Parasitological and new molecular-phylogenetic characterization of the malaria parasite Plasmodium tejerai in South American penguins

Parasitology International 62 (2013) 165–171

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Parasitology International

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Parasitological and new molecular-phylogenetic characterization of the malariaparasite Plasmodium tejerai in South American penguins

Patricia Silveira a, Nayara O. Belo a, Gustavo A. Lacorte a, Cristiane K.M. Kolesnikovas b, Ralph E.T. Vanstreels c,Mário Steindel d, José Luiz Catão-Dias c, Gediminas Valkiūnas e, Érika M. Braga a,⁎a Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazilb Centro de Triagem de Animais Silvestres, Instituto Brasileiro de Meio ambiente e Recursos Naturais Renováveis (IBAMA) e Associação Animal R3, Florianópolis, Brazilc Laboratório de Patologia Comparada de Animais Selvagens, Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazild Laboratório de Protozoologia, Departamento de Microbiologia e Parasitologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazile Nature Research Centre, Akademijos 2, Vilnius, Lithuania

⁎ Corresponding author. Tel.: +55 31 34092876; fax:E-mail address: [email protected] (É.M. Braga).

1383-5769/$ – see front matter © 2012 Elsevier Irelandhttp://dx.doi.org/10.1016/j.parint.2012.12.004

a b s t r a c t

Article history:Received 22 August 2012Received in revised form 19 October 2012Accepted 13 December 2012Available online 23 December 2012

Keywords:Avian malariaPlasmodium tejeraiPenguinsMorphologic analysesPhylogenetic analyses

This study is the first report on mortality of Spheniscus magellanicus, penguin of South America, caused byPlasmodium tejerai, which was identified usingmorphological andmolecular analyses. Blood stages (trophozoites,meronts and gametocytes) were reported and illustrated. The necropsy revealed marked splenomegaly and pul-monary edema, as well as moderate hepatomegaly and hydropericardium. The histopathology revealed the pres-ence of tissue meronts in the macrophages and endothelial cells of multiple organs. The molecular analysesshowed 5.6% of genetic divergence in cytochrome b gene between P. tejerai and Plasmodium relictum. Morphologyof blood and tissue stages of P. tejerai is similar to P. relictum; the distinction between these two species requiresexperience in the identification of avian Plasmodium species. Molecular studies associated with reliably identifiedmorphological species are useful for barcoding and comparisons with previous studies of wildlife malaria infec-tions as well as for posterior phylogenetic and phylogeographic studies. S. magellanicus is a new host record ofP. tejerai, which is the virulent parasite and worthmore attention in avian conservation and veterinary medicineprojects in South America.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The Magellanic penguin (Spheniscus magellanicus) is a SouthAmerican penguin that breeds along the coasts of Argentina, Chileand Falkland Islands [1]. From March to September, these seabirdsmigrate from their Argentinean and Falkland breeding colonies to-wards the coasts of northern Argentina, Uruguay, and southern Brazil[1,2]. During this migration, weakened penguins may wash ashoreBrazilian beaches and are usually taken into rehabilitation centersand zoos [3]. In these institutions, they are often kept in open-air fa-cilities without mosquito control, a condition known to permit thetransmission of malarial parasites [4–7].

Malaria parasites of the genus Plasmodium are cosmopolitanvector-transmitted; they have been reported in birds belonging tomany families and orders around the world, except Antarctica [8].Avian malaria is a serious infectious diseases and the major cause ofmortality in captive penguins [4,6,7,9,10]. Several cases of fatal infec-tions in captive penguins were attributed to malaria caused mainly byPlasmodium relictum and Plasmodium elongatum [4,6,9,11–13]. The

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infected penguins with P. relictum or P. elongatum developed severeclinical signs but none was specific [9,14].

The identification of Plasmodium parasites in birds is not alwayspossible because of low parasitemia or the great diversity of speciesand frequent cases of co-infections [15]. Nowadays, two methods forhemosporidian parasites diagnostic – PCR-based and microscopy –

have been used together to remarkably improve the parasites identi-fication and detection of parasitemia in wild birds [8,16–19]. Theidentification of Plasmodium parasites from themicroscopic observa-tion is basedmainly onmorphologic characteristics of the intracellu-lar stages [8,20,21] while the molecular approaches are basicallybased on sequence similarity analyses providing high congruencewith data of traditional parasitology.

We had the opportunity to evaluate two penguins, which havesuddenly died in a rehabilitation center in South Brazil. The aims ofthe study were (1) to identify the Plasmodium species causing mortal-ity; (2) to describe the pathology associated with the infection causedby this parasite; (3) to determine mitochondrial cytochrome b (cyt b)gene sequences that can be used for molecular characterization of thisparasite; and (4) to infer the phylogenetic relationships among thePlasmodium species identified in this study and other describedavian malaria parasites.

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2. Material and methods

2.1. Sample collection and deposition

Injured Magellanic penguins found along the beaches of SantaCatarina State, Brazil, are often taken to the Screening Center ofWild Animals in Florianópolis city, maintained by the EnvironmentalMilitar Police of Santa Catarina State, the Brazilian Institute of Envi-ronment and Renewable Natural Resources (IBAMA) and AssociaçãoR3 Animal (27°34′S 48°25′W). These seabirds were kept in open facil-ities without mosquito control, in an area of moist semi-deciduousAtlantic forest and near a pond of brackish water.

Two penguins (A and B) were under rehabilitation since late No-vember 2008 when they suddenly died in late March 2009. Thebirds were subjected to gross necropsy. Samples of lung, spleen,heart, adrenal, testicles, kidney, duodenum, ventriculus, and liverwere collected; they were fixed in 10% buffered formalin and sentto the Laboratory of Wildlife Comparative Pathology, Department ofPathology, School of Veterinary Medicine and Animal Sciences, Uni-versity of São Paulo (LAPCOM-FMVZ-USP) for histopathological ex-amination. Blood samples were also collected. Thin blood smearswere prepared from fresh blood, air dried, fixed in absolute methanolfor 3 min and stained with 10% Giemsa for 45 min. Blood sampleswere also stored in EDTA tubes and frozen (−20 °C). These sampleswere sent to the Laboratory of Malaria at the Federal University ofMinas Gerais, Brazil, for molecular and morphological analyses.

The samples of whole blood from the Magellanic penguins (origi-nal field numbers are 93, 506 and 520), were deposited in Érika M.Braga's laboratory (Laboratório de Malária, Bloco E, sala 177,Departamento de Parasitologia, Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil).

2.2. Microscopic analysis of blood smears

Blood smears were examined for 10–15 min at low magnification(×400), and then 200 fields were studied at high magnification(×1000) using Olympus Microscope CX31 (Olympus Corporation,Tokyo, Japan). Intensity of infection was estimated as a percentageby counting the number of parasites per 10,000 erythrocytes [22].The parasites were identified by microscopy according to morpholog-ic characteristics of blood stages [8,23]. The characters used to identi-fy Plasmodium tejerai are discussed in detail in the Results section. AnOlympus BX51 light microscope equipped with Olympus DP12 digitalcamera and imaging software DP-SOFT was used to prepareillustrations.

2.3. Histopathology

Formalin-fixed tissues were embedded in paraffin; sections of5 μmwere obtained, stained with hematoxylin & eosin and examinedunder light microscope [24].

2.4. DNA extraction, PCR amplification and sequencing

For the DNA extraction, approximately 20 μL of blood sample wasstored at room temperature (22–25 °C) in cell lysis solution(PROMEGA®, Madison, Wisconsin, USA) for approximately one dayprior to DNA extraction. DNA from blood samples was extracted withWizard Genomic DNA Purification Kit (PROMEGA®, Madison, Wiscon-sin, USA) according to the manufacturer's protocol. The DNA pellet wassuspended in 30 μL of hydration solution and kept at −20 °C until use.

In order to determine DNA sequences, we amplified a 479 bp frag-ment of the mitochondrial cytochrome b (cyt b) gene using a nestedPCR protocol [25]. The primers HaemNFI and HaemR3 were used inthe first PCR for general hemosporidian species fragments amplifica-tion (Plasmodium, Haemoproteus and Leucocytozoon). For the second

PCR, we used the primers HaemF and HaemR2, which are specific toPlasmodium and Haemoproteus spp. PCR products were purifiedfrom 1.5% agarose gel using the QIAquick gel extraction kit (Qiagen).Bi-directional sequencing with dye-terminator fluorescent labelingwas performed in an ABI Prism 3100 automated sequencer (AppliedBiosystems, Inc.). The positive control consisted of genomic DNAfrom Plasmodium gallinaceum (obtained from experimentally infectedchickens). Negative controls were DNA samples obtained from2-day-old non-infected domestic chickens SPF (specific pathogenfree) maintained at the Veterinary School (UFMG). The P. tejerai se-quences were deposited in the GenBank database with accessionnumbers JX272844 and HQ591361. New sequences in MalAvi systemwere named as pSPMAG01 and pSPMAG02.

2.5. Phylogenetic analysis

Phylogenetic relationships among the lineages of P. tejerai identi-fied in this study and related hemosporidian parasites were inferredby using cyt b gene sequences from MalAvi database [26], since theGenBank contains misidentified sequences of avian hemosporidians[62]. We selected those sequences for which species were identifiedbased on targeting studies using both morphological and molecularevidences (Table S1). In addition, we included Plasmodium sp. line-ages recovered in different birds South American [27]; as well the lin-eages obtained from Galapagos penguins (Spheniscus mendiculus)[28] (Table S2).

The phylogenetic tree was constructed using the Bayesian inferencemethod implemented in MrBayes v3.0b4 [29]. Bayesian inference wasperformed with two Markov chains that run simultaneously for 3 mlngenerations, with trees sampled every 100 generation for a total of30,000 trees. The first 7500 trees were discarded as “burn-in” step andthe remaining trees were used to calculate the posterior probabilities.The sequence divergence among different lineages of Plasmodiumspp. was calculated with the Jukes–Cantor model of substitution,implemented in MEGA 5 [30].

3. Results

3.1. Clinical signs and pathology

No clinical signs were observed before bird deaths. Macroscopicevaluation revealed marked splenomegaly and pulmonary edema,as well as moderate hepatomegaly and hydropericardium.

Histopathology revealed the presence of tissue meronts in themacrophages and endothelial cells of multiple organs (Fig. 1).Meronts were seen with high intensity in the heart and kidneys, mod-erate intensity in the lungs, and were rare in the spleen, testicles andliver. Tissue meronts were highly variable in form, adapting to as wellas distorting the contour of the infected cell, ranging between 10 and80 μm in largest diameter, most frequently from 15 to 30 μm. The en-velope of tissue meronts was mildly eosinophilic, thin and poorly de-fined. Each meront contained tens to hundreds visible merozoites,which were round and densely stained with size of approximately1 μm in diameter (Fig. 1).

Other major pathological processes included moderate to severediffuse interstitial granulocytic pneumonia, moderate pulmonaryedema and congestion, severe acute necrotizing splenitis, moderatemultifocal to coalescent mixed necrotizing hepatitis and nephritisand mild diffuse granulocytic myocarditis with multifocal to coales-cent areas of cardiomyolisis. Death probably resulted due to cardiore-spiratory insufficiency secondary to pneumonia.

3.2. Identification of the parasite

The reported blood parasite belongs to the genus Plasmodium dueto the presence of merogony in blood and malarial pigment

Fig. 1. Tissuemeront (arrow) of Plasmodium tejerai in the endothelial cell of myocardiumin Magellanic penguin Spheniscus magellanicus. Hematoxylin–eosin.

167P. Silveira et al. / Parasitology International 62 (2013) 165–171

(hemozoin) in all its blood stages (trophozoites, meronts and game-tocytes). One Plasmodium species was identified in blood smearsfrom the two evaluated penguins, with parasitemia of 1.5% (penguinA) and 62% (penguin B). This Plasmodium species belongs to the sub-genus Haemamoeba, which is characterized by the presence of largeroundish erythrocytic meronts and gametocytes which size exceedsthe nuclei of infected erythrocytes. By comparing this Plasmodiumparasite with other species of the subgenus Haemamoeba we con-clude that it is P. tejerai (Fig. 2). Because this malaria parasite wasreported for the first time after its original description in turkeyMeleagris gallopavo and penguins are new host reports of this para-site, we prove morphological description of blood stages, which is es-sential for species identification.

3.3. Morphology of blood stages of P. tejerai in Magellanic penguins

Trophozoites and erythrocytic meronts were present mainly inmature erythrocytes, but also seen in polychromatic erythrocytes(Fig. 2). Gametocytes were seen only in mature red blood cells, inwhich nuclei are markedly displaced and changed in morphology.The nuclei of infected erythrocytes are rounded and compressed incomparison to nuclei of uninfected erythrocytes (see Fig. 2, h and i).Erythrocytic merogony is not synchronized; all blood stages were ob-served simultaneously in blood smears. Polyparasitism of erythro-cytes was detected more frequently during heavy parasitemia (62%,penguin B).

Trophozoites (Fig. 2, a–c) were usually seen in polar and sub-polarpositions in infected erythrocytes. Young trophozoites are oval orroundish, usually with smooth or slightly amoeboid margins; asmall nucleus and a small vacuole can be readily distinguished(Fig. 2, a). Growing trophozoites are of even outline; they are nearlycircular in shape (Fig. 2, b and c), which is a characteristic feature ofthis species development. Each growing trophozoite possesses alarge conspicuous vacuole (frequently >2 μm in diameter), which islocated centrally (Fig. 2, b and c), a characteristic feature of this spe-cies development. The parasite nucleus locates at one side of tropho-zoite (Fig. 2, c). Pigment granules small (b0.5 μm), locate aroundvacuole and tent to clamp together close to edge of trophozoites(Fig. 2, b and c). Earliest trophozoites usually do not displace thehost cell nucleus (Fig. 2, a), however as they became larger theymarkedly displace the nuclei and deform infected erythrocytes(Fig. 2, b and c). The largest trophozoites are roundish in shape;each possesses a large vacuole with brown pigment granules locatedaround this vacuole (Fig. 2, c). As trophozoites develop, vacuoles

became less evident (compare Fig. 2, b and c), and they gradually dis-appear in meronts. Outline of advanced trophozoites is even (Fig. 2, band c).

Erythrocytic meronts (Fig. 2, d–g) are roundish or oval bodies withplentiful cytoplasm; they markedly displace nuclei of infected eryth-rocytes from earliest stages of their development (Fig. 2, d), deforminfected cells (Fig. 2, e–g) and even can enucleate them. The cyto-plasm stains dark-blue. A clear vacuole-like pale-stained, centrally lo-cated space is present in early meronts (Fig. 2, d); this space graduallydisappears as parasites mature. Nuclei are prominent; tend to locateclose to edges of growing and maturing meronts (Fig. 2, d–f). Size ofnuclei markedly decreases as meronts mature (compare Fig. 2, d, eand Fig. 2, f). Pigment granules are of small size (b0.5 μm),dark-brown; they tend to group close to the edge of meronts (Fig. 2,d and e); they are aggregated into large compact masses in maturemeronts (Fig. 2, f and g). Numerous growing meronts assume theform of rosettes (Fig. 2, f). Mature meronts produce between 10 and16merozoites (most commonly 10 or 12). Merozoites locate random-ly in the cytoplasm around the nuclei of erythrocytes (Fig. 2, g). Ma-ture merozoites are of round, oval or irregular shape; eachpossesses a prominent nucleus and small portion of the cytoplasm.

Macrogametocytes (Fig. 2, h–k) are of markedly variable morpholo-gy; roundish, oval, elongated and lobulated (Fig. 2, h) forms present.Irregular (Fig. 2, k) and lobular-like (Fig. 2, h) shape of gametocytesare distinctive features of this parasite. The cytoplasm stainsdark-blue, homogenous. Mature gametocytes occupy much of theerythrocyte cytoplasm and cause marked displacement of erythrocytenuclei (Fig. 2, h–j). Parasite nucleus is prominent. Pigment granulesare roundish or slightly oval, of small size (b0.5 μm) tend to group to-gether (Fig. 2, j).

Microgametocytes (Fig. 2, l) present general configuration andmain features as for macrogametocytes with usual hemosporidiansexually dimorphic characters. Nucleus is large, locates centrally. Pig-ment granules gather close to edge of gametocyte, as is the case ingrowing trophozoites (compare Fig. 2, b and l).

3.4. Molecular analyses

The cyt b sequences recovered from Magellanic penguin parasites,which were identified as P. tejerai, formed a well-supported cladewith four South American different Plasmodium sp. lineages obtainedin birds from Guyana and Chile (Fig. 3, Table S2).

4. Discussion

Malaria in penguins is mostly caused by P. relictum andP. elongatum [4,9–13], as well as Plasmodium cathemerium [31] andPlasmodium juxtanucleare [7]. In Brazil, avian malaria outbreaks inpenguins have been reported at rehabilitation centers in Rio Grande[32] and São Paulo [6]. P. relictum and P. elongatum are two speciescommonly present in passeriform birds [33–36] while P. juxtanucleareis a common hemoparasite in species of Galliformes [37–39]. Due tolow natural resistance, the penguins can have increased susceptibilityto malaria parasites when compared to other birds of tropical andtemperate environment [59–61].

Similar to the observed in Magellanic penguin, the clinical featuresof avian malaria infection in penguins reported in the literature usu-ally include nonspecific signs such as anorexia, prostration, and lym-phocytosis [4,6,9,11,13,14,61]. The gross and histopathologicalfindings were also similar to those described for penguins infectedby P. relictum and P. elongatum [4,6,9,11,14,32], suggesting similarpathogenesis. While many questions are raised on the pathogenesisof this disease and the reason for its unusual severity in penguins re-main unanswered, our findings suggest that the gross pathogenesisand lesion patterns of avian malaria in penguins may be relativelyconsistent in different Plasmodium species.

Fig. 2. Plasmodium tejerai from theMagellanic penguin Spheniscus magellanicus. a–c: trophozoites; d–g: erythrocytic meronts; h–k: macrogametocytes; l: microgametocyte. Arrow—

vacuole. Arrowhead — pigment granules. Giemsa stained thin blood films. Scale bar=10 μm. (For interpretation of the references to color in the text with the citation of this figure,the reader is referred to the web version of this article.)

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The absence of vectors control in penguins environment may havebeen the most important factor for the infection of Magellanic pen-guins. Graczyk et al. [13] monitored a group of penguins hatched incaptivity and exposed to an environment without protection frommosquito vectors for 24 weeks. During that study, the prevalence ofPlasmodium spp. infection in the exposed penguins was 62.1%; mor-tality was associated to primary infection caused by the avian malariaparasites in all cases.

The pathogens are one of the main causes of extinction of wildlifepopulations, so their role as cause of infectious diseases in biologicalconservation has been largely recognized [48–50]. A special case ofhigh impact of parasitism in wildlife is due to recent association be-tween parasites and vertebrate hosts that have no common evolu-tionary history [7,39,51]. The costs of the infection for the

immunologically naïve host populations are generally severe; infec-tions can lead to individual death within a short-term [8,52,53].

Linkage of morphological and DNA sequence information is an im-portant step to the better understanding of the biology of Plasmodiumspecies [15,19,54,55]. Moreover, linkage of Plasmodium spp. molecu-lar lineages with morphospecies is a fair way to ensure that theinformation deposited at GenBank and other databases is reliableand suitable for further comparative studies, particularly inphylogeographic, phylogenetic and biodiversity investigations [15].However, such combined PCR-based and microscopic identificationof avian malaria parasites remains scarce, and avian malaria infec-tions have often been detected and identified mainly and solely byPCR-based methods and sequencing. PCR-based approach in malariadiagnostics is relatively fast and sensitive in determining some

Fig. 3. Bayesian majority-rule consensus tree of 39 mitochondrial cytochrome b lineages of avian hemosporidians, with Leucocytozoon schoutedeni as outgroup. Plasmodium tejerailineages recovered in this study are presented in bold; closely related Plasmodium sp. lineages recovered in South American birds and Galapagos penguins are shown. Values nearthe nodes correspond the Bayesian posterior probabilities (in percentage). Branch lengths are drowning proportionally to the amount of changes (scale bar is shown).

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parasite lineages, but it might markedly underestimate these para-sites' diversity [19,56,57]. Combination of PCR-based methods[16–18,26,57] and microscopic approaches are essential in currentwildlife malaria studies [19,57]. Identification of barcodes for detec-tion of certain Plasmodium species is an important step in such re-search [58].

In this study we report P. tejerai infections leading to the mortalityof Magellanic penguins undergoing rehabilitation in South Brazil. Toour knowledge, this is the first report of P. tejerai infecting penguins(Aves: Sphenisciformes) and first morphologically documented re-cord of this parasite after its discovery [23]. P. tejerai was described

from naturally infected turkeys in 1977 [23]. Further experimentalstudies revealed that P. tejerai infected broad range of species belong-ing to the Galliformes, Anseriformes and Columbiformes, but canaryseems to be resistant [23]. The present study adds penguins to thelist of avian hosts of this malaria parasite.

After the description of P. tejerai in Venezuela, this malaria para-site was not observed or reported anymore. However, studies on oc-currence of bird's hemoparasites in South America are scarce andmajority of them were performed by morphological analyses[40–45]. Low parasitemia frequently observed in wild birds and scar-city of PCR-based studies for detection of hemoparasites in South

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America could explain the lack of records of P. tejerai in this region.Studies using PCR screening techniques in South America showedhigh prevalence of blood parasites lineages [27,46,47]. Furthermore,the phylogenetic analysis revealed that some Plasmodium sp. lineagesobtained in passerines from Guyana and Chile [27,63], are closely re-lated to P. tejerai lineages reported in this study. This finding raisesthe hypothesis that parasite lineages, which are phylogeneticallyclose to P. tejerai occur in other South American birds. However,blood stages of these Plasmodium lineages have not been described,so comparisons with P. tejerai is currently impossible. We understandthat the morphological identification of avian blood parasites speciesis a complex matter for two basic reasons. First, parasitemia is usuallylight in the majority of naturally infected wild birds; additionally, thebirds are often co-infected with different hemosporidian species [19].Second, the examination of blood smears is time consuming and re-quires expertise of taxonomists [15]. However, species identificationis fundamental for parasitology studies.

This is the first study reporting cyt b gene sequences fromP. tejerai. Molecular studies associated with reliably identified mor-phological species are useful for barcoding and comparisons with pre-vious studies of wildlife malaria infections as well as for posteriorphylogenetic and phylogeographic studies. We predict that molecularcharacterization of this malaria parasite should stimulate epidemio-logical studies of P. tejerai-malaria in South America, where it mightbe endemic.

Our findings illustrate the importance of applying molecular tech-niques for characterization of Plasmodium infections. Blood stages ofP. tejerai are similar to P. relictum. The distinction between these twospecies requires presence of growing trophozoites, which possessprominent centrally-located vacuoles (Fig. 2, a and b) and maturegametocytes in blood films (irregular-shaped and lobular-like forms,Fig. 2, h and l) and experience in the identification of avian Plasmodiumspecies. However, the molecular evidence provided by the cyt bsequencing shows that these two species are clearly distinct genetically(5.6% of genetic divergence), making it easier to distinguish these twospecies even at light parasitemia by using barcoding methods.

In conclusion, we have reported the presence of high parasitemiaby P. tejerai in penguins for the first time and redescribed blood stagesof this parasite 35 years after its original description. Additionally weidentified phylogenetic relationships of P. tejeraiwith other avian ma-laria parasites and deposited the cyt b sequence of its DNA inGenBank. P. tejerai is worth more attention in avian conservationand veterinary medicine projects in South America due to its highpathogenic potential.

Acknowledgments

We wish to thank Dr. Vaidas Palinauskas (Nature Research Centre,Vilnius) for the help in preparing Fig. 2. Dr. Tatjana A. Iezhova, Dr. AstaKrižanauskienė (Nature Research Centre, Vilnius), and Sandro Sandri(IBAMA/R3) are gratefully acknowledged for their technical assistancein the laboratory. Érika M. Braga and José Luiz Catão-Dias are recipientsof a scholarship by the CNPq. CNPq, CAPES, FAPEMIG and FAPESPsupported the study.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.parint.2012.12.004.

References

[1] Williams TD, Boersma PD. Magellanic penguins. In: Perrins CM, Bock WJ, KikkawaJ, editors. The penguins: bird families of the world. New York: Oxford UniversityPress; 1995. p. 249–57.

[2] Sick H. Ornitologia Brasileira. Rio de Janeiro: Editora Nova Fronteira; 1997.

[3] García-Borboroglu P, Boersma PD, Ruoppolo V, Reyes L, Rebstock GA, Griot K, et al.Chronic oil pollution harms Magellanic penguins in the Southwest Atlantic.Marine Pollution Bulletin 2006;52:193–8.

[4] Graczyk TK, ShawML, Cranfield MR, Beall BF. Hematologic characteristics of avianmalaria cases in African black-footed penguins (Spheniscus demersus) during thefirst outdoor exposure season. Journal of Parasitology 1994;80:302–8.

[5] Brossy JJ, Plös AL, Blackbeard JM, Kline A. Diseases acquired by captive penguins:what happens when they are released into the wild? Marine Ornithology1999;27:185–6.

[6] Bueno MG, Lopez RP, de Menezes RM, Costa-Nascimento MJ, Lima GF, Araújo RAS,et al. Identification of Plasmodium relictum causing mortality in penguins (Spheniscusmagellanicus) from São Paulo Zoo, Brazil. Veterinary Parasitology 2010;173:123–7.

[7] Grim KC, van der Merwe E, Sullivan M, Parsons N, Mc Cutchan TF, Cranfield M.Plasmodium juxtanucleare associated with mortality in black-footed penguin(Spheniscus demersus) admitted to a rehabilitation center. Journal of Zoo andWildlife Medicine 2003;34:250–5.

[8] Valkiūnas G. Avian malaria parasites and other haemosporidia. Florida: CRC PressBoca Raton; 2005.

[9] Fix AS, Waterhouse C, Greiner EC, Stoskopf MK. Plasmodium relictum as a cause ofavian malaria in wild-caught Magellanic penguins (Spheniscus magellanicus).Journal of Wildlife Diseases 1988;24:610–9.

[10] Graczyk TK, Cranfield MR, Brossy JJ, Cockrem JF, Jouventin P, Seddon PJ. Detectionof avian malaria infections in wild and captive penguins. Journal of theHelminthological Society of Washington 1995;62:135–41.

[11] Bak UB, Park JC, Lim YJ. An outbreak of malaria in penguins at the Farm-land Zoo.Kisaengch'unghak Chapchi 1984;22:267–72.

[12] Cranfield MR, Graczyk TK, Beall FB, Iallegio DM, ShawML, SkjoldagerML. Subclinicalavian malaria infection and induction of parasite recrudescence. Journal of WildlifeDiseases 1994;30:372–6.

[13] Graczyk TK, Cranfield MR, McCutchan TF. Characteristics of naturally acquiredavian malaria infections in naive juvenile African black-footed penguins(Spheniscus demersus). Parasitology Research 1994:634–7.

[14] Fleischman RW, Squire RA. Pathologic confirmation of malaria (Plasmodiumelongatum) in African penguins (Spheniscus demersus). Bulletin of the WildlifeDisease Association 1968;4:133–5.

[15] Braga EM, Silveira P, Belo NO, Valkiūnas G. Recent advances in the study of avianmalaria: an overview with an emphasis on the distribution of Plasmodium spp. inBrazil. Memórias do Instituto Oswaldo Cruz 2011;106:3–11.

[16] Bensch S, Stjernman M, Hasselquist D, Ostman O, Hansson B, Westerdahl H, et al. Hostspecificity in avianbloodparasites: a studyofPlasmodium andHaemoproteusmitochon-drial DNA amplified from birds. Proceedings of the Royal Society B 2000;267:1583–9.

[17] Perkins SL, Schall JJ. A molecular phylogeny of malaria parasites recovered fromcytochrome b gene sequences. Journal of Parasitology 2002;88:972–8.

[18] Waldenström J, Bensch S, Hasselquist D, Ostman O. A new nested polymerasechain reaction method very efficient in detecting Plasmodium and Haemoproteusinfections from avian blood. Journal of Parasitology 2004;90:191–4.

[19] Valkiūnas G, Bensch S, Iezhova TA, Krizanauskienė A, Hellgren O, Bolshakoz CV.Nested cytochrome b polymerase chain reaction diagnostics underestimatemixed infections of avian blood haemosporidian parasites: microscopy is still es-sential. Journal of Parasitology 2006;92:418–22.

[20] Garnham PCC. Malaria parasites and other haemosporidia. Oxford: Blackwell Sci-entific Public; 1966.

[21] Martinsen ES, Paperna I, Schall JJ. Morphological versus molecular identificationof avian haemosporidia: an exploration of three species concepts. Parasitology2006;133:279–88.

[22] Godfrey RD, Fedynich AM, Pence DB. Quantification of hematozoa in bloodsmears. Journal of Wildlife Diseases 1987;23:558–65.

[23] Galbadón A, Ulloa G. Plasmodium (Haemamoeba) tejerai sp. n. en pavo domestico(Melleagris gallopavo) del Venezuela. Boletín de la Dirección de Malariología ySaneamiento Ambiental 1977;17:255–73.

[24] Luna LG. Histopathologic methods and color atlas of special stains and tissue arti-facts. Gaithersburg, USA: American Histolabs; 1992.

[25] Hellgren O, Waldenström J, Bensch S. A new PCR assay for simultaneous studies ofLeucocytozoon, Plasmodium and Haemoproteus from avian blood. Journal of Parasi-tology 2004;90:797–802.

[26] Bensch S, Hellgren O, Pérez-Tris J. MalAvi: a public database of malaria parasitesand related haemosporidians in avian hosts based on mitochondrial cytochromeb lineages. Molecular Ecology Resources 2009;9:1353–8.

[27] Durrant K, Beadell JS, Ishtiaq F, Graves GR, Olson SL, Gering E, et al. Avianhematozoa in South America: a comparison of temperate and tropical zones.Ornithological Monographs 2006;60:98–111.

[28] Levin II, Outlaw DC, Vargas HF, Parker PG. Plasmodium blood parasite found in en-dangered Galapagos penguins (Spheniscus mendiculus). Biological Conservation2009;142:3191–5.

[29] Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogeny. Bioinfor-matics 2001;17:754–5.

[30] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance,and maximum parsimony methods. Molecular Biology and Evolution 2011;28:2731–9.

[31] Bennett GF, Bishop MA, Peirce MA. Checklist of the avian species of PlasmodiumMarchiafava & Celli, 1885 (Apicomplexa) and their distribution by avian familyand Wallacean life zones. Systematic Parasitology 1993;26:171–9.

[32] Ruoppolo V, Silva-Filho RP, Adornes AC, Catão-Dias JL. Occurrence of malaria inMagellanic penguins (Spheniscus magellanicus) in a rehabilitation center in South-ern Brazil. Vth International Penguin Conference; 2004 [Ushuaia, Uruguay].

171P. Silveira et al. / Parasitology International 62 (2013) 165–171

[33] Kilpatrick AM, LaPoint DA, Atkinson CT, Woodworth BL, Lease JK, Reiter ME, et al.Effects of chronic avian malaria (Plasmodium relictum) infection on reproductivesuccess of Hawaii Amakihi (Hemignathus virens). The Auk 2006;123:764–74.

[34] Valkiūnas G, Zehtindjiev P, Dimitrov D, Križanauskiené A, Iezhova TA, Bensch S.Polymerase chain reaction-based identification of Plasmodium (Huffia) elongatum,with remarks on species identity of haemosporidian lineages deposited inGenBank. Parasitology Research 2008;102:1185–93.

[35] Palinauskas V, Valkiūnas G, Križanauskiené A, Bensch S, Bolshakov CV. Plasmodi-um relictum (lineage P-SGS1): further observation of effects on experimentallyinfected passeriform birds, with remarks on treatment with Malarone™. Experi-mental Parasitology 2009;123:134–9.

[36] Palinauskas V, Valkiūnas G, Bolshakov CV, Bensch S. Plasmodium relictum (lineageSGS1) and Plasmodium ashfordi (lineage GRW2): the effects of the co-infection onexperimentally infected passerine birds. Experimental Parasitology 2011;127:527–33.

[37] Santos-Prezoto HH, D'Agosto M, Daemom E. Prevalência e variação dos estádioseritrocíticos do Plasmodium (Novyella) juxtanucleare em Gallus gallus sobcondições naturais, no período de um ano. Parasitología latinoamericana2004;59:14–20.

[38] Silveira P, Damatta RA, D'Agosto M. Hematological changes of chickens experi-mentally infected with Plasmodium (Bennettinia) juxtanucleare. Veterinary Parasi-tology 2009;162:257–62.

[39] Murata K, Nii R, Sasaki E, Ishikawa S, Sato S, Kyoko S, et al. Plasmosium (Bennettinia)juxtanucleare in a captive white eared-pheasant (Crossoptilon crossoptilon) at aJapanese zoo. Journal of Veterinary Medical Science 2008;70:203–5.

[40] Bennett G, Lopes OS. Blood parasites of some birds from São Paulo State, Brazil.Memórias do Instituto Oswaldo Cruz 1980;75:117–34.

[41] Woodworth-Lynas CB, Caines JR, Bennett GF. Prevalence of avian haematozoa inSão Paulo state, Brazil. Memórias do Instituto Oswaldo Cruz 1989;84:515–26.

[42] Rodríguez OA, Matta NE. Blood parasites in some birds from eastern plains of Co-lombia. Memórias do Instituto Oswaldo Cruz 2001;96:1173–6.

[43] Valkiūnas G, Salaman P, Iezhova TA. Paucity of haematozoa in Colombian birds.Journal of Wildlife Diseases 2003;39:445–8.

[44] Sebaio F, Braga EM, Branquinho F, Manica LT, Marini MA. Blood parasites in Bra-zilian Atlantic Forest birds: effects of fragment size and habitat dependency.Bird Conservation International 2010;20:432–9.

[45] Fecchio A, Lima MR, Silveira P, Braga EM, Marini MA. High prevalence of bloodparasites in social birds from a neotropical savanna in Brazil. Emu — Austral Orni-thology 2011;111:132–8.

[46] Belo NO, Pinheiro RT, Reis SE, Ricklefs RO, Braga EM. Host species and parasite lin-eage diversity of haemosporidians in three different environments with distinctlevels of disturbance. PloS One 2011;6:17654.

[47] Belo NO, Rodríguez-Ferraro A, Braga EM, Ricklefs ER. Diversity of avianhaemosporidians in arid zones of northern Venezuela. Parasitology 2012;139:1021–8.

[48] Smith KF, Sax DF, Lafferty KD. Evidence for the role of infectious disease in speciesextinction and endangerment. Conservation Biology 2006;20:1349–57.

[49] Smith KF, Acevedo-Whitehouse K, Pedersen AB. The role of infectious diseases inbiological conservation. Animal Conservation 2009;12:1–12.

[50] Tompkins DM, Dunn AM, Smith MJ, Telfer S. Wildlife diseases: from individual toecosystems. Journal of Animal Ecology 2011;80:19–38.

[51] van Riper III C, van Riper SG, Goff ML, Laird M. The epizootiology and ecologicalsignificance of malaria on the birds of Hawaii. Ecological Monographs 1986;56:327–44.

[52] AtkinsonCT, vanRiper III C. Pathogenicity and epizootiologyof avianhaematozoa: Plas-modium, Leucocytozoon, andHaemoproteus. In: Loye JE, ZukM, editors. Bird–parasite in-teractions: ecology, evolution, and behaviour. Oxford: University Press; 1991. p. 19–48.

[53] Atkinson CT, Dusek RJ, Lease JK. Serological responses and immunity to superin-fection with avian malaria in experimentally-infected Hawaii Amakihi. Journalof Wildlife Diseases 2001;37:20–7.

[54] Valkiūnas G, Iezhova TA, Križanauskiené A, Palinauskas V, Sehgal RNM, Bensch S.A comparative analysis of microscopy and PCR-based detection methods for bloodparasites. Journal of Parasitology 2008;94:1395–401.

[55] Garamszegi LZ. The sensitivity of microscopy and PCR-based detection methodsaffecting estimates of prevalence of blood parasites in birds. Journal of Parasitol-ogy 2010;96:1197–203.

[56] Loiseau C, Iezhova T, Valkiūnas G, Chasar A, Hutchinson A, Buermann W, et al.Spatial variation of haemosporidian parasite infection in African rainforest birdspecies. Journal of Parasitology 2010;96:21–9.

[57] Zehtindjiev P, Križanauskienė A, Bensch S, Palinauskas V, Asghar M, Dimitrov D,et al. A new morphologically distinct avian malaria parasite that fails detectionby established PCR-based protocols for amplification of the cytochrome b gene.Journal of Parasitology 2012;98:657–65.

[58] Hajibabaei M, Singer GA, Hebert PD, Hickey DA. DNA barcoding: how it comple-ments taxonomy, molecular phylogenetics and population genetics. Trends in Ge-netics 2007;23:167–72.

[59] Jones HI, Shellam GR. The occurrence of blood-inhabiting protozoa in captive andfree living penguins. Polar Biology 1999;21:5–10.

[60] Jones HI, Shellam GR. Blood parasites in penguins, and their potential impact onconservation. Marine Ornithology 1999;27:181–4.

[61] Cranfield RM. Sphenisciformes (penguins). In: Fowler ME, Miller RE, editors. Zoo& wild animal medicine. Philadelphia: W.B. Saunders Company; 2003. p. 103–10.

[62] Valkiūnas G, Atkinson CT, Bensch S, Sehgal RN, Ricklefs RE. Parasite misidentifica-tions in GenBank: how to minimize their number? Trends in Parasitology2008;24:247–8.

[63] Merino S, Moreno J, Vásquez RA, Martínez J, Sánchez-Monsálvez I, Estades CF,et al. Haematozoa in forest birds from southern Chile: latitudinal gradients inprevalence and parasite lineage richness. Austral Ecology 2008;33:329–40.