Angélica María Sánchez-Sarmiento

Lacaziosis-like disease in Tursiops truncatus from Brazil: a histopathological and immunohistochemical approach

Cetacean lacaziosis-like disease or lobomycosis-like disease (LLD) is a chronic skin condition caused by a non-cultivable yeast of the order Onygenales, which also includes Lacazia loboi, as well as Paracoccidioides brasiliensis and P. lutzii, respectively responsible for lacaziosis and paracoccidioidomycosis in humans. Complete identification and phylogenetic classification of the LLD etiological agent still needs to be elucidated, but preliminary phylogenetic analyses have shown a closer relationship of the LLD agent to Paracoccidioides spp. than to L. loboi. Cases of LLD in South American cetaceans based on photographic identification have been reported; however, to date, only 3 histologically confirmed cases of LLD have been described. We evaluated multiple tissue samples from 4 Tursiops truncatus stranded in the states of Santa Catarina (n = 3) and Rio Grande do Sul (n = 1), southern Brazil. Macroscopically, all animals presented lesions consistent with LLD. Hematoxylin-eosin, periodic acid-Schiff, Grocotts methenamine silver, and Mayers mucicarmin stains were used for histological evaluation. Microscopically, numerous refractile yeasts (4-9 µm in diameter) were observed in skin samples (4/4), and for the first time in dolphins, also in a skeletal muscle abscess (1/4). Immunohistochemistry using anti-P. brasiliensis glycoprotein gp43 as a primary antibody, which is known to cross-react with L. loboi and the LLD agent, was performed and results were positive in all 4 cases. We describe 3 new cases of LLD in cetaceans based on histopathology and immunohistochemistry. This is the first report of LLD in the muscle of cetaceans.

Prenatal exposure to fipronil disturbs maternal aggressive behavior in rats.

Fipronil is a second-generation phenilpirazol insecticide that is used in agriculture and veterinary medicine for protection against fleas, ticks, ants, cockroaches and other pests. The insecticide blocks the chloride channels associated with the gamma-amino butyric acid (GABA) receptors in mammals and the chloride channels associated with the GABA and glutamate (Glu) receptors in insects. In this study, a commercial product that contain fipronil was administered orally to pregnant Wistar rats at dosages of 0.1, 1.0, or 10.0 mg/kg/day from the 6th to the 20th day of gestation (n=10 pregnant rats/group) to assess the maternal aggressive behavior (on the 6th day of lactation) and the histopathology of the ovaries and the thyroid gland of the dams. The fipronil caused a disturbance of the maternal aggressive behavior; the aggression against a male intruder decreased at the lowest dose, but increased at the highest dose, without interfering with the general activity of the dams in the open field test at either dose. The histopathological analysis revealed no abnormalities. The differential effects of fipronil behavior appeared to be a consequence of actions on central nervous system areas that control these behaviors. We suggest that fipronil acts on maternal aggressive behavior through GABA(A) receptors.

Challenges in Evaluating the Severity of Fibropapillomatosis: A Proposal for Objective Index and Score System for Green Sea Turtles (Chelonia mydas) in Brazil.

Fibropapillomatosis (FP) is a neoplastic disease that affects marine turtles worldwide, especially green sea turtles (Chelonia mydas). FP tumors can develop on the body surface of marine turtles and also internally in the oral cavity and viscera. Depending on their quantity, size and anatomical distribution, these tumors can interfere with hydrodynamics and the ability to feed, hence scoring systems have been proposed in an attempt to quantify the clinical manifestation of FP. In order to establish a new scoring system adapted to geographic regions, we examined 214 juvenile green sea turtles with FP caught or rescued at Brazilian feeding areas, counted their 7466 tumors and classified them in relation to their size and anatomical distribution. The patterns in quantity, size and distribution of tumors revealed interesting aspects in the clinical manifestation of FP in specimens studied in Brazil, and that FP scoring systems developed for other areas might not perform adequately when applied to sea turtles on the Southwest Atlantic Ocean. We therefore propose a novel method to evaluate the clinical manifestation of FP: fibropapillomatosis index (FPI) that provides the Southwest Atlantic fibropapillomatosis score (FPSSWA). In combination, these indexing and scoring systems allow for a more objective, rapid and detailed evaluation of the severity of FP in green sea turtles. While primarily designed for the clinical manifestation of FP currently witnessed in our dataset, this index and the score system can be adapted for other areas and compare the characteristics of the disease across regions. In conclusion, scoring systems to classify the severity of FP can assist our understanding on the environmental factors that modulate its development and its impacts on the individual and population health of green sea turtles.

Immune Response of Green Sea Turtles with and without Fibropapillomatosis: Evaluating Oxidative Burst and Phagocytosis via Flow Cytometry

Abstract Fibropapillomatosis (FP) has a complex etiology, involving genetic and environmental factors, and is considered a threat to green sea turtles (Chelonia mydas). The goals of this study were to evaluate phagocytosis and oxidative burst in blood samples of green sea turtles with and without FP. We analyzed samples from 38 specimens (27 with FP) captured at a feeding area in Brazil. No differences were observed between specimens with and without FP regarding leukocyte activity; nevertheless, the analyses revealed there were significant differences among leukocyte populations of animals with FP, lymphocytes and monocytes had higher phagocytic activity than did granulocytes, and lymphocytes had lower oxidative burst activity than did granulocytes and monocytes. This study described an efficient method to assess leukocyte activity through flow cytometry and revealed important characteristics of white blood cells from green sea turtles with FP.

Hematological parameters and the variations resulting from stress of Alouatta caraya during a wildlife rescue program in Brazil

Deforestation and habitat fragmentation are major threats to the conservation status of New World primates, such as the howler monkey (Alouatta caraya) in Brazil, where vegetation destruction is often associated with projects such as Small Hydro Power Plant (SHP) construction. The resulting stress from the implementation of this type of enterprise may be a factor that influences individual susceptibility against pathogens and can determine the success or failure of mitigation measures proposed by responsible and/or requested companies by environmental agencies. To perform health monitoring and to understand physiological changes that ensued from the stress of capturing and keeping primates in captivity, we determined the blood profile of A. caraya rescued at the time of capture (N = 15) and in captivity (N = 11). Complete blood counts (CBCs) performed at the time of capture showed hematological changes compatible with acute stress, such as evident neutrophilic leukocytosis (WBCs; females = 20.48 ± 7.26; males = 18.78 ± 10.39 × 109/L). In captivity, monocytosis was observed in both sexes (females = 1.34 ± 0.26; males = 0.39 ± 0.32 × 109/L; U‐test, P = 0.01), suggesting chronic stress. In females, there was also statistically significant eosinopenia (0.28 ± 0.10 × 109/L; U‐test, P = 0.01) and hematocrit increases (39.00 ± 1.41%; U‐test, P = 0.01). Thus, data shows the impact of both capture related acute stress as well as captivity chronic stress, suggesting that primate management in the implementation of these projects causes significant changes in physiological parameters and, consequently, animal health. Whereas chronic stress is an inducer of immunosuppression and susceptibility to pathogen factors, monitoring hematological parameters in captive animals can act as an indicator of health status, contributing to the success of management and conservation wildlife programs. Am. J. Primatol. 77:246–253, 2015.

Concentrations of polycyclic aromatic hydrocarbons in liver samples of juvenile green sea turtles from Brazil: Can these compounds play a role in the development of fibropapillomatosis?

Fibropapillomatosis (FP) poses a significant threat to the conservation of green sea turtles (Chelonia mydas). Polycyclic aromatic hydrocarbons-PAHs are considered mutagenic, carcinogenic and toxic, and can act as cofactor of this disease. In order to evaluate possible differences between green sea turtles with and without FP, we monitored 15 PAHs in liver samples of 44 specimens (24 with FP) captured in Brazil. We detected eight PAHs and quantified phenanthrene in all green sea turtles with FP. Specimens without FP presented lower values than the tumored ones (1.48 ng g-1 and 17.35 ng g-1, respectively; p < 0.0001). There were no significant differences between tumored and non-tumored specimens, among studied areas, or Southwest Atlantic Fibropapillomatosis Score. Even though we found higher concentrations in the liver samples of green sea turtles with FP, further studies are necessary to confirm if these pollutants are involved in the pathogenesis of the disease.

Pulmonary and systemic fungal infections in an Atlantic spotted dolphin and a Bryde’s whale, Brazil

We report the gross and microscopic findings and molecular identification of 2 cases of hyphate fungal infection in cetaceans from Brazil. The first case involved an adult male Atlantic spotted dolphin Stenella frontalis with localized pulmonary disease characterized by pyogranulomatous and necrotizing bronchopneumonia with intralesional hyphae. The second case involved an adult male Brydes whale Balaenoptera edeni with orchitis, periorchitis, mesenteric lymphadenitis and pyogranulomatous bronchopneumonia with intralesional hyphae. PCR analysis from the dolphins lung yielded Aspergillus fumigatus, and the fungus from the whales mesenteric lymph node showed the greatest identity to Nanniziopsis obscura and Stagonosporopsis cucurbitacearum These cases represent the first reports of pulmonary aspergillosis by A. fumigatus in an Atlantic spotted dolphin and systemic mycosis by a possibly novel Onygenales in marine mammals.

Molecular identification and microscopic characterization of poxvirus in a Guiana dolphin and a common bottlenose dolphin, Brazil

The poxviruses identified in cetaceans are associated with characteristic tattoo or ring skin lesions. However, little is known regarding the prevalence and progression of these lesions and the molecular characterization of cetacean poxviruses in the Southern Hemisphere. This manuscript describes the progression of poxvirus-like skin lesions in 5 free-ranging Guiana dolphins Sotalia guianensis. Additionally, 151 skin samples from 113 free-ranging cetaceans from Brazil, including 4 animals with tattoo skin lesions, were selected for poxvirus testing. Poxviral DNA polymerase gene PCR amplification was used to detect the virus in β-actin-positive samples (145/151). DNA topoisomerase I gene PCR was then used in Cetaceanpoxvirus (CePV)-positive cases (n = 2), which were further evaluated by histopathology and electron microscopy. Based on photo-identification, adult Guiana dolphins presented regressing or healed poxvirus-like lesions (2/2), while juveniles presented persistent (2/3) or healed and progressive lesions (1/3). CePV DNA was amplified in a common bottlenose dolphin Tursiops truncatus and in a Guiana dolphin. Intracytoplasmic inclusion bodies and viral particles consistent with poxvirus were identified by histology and electron microscopy, respectively. CePV-specific amino acid motifs were identified through phylogenetic analysis. Our findings corroborate previous studies that suggest the placement of poxviruses from cetaceans within the novel CePV genus. This is the first molecular identification of poxvirus in South American odontocetes.

Evaluation of Prolonged Exposure to Varenicline in Adult Rats: Hematological, Biochemical and Anatomopathological Studies

Varenicline is a synthetic chemical substance produced from the alkaloid cytisine, used for smoking treatment, which acts as a partial agonist for α4β2 and α3β4 nicotinic cholinergic receptors and as a total agonist for α7 receptor. While there are studies regarding vareniclines non‐smoking‐related effects, as in treatment for drug dependence, there are no studies in the literature evaluating the long‐term toxicity of varenicline through a physiological approach. Thus, the aim of this study was to evaluate possible toxicity through haematological, biochemical and anatomopathological parameters of prolonged exposure (30 days) to varenicline in rats. Three doses of varenicline were used: 0.03 (therapeutic dose for human beings), 0.1 and 0.3 mg/kg orally (gavage). Body‐weight, water and food intake were measured weekly during treatment. On the 30th treatment day, blood and various organs were collected for haematological, biochemical and anatomopathological evaluations. The results show a decrease in some biochemical parameters in animals from the 0.1 and 0.3 mg/kg group, although the values are within the normal range of the species. There were no changes in the other evaluations performed. Together, these data indicate that prolonged exposure of rats to different doses of varenicline was not able to alter haematological, biochemical and anatomopathological parameters.

Fatal haemorrhage and neoplastic thrombosis in a captive African lion ( Panthera leo ) with metastatic testicular sex cord–stromal tumour

BackgroundThe study of neoplasia in wildlife species contributes to the understanding of cancer biology, management practices, and comparative pathology. Higher frequencies of neoplasms among captive non-domestic felids have been reported most commonly in aging individuals. However, testicular tumours have rarely been reported. This report describes a metastatic testicular sex cord–stromal tumour leading to fatal haemorrhage and thrombosis in a captive African lion (Panthera leo).Case presentationDuring necropsy of a 16-year-old male African lion, the left testicle and spermatic cord were found to be intra-abdominal (cryptorchid), semi-hard and grossly enlarged with multiple pale-yellow masses. Encapsulated haemorrhage was present in the retroperitoneum around the kidneys. Neoplastic thrombosis was found at the renal veins opening into the caudal vena cava. Metastases were observed in the lungs and mediastinal lymph nodes. Histology revealed a poorly differentiated pleomorphic neoplasm comprised of round to polygonal cells and scattered spindle cells with eosinophilic cytoplasm. An immunohistochemistry panel of inhibin-α, Ki-67, human placental alkaline phosphatase, cytokeratin AE1/AE3, cKit, vimentin and S100 was conducted. Positive cytoplasmic immunolabeling was obtained for vimentin and S100.ConclusionsThe gross, microscopic and immunohistochemical findings of the neoplasm were compatible with a poorly differentiated pleomorphic sex cord–stromal tumour. Cause of death was hypovolemic shock from extensive retroperitoneal haemorrhage and neoplastic thrombosis may have contributed to the fatal outcome. To our knowledge, this is the first report of sex cord–stromal tumour in non-domestic felids.