Establishing hematologic and biochemical reference intervals for the endangered Lear's macaw (Anodorhynchus leari).

BACKGROUND: The Lear's macaw, Anodorhynchus leari (Psittaciformes, Psittacidae), is an endemic species of the Caatinga region in Brazil. With only a few free-living individuals that are constantly under threat from agricultural clearance, hunting, and trapping, A. leari is classified as "endangered" by the Red Book of Endangered Brazilian Fauna and by the International Union for Conservation of Nature. At the same time, the number of individuals in captivity has been growing due to the efforts of biologists and veterinarians in zoological parks to reproduce this species. Thus, having species-specific reference intervals for hematologic and biochemical measurands is essential to assess the health status of these captive and wild birds and guarantee their reproductive success. OBJECTIVES: This study aimed to determine reference intervals for hematologic and biochemical measurands for A. leari kept in captivity in São Paulo Zoo, Brazil. METHODS: Blood samples from 32 individuals were selected based on inclusion and exclusion criteria. Only samples from adults and healthy individuals were included in this study. All statistical analyses were performed according to the American Society for Veterinary Clinical Pathology guidelines and available literature. RESULTS: Reference values and reference intervals were determined for the captive population of Anodorhynchus leari. CONCLUSIONS: This is the first study to establish reference intervals for A. leari, which can aid the veterinarian in the interpretation of laboratory test results, helping in the diagnosis, treatment, and monitoring of diseases of this endangered species of Brazilian fauna.

DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna.

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.

Hematologic and biochemical reference intervals of brown-throated sloths (Bradypus variegatus).

The brown-throated sloth, Bradypus variegatus, is a common species endemic to South and Central America. Nonetheless, maintaining these animals in captivity can be challenging, and very few institutions manage to do so. The São Paulo Zoological Park Foundation is in a remnant of the Atlantic rainforest in the middle of São Paulo, the largest city in Brazil. This forest fragment has a population of B. variegatus that is geographically isolated and yet to be studied. Assessing the health status of individuals remains difficult due to the lack of reference intervals (RIs) for hematologic and biochemical variables for this species. We aimed to establish hematologic and biochemical RIs in a population of wild B. variegatus living in the largest remnant of Atlantic rainforest in São Paulo city, Brazil. Blood samples from 25 individuals of wild B. variegatus were collected and analyzed for 20 hematologic and 21 biochemical variables, using standard laboratory techniques. Each variable was statistically analyzed according to the American Society for Veterinary Clinical Pathology guidelines. The results obtained for each variable were statically analyzed, making it possible to obtain descriptive statistics for all hematologic and biochemical variables. RIs were determined for 16 hematologic variables. During the microscopic analysis, we observed anisocytosis, polychromatophils, Howell-Jolly bodies, macroplatelets, and reactive lymphocytes. The RIs and descriptive statistics described here establish important baseline numbers that could be essential for the management and treatment of both captive and wild B. variegatus sloths.

Assessing Diversity, Plasmodium Infection and Blood Meal Sources in Mosquitoes (Diptera: Culicidae) from a Brazilian Zoological Park with Avian Malaria Transmission.

Avian malaria parasites are widespread parasites transmitted by Culicidae insects belonging to different genera. Even though several studies have been conducted recently, there is still a lack of information about potential vectors of Plasmodium parasites, especially in Neotropical regions. Former studies with free-living and captive animals in São Paulo Zoo showed the presence of several Plasmodium and Haemoproteus species. In 2015, a pilot study was conducted at the zoo to collect mosquitoes in order to find out (i) which species of Culicidae are present in the study area, (ii) what are their blood meal sources, and (iii) to which Plasmodium species might they be potential vectors. Mosquitoes were morphologically and molecularly identified. Blood meal source and haemosporidian DNA were identified using molecular protocols. A total of 25 Culicidae species were identified, and 6 of them were positive for Plasmodium/Haemoproteus DNA. Ten mosquito species had their source of blood meal identified, which were mainly birds, including some species that were positive for haemosporidian parasites in the former study mentioned. This study allowed us to expand the list of potential vectors of avian malaria parasites and to improve our knowledge of the evolutionary and ecological relationships between the highly diverse communities of birds, parasites, and vectors present at São Paulo Zoo.

Comparative analyses of coproscopical techniques to diagnose enteroparasites in a group of captive Indian peafowl (Pavo cristatus).

Captive animals commonly have infections by direct life cycle parasites, since they are easily transmitted between individuals. However, diagnosing these infections in the laboratory is challenging due to the wide variety of parasite, their life stages and to the variety of available diagnose techniques, being difficult to choose the best one. The present study sampled a group of captive Indian peafowl (Pavo cristatus) from São Paulo Zoological Park Foundation, São Paulo, Brazil, to test and compare different coproscopical techniques commonly applied in veterinarian clinical analysis laboratories: direct smear, concentrations by sodium chlorite, sucrose, zinc sulphate, faecal sedimentation and formalin-ether followed by modified Ziehl-Neelsen staining. Sensitivity, specificity, predictive values (positive and negative) and Cohen's kappa index were calculated. In total 108 samples were processed and parasites found were: nonsporulated coccidian oocysts (91.7%), Capillarinae eggs (89.8%), unidentified nematode larvae (75%), Ascarididae eggs (63%), unidentified nematode adults (60.2%), unidentified nematode eggs (42.6%), strongylid-like eggs (42.6%), Cryptosporidium spp. (28.7%), flagellated (15.7%) and ciliated (10.2%) protozoans, trematode eggs (0.9%), Acanthocephala eggs (0.9%), Adeleidae oocysts (0.9%) and Cruzia sp. eggs (0.9%). Sensitivity and specificity varied considerably between parasite groups. Cohen's Kappa index reinforces the recommendation of applying more than one technique to diagnose enteroparasites infections.

Wing Geometric Morphometrics as a Tool for the Identification of Culex Subgenus Mosquitoes of Culex (Diptera: Culicidae).

Culex is the largest subgenus within the genus Culex that includes important vectors of diseases. The correct identification of mosquitoes is critical for effective control strategies. Wing geometric morphometrics (WGM) has been used to identify mosquito species alongside traditional identification methods. Here, WGM was used for eleven Culex species from São Paulo, Brazil, and one from Esquel, Argentina. Adult mosquitoes were collected using CDC (Centers for Disease Control) traps, morphologically identified and analyzed by WGM. The canonical variate analysis (CVA) was performed and a Neighbor-joining (NJ) tree was constructed to illustrate the patterns of species segregation. A cross-validated reclassification test was also carried out. From 110 comparisons in the cross-validated reclassification test, 87 yielded values higher than 70%, with 13 comparisons yielding 100% reclassification scores. Culexquinquefasciatus yielded the highest reclassification scores among the analyzed species, corroborating with the results obtained by the CVA, in which Cx. quinquefasciatus was the most distinct species. The high values obtained at the cross-validated reclassification test and in the NJ analysis as well as the segregation observed at the CVA made it possible to distinguish among Culex species with high degrees of confidence, suggesting that WGM is a reliable tool to identify Culex species of the subgenus Culex.

Naturally Acquired Humoral Immunity against Malaria Parasites in Non-Human Primates from the Brazilian Amazon, Cerrado and Atlantic Forest.

Non-human primates (NHPs) have been shown to be infected by parasites of the genus Plasmodium, the etiological agent of malaria in humans, creating potential risks of zoonotic transmission. Plasmodium brasilianum, a parasite species similar to P. malariae of humans, have been described in NHPs from Central and South America, including Brazil. The merozoite surface protein 1 (MSP1), besides being a malaria vaccine candidate, is highly immunogenic. Due to such properties, we tested this protein for the diagnosis of parasite infection. We used recombinant proteins of P. malariae MSP1, as well as of P. falciparum and P. vivax, for the detection of antibodies anti-MSP1 of these parasite species, in the sera of NHPs collected in different regions of Brazil. About 40% of the NHP sera were confirmed as reactive to the proteins of one or more parasite species. A relatively higher number of reactive sera was found in animals from the Atlantic Forest than those from the Amazon region, possibly reflecting the former more intense parasite circulation among NHPs due to their proximity to humans at a higher populational density. The presence of Plasmodium positive NHPs in the surveyed areas, being therefore potential parasite reservoirs, needs to be considered in any malaria surveillance program.

Wing geometric morphometrics as a tool for the Identification of culex subgenus mosquitoes of culex (Diptera: Culicidae)

Culex is the largest subgenus within the genus Culex that includes important vectors of diseases. The correct identification of mosquitoes is critical for effective control strategies. Wing geometric morphometrics (WGM) has been used to identify mosquito species alongside traditional identification methods. Here, WGM was used for eleven Culex species from São Paulo, Brazil, and one from Esquel, Argentina. Adult mosquitoes were collected using CDC (Centers for Disease Control) traps, morphologically identified and analyzed by WGM. The canonical variate analysis (CVA) was performed and a Neighbor-joining (NJ) tree was constructed to illustrate the patterns of species segregation. A cross-validated reclassification test was also carried out. From 110 comparisons in the cross-validated reclassification test, 87 yielded values higher than 70%, with 13 comparisons yielding 100% reclassification scores. Culexquinquefasciatus yielded the highest reclassification scores among the analyzed species, corroborating with the results obtained by the CVA, in which Cx. quinquefasciatus was the most distinct species. The high values obtained at the cross-validated reclassification test and in the NJ analysis as well as the segregation observed at the CVA made it possible to distinguish among Culex species with high degrees of confidence, suggesting that WGM is a reliable tool to identify Culex species of the subgenus Culex.

Giardia spp., ten years of parasitological data in the biggest zoo of Latin America

Giardia spp. (Diplomonadida: Hexamitidae) is an important and widely studied protozoan parasite with worldwide distribution. Nowadays have six described species, and the most important probably is Giardia duodenalis due to the zoonotical potential that some assemblages have. Many studies analysing samples from wild animals have detected Giardia in captive environment, including the zoonotic type. The aim of this study was to determine the prevalence of Giardia sp. in wild captive animals at São Paulo Zoo, using conventional parasitological techniques (direct smear, passive flotation with saturated sodium chloride solution and simple gravity sedimentation), from 2006 to 2016. In total, 7066 coprological exams were performed during this period with samples from mammals, birds, reptiles and amphibians. The prevalence of Giardia infections was of 1.5% (103/7066). Mammals had the higher prevalence of infections with 2% (77/3872), followed by birds with 1.1% (25/2186) and reptiles with only one positive sample (1/894). All samples from amphibians were negative. Representatives of thirteen families presented positive results for this parasite: Dromaidae, Phasianidae, Ramphastidae, Cervidae, Giraffidae, Canidae, Felidae, Herpestidae, Myrmecophagidae, Callithrichidae, Cebidae, Hylobatidae and Dipsadidae. This study presents the first report of Giardia sp. in Pavo muticus and Brachyteles arachnoides. Infections were prevalent in Cebidae and Ramphastidae species. The findings of this study highlight the importance of identifying which Giardia assemblage are involved in the infections and if they may have a zoonotic potential.

Molecular and morphological characterization of Hepatozoon spp. in Brazilian snakes

The genus Hepatozoon represents one of six genera in the hemogregarine group. Some studies in snakes indicated effects in the host, from slight influences on fitness to severe effects on growth rate, reproduction and offspring survival rates. Diagnosis and identification are usually through blood smear analyses; but not all infected animals show parasitemia. Based on this, the present study established an adapted molecular protocol to identify Hepatozoon spp. to be used as a complementary test for routine diagnoses at the Clinical Analysis Laboratory at São Paulo Zoological Park Foundation. The study was conducted with 113 individuals. Microscopical analysis and molecular techniques were used to identify the parasite. Microscopic analyses showed 13.3% of the samples to be positive. The first pair of primers, targeting 18S rRNA gene, amplified parasite DNA in 6.3% of the samples. The second pair of primers, targeting Apicoplast fragment, were used only on samples that were identified microscopically as being positive, detecting the presence of parasite DNA in 93.3% of these. Phylogenetic analysis of the resulting sequences found five clusters for the 18S gene and five clusters for the Apicoplast fragment. Studies involving Hepatozoon spp. are still scarce and limited, mainly in snakes and the impacts of this parasite on the vertebrate host, so diagnostic studies are essential for wildlife conservation, especially in ex situ work.

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil / Carrapatos infestando espécies de animais silvestres cativas e de vida livre no Zoológico de São Paulo, São Paulo, Brasil

Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.(AU)

Os carrapatos são ectoparasitas da classe Arachnida, conhecidos como importantes vetores de agentes infecciosos para animais e humanos. A Fundação Parque Zoológico de São Paulo (FPZSP) localizada em uma Unidade de Conservação do bioma Mata Atlântica e inserida na cidade de São Paulo, mantém mais de 3.000 animais selvagens cativos, abrigando ainda diversas espécies silvestres nativas da Mata Atlântica. Devido à importância do conhecimento dos carrapatos que parasitam a fauna selvagem, o objetivo deste trabalho foi identificar a coleção de carrapatos da FPZSP e analisar a diversidade desses parasitas em animais cativos e de vida livre. O acervo de carrapatos mantidos pela FPZSP contém 523 exemplares que foram coletados de diferentes espécies hospedeiras, desde 1990 até 2017. Dez espécies de carrapatos foram encontradas. Além dos animais silvestres, foram encontrados Amblyomma aureolatum (Pallas) em gatos domésticos errantes que circulam nas áreas da FPZSP. Este trabalho demonstra a baixa ocorrência de parasitismo em animais cativos e a alta diversidade de carrapatos nos hospedeiros deste fragmento de Mata Atlântica, contribuindo com informações sobre a relação parasita-hospedeiro e potenciais vetores de doenças zoonóticas, já que os vetores da Febre Maculosa Brasileira A. aureolatum e Amblyomma sculptum Berlese, foram encontrados em alguns hospedeiros.(AU)

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil / Carrapatos infestando espécies de animais silvestres cativas e de vida livre no Zoológico de São Paulo, São Paulo, Brasil

Abstract Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.

Resumo Os carrapatos são ectoparasitas da classe Arachnida, conhecidos como importantes vetores de agentes infecciosos para animais e humanos. A Fundação Parque Zoológico de São Paulo (FPZSP) localizada em uma Unidade de Conservação do bioma Mata Atlântica e inserida na cidade de São Paulo, mantém mais de 3.000 animais selvagens cativos, abrigando ainda diversas espécies silvestres nativas da Mata Atlântica. Devido à importância do conhecimento dos carrapatos que parasitam a fauna selvagem, o objetivo deste trabalho foi identificar a coleção de carrapatos da FPZSP e analisar a diversidade desses parasitas em animais cativos e de vida livre. O acervo de carrapatos mantidos pela FPZSP contém 523 exemplares que foram coletados de diferentes espécies hospedeiras, desde 1990 até 2017. Dez espécies de carrapatos foram encontradas. Além dos animais silvestres, foram encontrados Amblyomma aureolatum (Pallas) em gatos domésticos errantes que circulam nas áreas da FPZSP. Este trabalho demonstra a baixa ocorrência de parasitismo em animais cativos e a alta diversidade de carrapatos nos hospedeiros deste fragmento de Mata Atlântica, contribuindo com informações sobre a relação parasita-hospedeiro e potenciais vetores de doenças zoonóticas, já que os vetores da Febre Maculosa Brasileira A. aureolatum e Amblyomma sculptum Berlese, foram encontrados em alguns hospedeiros.

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil.

Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.

Diversity and distribution of avian malaria and related haemosporidian parasites in captive birds from a Brazilian megalopolis.

BACKGROUND: The role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited. METHODS: In 2011-2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections. RESULTS: The overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals). In all, infections with parasites of the genus Plasmodium (overall prevalence 97.6%) predominated when compared to those of the genus Haemoproteus (2.4%). In total, 14 cytochrome b (cytb) lineages of Plasmodium spp. and 2 cytb lineages of Haemoproteus spp. were recorded. Eight lineages were new. One of the reported lineages was broad generalist while others were reported in single or a few species of birds. Molecular characterization of Haemoproteus ortalidum was developed. CONCLUSION: This study shows that many species of birds are at risk in captivity. It is difficult to stop haemosporidian parasite transmission in zoos, but is possible to reduce the infection rate by treating the infected animals or/and while keeping them in facilities free from mosquitoes. Protocols of quarantine should be implemented whenever an animal is transferred between bird maintaining institutions. This is the first survey of haemosporidians in captive birds from different orders maintained in zoos. It is worth emphasizing the necessity of applying practices to control these parasites in management and husbandry of animals in captivity.

Parasitological surveillance in a rat (Rattus norvegicus) colony in São Paulo Zoo animal house

Rattus norvegicus (Mammalia: Rodentia) is a widespread and synanthropic rodent, broadly used in medical experiments. It can also be used for feeding captive animals in zoos. Parasitological surveys are important to guarantee the health of both the animals and the staff responsible for their management. The aim of this study was to identify intestinal parasites of Rattus norvegicus offered as food to captive animals from São Paulo Zoo, and demonstrate the importance of sanitary hurdling, disease control and biosecurity. The identified protozoan parasites were Eimeria sp., Entamoeba sp., Spironucleus sp., Giardia sp., Tritrichomonas sp., Chilomastix sp., unidentified cysts and non-sporulated coccidians oocysts (Isospora/Eimeria). The following helminths were found: Syphacia muris, Rodentolepis nana and Aspiculuris tetraptera.

Hemosporidian parasites of free-living birds in the São Paulo Zoo, Brazil.

Numerous studies addressed the diversity of bird Plasmodium and Haemoproteus parasites. However, a few have been carried out in continental avian hotspot regions such as Brazil, a country with markedly different biomes, including Amazon, Brazilian Savanna, Atlantic Forest, Caatinga, Pantanal, and Pampas. We present the first study on hemosporidian (Haemosporida) parasites in free-living birds from an Atlantic Forest fragment where more than 80 avian species have been reported. Within this area, the São Paulo Zoo locates, and it is the fourth largest zoo in the world and the largest in Latin America. A total of 133 free-living bird samples representing 12 species were collected in the zoo, with the overall hemosporidian prevalence of 18 % by PCR-based diagnostics. Twenty-four positive PCR signals were reported from four different bird species, including migratory ones. Columba livia, an urban species, considered nowadays a pest in big cities, showed 100 % prevalence of Haemoproteus spp., mainly Haemoproteus columbae. We discuss the epidemiological importance of new parasites introduced by migratory birds in the São Paulo Zoo area and the risk it poses to the captive species, which are natives or exotics. We also warn about the influence these parasites can have on the biodiversity and the structure of host populations by altering the competitive interaction between the free-living and the captive birds.

Plasmodium (Novyella) nucleophilum from an Egyptian Goose in São Paulo Zoo, Brazil: microscopic confirmation and molecular characterization.

Plasmodium (Novyella) nucleophilum was identified using microscopy and PCR, in an Egyptian Goose (Alopochen aegyptiacus) that died in São Paulo Zoo, Brazil. This parasite is characterized by elongated gametocytes, small meronts with scant cytoplasm, less than eight merozoites and mainly for having all the stages appressed to the nuclei of infected erythrocytes. Additionally, Plasmodium (Haemamoeba) sp. was identified by microscopy in the same blood sample. The latter parasite lacks nucleophilic blood stages and is characterized by large roundish trophozoites, each with a large prominent centrally collated vacuole. This co-infection was not confirmed by PCR amplification of the mitochondrial cytochrome b (cytb) gene and sequencing; only one Plasmodium sp. cytb sequence was detected in the blood sample. Since parasitemia of P. nucleophilum (2.4%) was much higher than that of P. (Haemamoeba) sp. (0.2%), PCR may have favored the amplification of the cytb sequence of the former. Phylogenetic analysis is in agreement with this conclusion because the reported cytb sequence was positioned in the same branch of sequences of several Novyella species. This is the first assignment of the mitochondrial cytb gene sequence to P. nucleophilum. The P. (Haemamoeba) parasite is particularly similar to Plasmodium (Haemamoeba) tejerai, because its advanced trophozoites and young erythrocytic meronts possess a large vacuole with prominent pigment granules arranged around it, the characteristic features of development in this species. For definitive identification of P. (Haemamoeba) species, mature meronts and gametocytes are required; however, these were absent from the thin blood smear. Representative images of the blood stages of P. nucleophilum and P. (Haemamoeba) sp. are provided. Together with microscopy data, the P. nucleophilum cytb sequence will assist in molecular identification (barcoding) of this Plasmodium species in other birds.