infection with P. relictum (Table 2, Supplemental file 2). Nine mosquitoes with apparent blood meals were collected (Table 1): 2 Culex (Melanoconion) Melanoconion section; 3 Aedes albopictus using Shannon trap; and 4 Anopheles evansae using a Nasci aspirator. Using this methodology for the first time in Brazil, blood meal sources were successfully identified by DNA sequencing of the cytb fragment in 5 out of 9 mosquitoes (two Cx. (Mel.) Melanoconion section and three Ae. albopictus collected using the Shannon trap). One species was identified as avian (Nycticorax nycticorax) and one species as mammalian (Hippopotamus amphibius) hosts for Culex mosquitoes. N. nycticorax, popularly known as Socó, is an opportunistic bird that does not belong to the Zoo, but lives in the neighborhood and feeds on the leftover portions of food of the captive animals, mainly marine lions and penguins. All the blood meals identified from Ae. albopictus were human-derived. For all the An. evansae collected with apparent blood meals (44.4% of the total number of mosquitoes examined), it was not possible identify the blood meal source, since visible amplification products were not obtained. Using other methodologies, some authors were unable to identify the blood meal source in 37.5% of An. evansae tested (Forattini et al., 1987). The blood meal begins to be digested soon after ingestion and as a
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consequence, the DNA is degraded; thus, the longer the post-ingestion time, the lower the possibility of DNA detection (Oshaghi et al., 2006). Moreover, it is possible that the females had fed on sugar substances and not blood. Mosquitoes of both sexes seek out energy substances, generally carbohydrates, such as the nectar of flowers, dew and fruits, to fulfill functions that involve expending energy, including flight and dispersion (Foster, 1995). Although the study of avian malaria transmission by mosquitoes was not entirely conclusive, since the blood meal sources identified did not match with the penguins, the study showed that Culex mosquitoes could be the vectors of avian malaria in the park given the fact that the blood of one species of bird was successfully identified in these mosquitoes. Penguins from São Paulo Zoo were maintained in this location for at least 2 years without presenting malaria; however, they always had been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were allowed to move freely at night for a short period. São Paulo Zoo is located in one of the last forest remnants of the city, an area of 824.529 m2 of original Atlantic forest that contains springs of the historical stream of the Ipiranga, which rise in the State Park and whose waters form three lakes. In the winter period and also in the beginning of the summer, the space destined for Zoo birds is shared by other migratory species, as well as opportunistic species that live freely in the area of the park. In light of the evidence presented here, captive penguins must be managed to avoid contact with mosquitoes at night. These birds must also be periodically tested for malaria and should receive malaria therapy in cases where it is impossible to avoid contact with the mosquitoes. Acknowledgments The authors are grateful to Robson de Almeida Zampaulo and Caroline Perez Girardelli for the collection and identification of the mosquitoes. They would also like to thank Almir Robson Ferreira (Instituto de Medicina Tropical de São Paulo) for drawing the map in Supplemental File 1 and the staff of the São Paulo Zoological Park Foundation (Fundac¸ão Parque Zoológico de São Paulo) for their support during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetpar. 2010.06.026. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bak, U.B., Park, J.C., Lim, Y.J., 1984. An outbreak of malaria in penguins at the Farm-land Zoo. Kisaengchunghak Chapchi 22, 267–272. Belo, N.O., Passos, L.F., Júnior, L.M., Goulart, C.E., Sherlock, T.M., Braga, E.M., 2009. Avian malaria in captive psittacine birds: detection by microscopy and 18S rRNA gene amplification. Prev. Vet. Med. 88, 220–224.
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