Ex situ population of the Harpy Eagle and its potential for integrated conservation.

A main priority in conservation is the protection of species in their natural habitat. However, ex situ management of threatened species is a recognised strategy of conservation. Harpy Eagles (Harpiaharpyja) are removed from the wild due to illegal capture, nest tree destruction, or other conflict sources. This study presents a review of the current ex situ Harpy Eagle populations in Brazil and worldwide, including information on the origin, sex, and year of entrance or year of birth under human care. Worldwide, until 2020 there were 205 Harpy Eagles in 77 different facilities in 16 countries, with 40 institutions in Brazil and 37 in other countries. The largest ex situ Harpy Eagle population is maintained in Brazil, with 139 individuals (75 females and 64 males) in 40 institutions. Of these institutions, there were 24 zoos, seven conservation breeding centres, six commercial breeders, two wildlife shelters, and one wildlife sorting centre. In Brazil, 62% (n = 86) of the individuals were hatched in the wild and 38% (n = 53) were bred in captivity under human care; for the wild individuals, only 73% (n = 64) have a known state of origin, with the majority from Pará state. This investigation provided relevant information to establish an ex situ demographic database. These individuals may potentially constitute a genetically and demographically viable safety population for future conservation strategies, as well as a source for research and education applied to Harpy Eagle integrated conservation.

Canine distemper virus and canine adenovirus type-2 infections in neotropical otters (Lontra longicaudis) from Southern Brazil.

All descriptions of infectious diseases affecting otters were published in the Northern Hemisphere, with no occurrence identified in neotropical otters (Lontra longicaudis). Consequently, a retrospective histopathological study using archival tissue samples from six free-living neotropical otters was done to investigate the possible occurrence of disease patterns associated with common viral infectious disease agents of the domestic dogs. Immunohistochemical (IHC) assays were designed to identify intralesional tissue antigens of canine distemper virus (CDV), and canine adenovirus-1 (CAdV-1) and canine adenovirus-2 (CAdV-2). The most frequent histopathological patterns diagnosed were interstitial pneumonia (83.33%; 6/5) and hepatocellular vacuolar degeneration (50%; 3/6). IHC identified intralesional intracytoplasmic immunoreactivity to CDV antigens in all otters evaluated, with positive immunolabeling occurring within epithelial cells of the lungs, stomach, kidneys, and liver, and skin. Intracytoplasmic CAdV-2 antigens were identified within epithelial cells of the peribronchial glands in four otters with interstitial pneumonia. These findings resulted in singular and simultaneous infections in these neotropical otters, represented the first report of concomitant infections by CDV and CAdV-2 in free-living neotropical otters from the Southern Hemisphere, and suggested that this mammalian species is susceptible to infections by viral disease agents common to the domestic dogs and may develop similar histopathologic disease patterns.

Immunohistochemical evidence of canine morbillivirus (canine distemper) infection in coatis (Nasua nasua) from Southern Brazil.

The pathological and immunohistochemical (IHC) findings associated with infection due to canine morbilivírus (canine distemper virus, CDV) are described in coatis (Nasua nasua). Tissue fragments of coatis (n = 13) that died at the Bela Vista Sanctuary, Paraná, Southern Brazil, were routinely processed for histopathology to identify the main histopathologic patterns as compared to that of the domestic dog. Selected formalin-fixed paraffin-embedded (FFPE) tissue fragments of the lungs, liver, urinary bladder and small intestine were used in IHC assays designed to identify the antigens of CDV, canine adenovirus (CAdV-1 and CAdV-2) and canine parvovirus type 2 (CPV-2). The main histopathologic patterns identified were interstitial pneumonia (n = 9), interstitial nephritis (n = 6), atrophic enteritis (n = 4) and ballooning degeneration of the uroepithelium (n = 3). Positive immunolabelling for intralesional antigens of CDV was identified in the lung with interstitial pneumonia (n = 3), in the intestine (n = 2) and in the degenerated epithelium of the urinary bladder (n = 2). Antigens of CPV-2, CAdV-1 and CAdV-2 were not identified in any FFPE tissue sections evaluated. These findings indicate that these wild carnivores were infected by a viral disease pathogen common to the domestic dog and develop similar histopathologic findings. Collectively, these findings suggest that these coatis were infected by CDV and can serve as a potential host for this infectious disease pathogen.

Immunohistochemical identification of antigens of canine distemper virus in neotropical felids from Southern Brazil.

The pathologic and immunohistochemical findings associated with infections due to canine distemper virus (CDV) are described in the cougar (Puma concolor), margay (Leopardus wiedii) and jaguarundi (Herpailurus yagouaroundi) from Southern Brazil. Tissue sections of the neotropical felids (n = 3) that died at the Bela Vista Sanctuary, Paraná, Southern Brazil were routinely processed for histopathology to identify possible histopathologic patterns associated with infections due to CDV. Selected formalin-fixed paraffin embedded tissue sections of the lungs and urinary bladder were used in immunohistochemical assays designed to identify the antigens of CDV. The main histopathologic patterns identified were interstitial pneumonia in the margay and jaguarundi, while ballooning degeneration of the transitional epithelium of the urinary bladder was observed in the cougar. Positive immunoreactivity to antigens of CDV was identified within intralesional sections of the lungs of the two wild felids with interstitial pneumonia and in the degenerated urothelium of the cougar. These findings indicate that these neotropical cats were infected by a viral infectious disease pathogen common to the domestic dog and add to the few documented descriptions of CDV-induced infections in wildlife from Brazil.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua).

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, 'Candidatus Mycoplasma turicensis', 'Candidatus Mycoplasma haemominutum', Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.

Occurrence of hemotropic mycoplasmas in non-human primates (Alouatta caraya, Sapajus nigritus and Callithrix jacchus) of southern Brazil.

Hemoplasmas, the erythrocyte-associated mycoplasmas, have been detected in several primates, causing mostly subclinical infection. This study aimed to determine the prevalence of hemoplasma infection in captive and free-ranging monkeys from southern Brazil, as well as factors and hematological abnormalities associated with infection. Blood samples from 40 non-human primates (NHP) were tested for hemoplasmas and coinfections. An overall of 10/40 (25.0%) NHP tested positive for hemoplasmas using PCR-based assays, including 9/14 (64.3%) black howler monkeys (Alouatta caraya) and 1/24 (4.2%) black-horned capuchin (Sapajus nigritus). Infection was not statistically associated with anemia, but wild-born monkeys and male black howler monkeys were more likely to be positive when compared with captive-born animals and female black howler monkeys, respectively. The sequences from the black howler monkey hemoplasma were similar (94% identity) to the squirrel monkey hemoplasma ("Candidatus Mycoplasma kahanei") and were phylogenetically located in a different cluster when compared to the human hemoplasma ("Candidatus Mycoplasma haemohominis").

Doppler ultrasonography of the pectinis oculi artery in harpy eagles (Harpia harpyja).

Twenty harpy eagles (Harpia harpyja) without systemic or ocular diseases were examined to measure blood velocity parameters of the pectinis oculi artery using Doppler ultrasonography. Pectinate artery resistive index (RI) and pulsatility index (PI) were investigated using ocular Doppler ultrasonography. The mean RI and PI values across all eyes were 0.44±0.10 and 0.62±0.20 respectively. Low RI and PI values found in the harpy eagle´s pectinis oculi artery compared with the American pekin ducks one and other tissue suggest indeed a high metabolic activity in pecten oculi and corroborates the hypothesis of a nutritional function and/or intraocular pressure regulation.

Chemical Restraint of Free-ranging South American Coatis ( Nasua nasua ) with a Combination of Tiletamine and Zolazepam.

We describe the use of a combination of tiletamine and zolazepam (Zoletil®) for chemical restraint of South American coatis ( Nasua nasua ) under field conditions. We immobilized 53 coatis from a free-ranging population at Iguaçu National Park, Brazil, with Zoletil. Males and females (1.0-8.7 kg) of different age groups participated in the study. Four dosage (milligram per kilogram body weight) groups were created based on quartiles as follows: 1) 4.76-6.68 mg/kg (n=13), 2) 6.83-7.71 mg/kg (n=13), 3) 7.72-8.68 mg/kg (n=18), and 4) 8.98-11.57 mg/kg (n=9). Variables analyzed were sex, age, body weight, dosage, induction time, handling time (HT: time elapsed between the onset of immobilization and first signs of recovery), time from injection to first stand-up posture after anesthesia, heart and respiratory rates, and body temperature. Mean (±SD) induction time was 2.9 (±1.4) min and was positively correlated with age. In dosage groups 1-4, mean HTs were 40.3 (±24.0), 64.5 (±19.1), 54.8 (±15.0), and 60.3 (±12.0), respectively. Handling time had a positive linear relationship with age and body weight, but the relationship between HT and dosage was nonlinear. Group 1 had a shorter HT compared to the other groups combined. Time from injection to first stand-up was 105.0 (±33.5) min. Zoletil was quick acting and safe for immobilization of coatis in the wild.

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil.

A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

The use of altrenogest to avoid hyperestrogenism after eCG-hCG ovulation induction in southern tigrina (Leopardus guttulus).

The goal of this study was to optimize an ovulation induction protocol for use with artificial insemination in the southern tigrina (Leopardus guttulus). The specific aims were to report the efficacy of using altrenogest, an oral progestin (Regumate, MSD Animal Health, Merck Animal Health), to suppress ovarian activity and prevent follicular hyperstimulation and hyperestrogenism after the administration of exogenous eCG and hCG. To monitor ovarian responses, fecal estrogen and progestogen metabolites were quantified by enzyme immunoassay in females before and after intramuscular administration of 200-IU eCG and 150-IU hCG in two trials, 4 months apart. During the first trial, there was no use of altrenogest, only the eCG-hCG ovulation induction protocol. In the second trial, the ovulation induction protocol was preceded by the administration of oral altrenogest for 14 days (minimum of 0.192 mg per kg per day). Altrenogest administration resulted in a suppression of follicular activity in three out of six females before eCG-hCG administration on the basis of lower mean estrogen concentrations (P < 0.05). It also resulted in four out of six females presenting lower fecal estrogen metabolite concentrations (P < 0.05) after ovulation induction, and two out of six individuals showed a reduction (P < 0.05) in postovulatory fecal progestogen metabolite concentrations, all when compared to the same female's cycles without the progestin. Fecal estrogen metabolite concentrations were closer to baseline in 50% of these individuals after altrenogest and eCG-hCG treatments when compared to basal concentrations before gonadotropins without the use of altrenogest. This study reported that use of altrenogest in southern tigrina can suppress ovarian activity and avoid hyperestrogenism after administration of eCG and hCG treatment. However, not all females responded uniformly, so more studies are needed to increase the efficacy of ovulation induction for use with artificial insemination in this species.

Hemotropic mycoplasma in a free-ranging black howler monkey (Alouatta caraya) in Brazil.

Hemotropic mycoplasmas are bacteria that infect erythrocytes and cause subclinical infections to life-threatening disease. We describe hemotropic mycoplasma infection in a free-ranging black howler monkey (Alouatta caraya). This is the first molecular detection of a hemotropic mycoplasma in a nonhuman primate from Brazil.

Serologic survey for Leptospira spp. in captive neotropical felids in Foz do Iguaçu, Paraná, Brazil.

Leptospirosis is a bacterial zoonosis of worldwide distribution and is endemic in tropical countries, where rodents and other wild mammals are abundant and may act as reservoirs. Leptospirosis has become a concern in captive wild animals, due mostly to their exposure to contaminated urine or environment. Although domestic cats (Felis catus) have been reported refractory to leptospirosis, serology and disease in captive wild felids is still unclear. In this study 57 adult, clinically healthy felids, including 1 Geoffroy's cat (Leopardus geoffroyi), 3 jaguarundis (Puma yagouaroundi), 17 margays (Leopardus wiedii), 22 little spotted cats (Leopardus tigrinus), and 14 ocelots (Leopardus pardalis) kept in captivity at the Sanctuary at the Itaipu Binacional hydroelectric power plant (Bela Vista Biological Sanctuary), Foz do Iguacu City, Paraná State, Brazil, were serologically surveyed for the presence of antibodies against 28 serovars of Leptospira spp. by microagglutination test (MAT). Two animals (3.5%) were seropositive: one male ocelot to the serovar Cynopteri (titer 100) and one female margay to Autumnalis (100) and Butembo (200). The captive-born, 5-yr-old ocelot had been solitary housed in an individual cage. The approximately 21-yr-old wild-caught margay was also kept individually. None of the tested animals showed signs ofleptospirosis. During a study conducted 4 yr previously in the same facility, this particular margay also tested positive for the same two serovars, among others. The present study indicates that the felids tested for Leptospira spp. by MAT were exposed to serovars, but did not demonstrate clinical signs of disease. Comparison with a previous study suggests that serovar titers may vary over time and that leptospirosis dynamics remains unclear in wild felids.

Mycoplasma ovis in captive cervids: prevalence, molecular characterization and phylogeny.

Hemotrophic mycoplasmas (hemoplasmas) are bacteria that attach to red blood cells of mammals, leading to acute and/or subclinical disease in infected animals. It has been suggested that Mycoplasma ovis, a hemoplasma that infects sheep and goats worldwide, may also infect deer. The aim of this study was to evaluate whether South American deer are infected with M. ovis. EDTA-anticoagulated blood samples from a herd of 32 captive South American deer were collected. DNA extraction of blood samples was performed followed by PCR amplification of the 16S and 23S rRNA genes, and sequencing of products. Using M. ovis PCR, 27/31 (87%) were positive, including 21/22 Mazama nana; 2/3 Mazama americana and 4/6 Blastocerus dichotomus. Sequencing of the nearly entire 16S rRNA gene of 26/27 positive samples showed 98.2-98.8% identity to M. ovis of sheep (GenBank, AF338268) and 98.6-99.4% identity to M. ovis-like of a fawn (FJ824847); the 23S rRNA gene from one of these isolates and the fawn's had 97.6% identity. The remaining isolate had just 94.9% identity to the 16S rRNA gene of M. ovis and only 89.4% identity to the 23S rRNA gene of the fawn's M. ovis. This is the first report of M. ovis in captive South American deer, revealing a high prevalence of hemoplasma infection in these animals.

Serological survey of Toxoplasma gondii in captive Neotropical felids from Southern Brazil.

Toxoplasma gondii is the causative intracellular protozoan of toxoplasmosis in human being and animals. Members of the Felidae family are considered the single definitive host for the infection; both wild and domestic cats are able to excrete oocysts in the environment. Wild cats maintained in captivity may serve as source of infection for other clinically susceptible animals in the same environment. The aim of this study was to determine the frequency of T. gondii IgG antibodies in 57 neotropical felids (1 Leopardus geoffroyi; 3 Puma yagouaroundi; 17 Leopardus wiedii; 22 Leopardus tigrinus; and 14 Leopardus pardalis) kept at the Bela Vista Biological Sanctuary, Itaipu Binacional, Southern Brazil, by the modified agglutination test (MAT) using titer 16 as cut-off point. Seropositivity was observed in 38/57 (66.67%; 95% CI 53.66-77.51%) samples, with higher frequency in ocelots (71.43%). Wild-caught felids were three times more likely to be infected when compared to zoo-born animals (P

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Detection of Plasmodium sp. in capybara.

In the present study, we have microscopically and molecularly surveyed blood samples from 11 captive capybaras (Hydrochaeris hydrochaeris) from the Sanctuary Zoo for Plasmodium sp. infection. One animal presented positive on blood smear by light microscopy. Polymerase chain reaction was carried out accordingly using a nested genus-specific protocol, which uses oligonucleotides from conserved sequences flanking a variable sequence region in the small subunit ribosomal RNA (ssrRNA) of all Plasmodium organisms. This revealed three positive animals. Products from two samples were purified and sequenced. The results showed less than 1% divergence between the two capybara sequences. When compared with GenBank sequences, a 55% similarity was obtained to Toxoplasma gondii and a higher similarity (73-77.2%) was found to ssrRNAs from Plasmodium species that infect reptile, avian, rodents, and human beings. The most similar Plasmodium sequence was from Plasmodium mexicanum that infects lizards of North America, where around 78% identity was found. This work is the first report of Plasmodium in capybaras, and due to the low similarity with other Plasmodium species, we suggest it is a new species, which, in the future could be denominated "Plasmodium hydrochaeri".

Pathology and first report of natural infections of the eye trematode Philophthalmus lachrymosus Braun, 1902 (Digenea, Philophthalmidae) in a non-human mammalian host.

The avian eye trematode Philophthalmus lachrymosus Braun, 1902 is for the first time referred naturally occurring in a non-human mammalian host. Previously, natural infections with P. lachrymosus and other species of Philophthalmus have been occasionally reported from man, with few data on experimental infections of non-human mammals. Results presented here are related to the report of two cases of philophthalmosis due to natural infections of wild Brazilian capybaras, Hydrochaeris hydrochaeris L., 1766 with P. lachrymosus and associated pathology. Clinical signs, gross and microscopic lesions as well as new morphometric data on the parasite are presented.