CHARACTERIZATION OF THE FUNGAL MICROBIOTA IN THE NOSTRILS AND RECTUM OF AMAZONIAN MANATEES (TRICHECHUS INUNGUIS) FROM A REHABILITATION PROGRAM IN BRAZIL.

The present study characterized the filamentous and yeast-like fungal microbiota of the nasal cavity and rectum of Amazonian manatees (Trichechus inunguis) undergoing rehabilitation at the Laboratory of Aquatic Mammals, National Institute of Amazonian Research, Manaus, Amazonas, and determined the antifungal susceptibility of these organisms. Nasal and rectal swabs were collected from 22 calves and three juveniles. The samples were seeded in Sabouraud agar supplemented with chloramphenicol 10%, incubated at 26°C, and observed daily for up to 7 d. The growth of different filamentous and yeast-like fungi was observed among the two anatomical sites. Filamentous fungi were categorized by macro- and microscopic characteristics of the colonies. Representatives of each group were selected for molecular identification based on the internal transcribed spacer region. Yeast identification was performed using MALDI-TOF MS and molecular analyses. Thirteen genera of filamentous fungi and six genera of yeasts were isolated and identified. The dominant filamentous species were Fusarium spp., Aspergillus spp., and Cochliobolus lunatus in the nostril samples and Aspergillus melleus in the rectal samples. Candida was the dominant genus among the identified yeasts at both anatomical sites. In the antifungal susceptibility test, 28 isolates showed resistance to fluconazole (78%), itraconazole (39%), and nystatin (42%). The knowledge of fungal microbiota composition of Amazonian manatees provides information that assists in monitoring the health status of individuals maintained in captivity, as these organisms can behave either as opportunists or as primary pathogens. Moreover, the composition and resistance of these organisms may vary among different rehabilitation institutions or different time frames of search, reinforcing the importance of constant in loco surveillance of these microorganisms. This study provides new perspectives on the fungal diversity in the microbiota of manatees and supports future studies concerning the clinical and epidemiological aspects and the impacts of these agents on the health of Amazonian manatees undergoing rehabilitation.

Ancestral chromosomal signatures of Paenungulata (Afroteria) reveal the karyotype of Amazonian manatee (Trichechus inunguis, Sirenia: Trichechidae) as the oldest among American manatees.

BACKGROUND: Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians. RESULTS: A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK). CONCLUSIONS: TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis.

Herpesvirus and adenovirus surveillance in threatened wild West Indian (Trichechus manatus) and Amazonian manatees (Trichechus inunguis), Brazil.

THE FAMILY TRICHECHIDAE (ORDER SIRENIA) COMPRISES THREE SPECIES: African (Trichechus senegalenses), West Indian (T. manatus), WIM)], and the Amazonian manatees (T. inunguis, AMM). Whereas WIM inhabits both riverine and coastal systems in the western Atlantic, AMM is the only exclusively freshwater sirenian, endemic to the Amazon River Basin. The study of infectious agents is essential to species conservation, especially considering that both species are classified as Vulnerable by the IUCN Red List and as Endangered by the Brazilian Red List. The current knowledge about viral agents in sirenians is scarce. Herpesviruses and adenovirus are DNA viruses able to infect and cause disease in a wide range of hosts. Herein, we used panPCR protocols to survey herpesvirus and adenovirus in blood samples of wild WIM (n = 23) and AMM (n = 26) under human care in Brazil. Herpesvirus DNA was detected in one juvenile female WIM (1/23; 4.3%; 95% CI -4.7 - 13.3) from Ceará state and in four AMM (two juvenile females, a juvenile male, and an adult female; 4/26; 15.4%; 95% CI 0.5 - 30.3) from Amazonas state. The two different gammaherpesvirus DNA polymerase sequence types identified (one per species, a sequence type in a WIM and another one in three AMM) were highly similar (99% nucleotide identity) to Trichechid herpesvirus 1, reported in West Indian manatees of Florida (USA), and 100% identical when translated into amino acids. A herpesviral glycoprotein B sequence was identified in two AMM. None of the samples was positive to adenovirus. To the authors' knowledge, this is the first herpesvirus detection in manatees from South America, expanding the herpesvirus geographical range, and the first in WIM and AMM worldwide. Our findings suggest (i) that West Indian and Amazonian manatees are possibly the natural hosts of the detected herpesvirus, and (ii) coevolution of that gammaherpesvirus with Trichechus. Future studies are necessary to characterize the obtained virus and elucidate potential pathological effects (if any) in these species.

Survey for Leptospira and Brucella in Amazonian manatees, Amazon river dolphins, and a tucuxi in the Brazilian Amazon.

Leptospirosis and brucellosis are zoonotic diseases with global distributions that represent severe hazards to humans and animals. We investigated exposure to Leptospira spp. and Brucella spp. in samples from Amazonian manatees Trichechus inunguis, Amazon river dolphins Inia geoffrensis, and a tucuxi Sotalia fluviatilis. The animals were free-ranging or undergoing in situ rehabilitation in the mid-Solimões River region, Brazilian Amazon. Serum samples from 19 Amazonian manatees were tested by microscopic agglutination test, Rose Bengal test, and 2-mercaptoethanol Brucella agglutination test. Antibodies against Leptospira spp. were detected in 63% of the manatees tested and serovar Patoc was considered the infecting serovar in all positive samples. Titers were generally low, indicating chronic exposure, but higher titers indicative of an active infection were detected in 3 animals. Anti-Brucella spp. antibodies were not detected. Tissue and/or body fluid samples from 12 Amazon river dolphins, a tucuxi, and 2 Amazonian manatees were investigated by multiplex PCR and bacteriology for Leptospira spp. and Brucella spp. All samples were negative. However, Enterococcus faecalis was isolated from uterine fluid, lymph node, and lung of 3 Amazon river dolphins. Bacillus spp. were isolated from milk and synovial fluid from 2 Amazon river dolphins and from a milk sample from 1 Amazonian manatee. Knowledge of the pathogens present in Amazonian manatees, Amazon river dolphins, and tucuxis is of great relevance to species conservation and environmental health. Although no clinical signs were noted, further research is needed to elucidate the clinical relevance of infection by Leptospira sp. serovar Patoc in Amazonian aquatic mammals.

Karyotypes of Manatees: New Insights into Hybrid Formation (Trichechus inunguis × Trichechus m. manatus) in the Amazon Estuary.

Great efforts have been made to preserve manatees. Recently, a hybrid zone was described between Trichechus inunguis (TIN) and the Trichechus manatus manatus (TMM) in the Amazon estuary. Cytogenetic data on these sirenians are limited, despite being fundamental to understanding the hybridization/introgression dynamics and genomic organization in Trichechus. We analyzed the karyotype of TMM, TIN, and two hybrid specimens ("Poque" and "Vitor") by classical and molecular cytogenetics. G-band analysis revealed that TMM (2n = 48) and TIN (2n = 56) diverge by at least six Robertsonian translocations and a pericentric inversion. Hybrids had 2n = 50, however, with Autosomal Fundamental Number (FNA) = 88 in "Poque" and FNA = 74 in "Vitor", and chromosomal distinct pairs in heterozygous; additionally, "Vitor" exhibited heteromorphisms and chromosomes whose pairs could not be determined. The U2 snDNA and Histone H3 multi genes are distributed in small clusters along TIN and TMM chromosomes and have transposable Keno and Helitron elements (TEs) in their sequences. The different karyotypes observed among manatee hybrids may indicate that they represent different generations formed by crossing between fertile hybrids and TIN. On the other hand, it is also possible that all hybrids recorded represent F1 and the observed karyotype differences must result from mechanisms of elimination.

Study of inorganic elements in different organs and tissues of Amazonian manatee (Trichechus inunguis) from Brazil.

Multielement concentrations (P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Rh) and total mercury (T-Hg) were analyzed in different organs and tissues of Amazonian manatee (Trichechus inunguis). Samples of 27 T. inunguis specimens, maintained in the collection of the Amazonian Center for the Research and Preservation of Aquatic Mammals, were used, situated in an area highly impacted by gold mining in the northern region of the Brazilian Amazon. Samples of aquatic plants used as food by the animals were also analyzed. The elements S, Cl, K, Cr, and Mn accumulated mainly in the musculature, while Fe and Cu were more concentrated in the liver. Trace elements, such as rubidium (Rb) and rhodium (Rh), not previously reported in the organs of animals of the family Trichechidae, were also identified. The averages for T-Hg in the skin, muscle, encephalon, liver, kidney, and lung samples were, respectively, 0.1540 ± 0.1332, 0.0593 ± 0.1044, 0.0517 ± 0.0467, 0.0486 ± 0.0543, 0.0237 ± 0.0336, and 0.0013 ± 0.0032 µg.g-1. The values obtained for the vibrissae samples were below the limit of quantification, which allows for the conclusion that this tissue cannot be used as a contamination marker. It was observed that even when kept in a conservation breeding site, these animals were exposed to non-essential trace elements. Differences in the accumulation of elements were observed between the different organs and tissues analyzed. The presence of contaminants in animals that live in a preservation center, even at low levels, deserves attention.

Abdominal ultrasound in amazonian manatee (Trichechus inunguis) (Natterer, 1883).

Morphophysiological species researches are fundamental, and diagnostic imaging is an excellent technique, already used in wild animals, with great application, not invasive and provide real-time information of each body. Amazonian manatees are on the list of endangered animals classified in the vulnerable category and knowledge of the normal pattern of ultrasound anatomy of organs and tissues is important for the maintenance and well-being of captive specimens contributing to reintroduction actions. The objective of the study was to standardize the examination technique and describe the ultrasound findings of the liver, gallbladder, stomach, urinary bladder and the subcutaneous tissue of the abdominal region in Trichechus inunguis, in order to contribute with the anatomical and sonographic knowledge and assist in the diagnosis and prognosis diseases. The study used 18 animals to describe the normal sonographic anatomy in the abdominal cavity of the Amazonian manatee. During abdominal scan, it was possible to visualize the features of the liver, gallbladder, stomach, urinary bladder obtained satisfactory results in this study. Therefore, other structures were not primarily identified by the reduced time, lots of fat and gases in intestines of animals.

Perfil metabólico de filhotes de peixe-boi da Amazônia (Trichechus inunguis) em cativeiro, alimentados com diferentes sucedâneos do leite materno / [Metabolic profile of captive Amazonian manatee calves (Trichechus inunguis), fed different substitutes for maternal milk]

Dez filhotes de peixe-boi da Amazônia criados em cativeiro foram amamentados com diferentes dietas lácteas em duas etapas. Na primeira, com sucedâneo 1 (S1), utilizado rotineiramente no Inpa e, na segunda, com sucedâneo 2 (S2), elaborado com base na composição centesimal do leite materno da espécie. Cada etapa durou 62 dias, realizando-se coleta de sangue ao final de cada uma para avaliação dos valores de proteínas totais (PT), albumina (ALB), globulinas (GLO), glicose (GLI), lipídeos totais (LT), colesterol (COL), triglicerídeos (TRI), cálcio (Ca), fósforo (P), magnésio (Mg), sódio (Na), potássio (K), hematócrito (Ht) e hemoglobina (Hb). Houve redução dos níveis séricos de PT, ALB, GLO, Ca, P, K, GLI, LT, COL e TRI, após a alimentação dos animais com S2 quando comparados aos valores obtidos com S1. Os níveis de Na foram maiores quando administrado o S2 (143,47±0,99mmol/L). Valores de Ht e Mg não diferiram entre os tratamentos. O perfil metabólico, em geral, esteve dentro dos níveis normais para a espécie.(AU)

Ten Amazonian manatee calves who live in captivity were fed with different substitutes for maternal milk in two phases. During the first phase, the calves were fed substitute 1 (S1), which is what was routinely used by INPA, and during the second phase they were fed substitute 2 (S2), which was formulated based on the nutritional composition of T. inunguis maternal milk. Each phase lasted 62 days, when blood samples were collected for analysis of values of total protein (PT), albumin (ALB), globulin (GLO), glucose (GLI), total lipids (LT), cholesterol (COL), triglycerides (TRI), calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), hematocrit (Ht) and hemoglobin (Hb). There was a decrease in PT, ALB, GLO, Ca, P and K after feeding the animals with S2 when compared to values obtained with S1. Sodium levels were higher when S2 was administered (148.16±1.15mmol/L) than when S1 was given (143.47±0.99 mmol/l). Values of Ht and Mg did not differ between treatments. The metabolic profile was within normal levels for this species.(AU)

A hybrid swarm of manatees along the Guianas coastline, a peculiar environment under the influence of the Amazon River plume.

The West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees have a sympatric occurrence at the mouth of the Amazon River. A result of this interspecific encounter is the occurrence of hybrids, which are frequently found along the coasts of Amapá state in Brazil, French Guiana and Guyana. Here we present new genetic evidence indicating the occurrence of a hybrid swarm along the Guianas Shield coastline, which is an interspecific hybrid zone that also separates T. manatus populations located east (Brazil) and west (Caribbean, Gulf of Mexico, Florida and Antilles). In addition, we suggest that this hybrid population occupies a peculiar mangrove-rich environment under strong influence of the Amazon River plume, which requires an independent management and should be considered a special conservation area.

Cutaneous mycobacteriosis in a captive Amazonian manatee Trichechus inunguis.

An adult male Amazonian manatee Trichechus inunguis under human care presented with 3 circular cutaneous lesions on the dorsal aspect of the rostrum and between the nostrils (plenum). Initially these lesions were superficial, hypopigmented, without warmth and non-painful. Microbiological cultures of skin swabs isolated Candida sp. and Pseudomonas aeruginosa, and topical treatment with antiseptic, antifungal, anti-inflammatory and antibiotic medication was instituted. This treatment strategy did not lead to any clinical improvement, and after 6 mo, the lesions progressed to a confluent abscess (5.0 × 3.0 cm) with increased temperature and obvious discomfort on palpation. An impression smear of a cutaneous biopsy was submitted for Ziehl-Neelsen staining and after detection of acid-fast bacilli, the cutaneous biopsy and a swab from the lesion were sent for histopathology, culture and sensitivity testing. After 5 d of incubation and through PCR-restriction analysis of the isolates, Mycobacterium fortuitum and M. abscessus were identified. Sensitivity testing indicated that the isolates were susceptible to ciprofloxacin and clarithromycin, and after draining of the lesion and administration of systemic antibiotic treatment, there was rapid clinical improvement. This report describes non-healing lesions in an aquatic animal and illustrates the importance of evaluating the presence of non-tuberculous mycobacteria, opportunistic pathogens which are ubiquitous in the aquatic environment, in protracted, non-responsive cases. We also highlight the importance of a correct diagnosis and treatment approach, and we review concerns that these bacteria are zoonotic agents and are frequently resistant to conventional antibiotics.

Bottlenecks in the migration routes of Amazonian manatees and the threat of hydroelectric dams / Gargalos na rota migratória do peixe-boi amazônico e a ameaça das barragens hidrelétricas

In a particular region within western Amazonia, Amazonian manatees (Trichechus inunguis) live in a floodplain environment that becomes inhospitable for them during the annual low-water season. To flee from it, they undergo a dangerous migration to a refuge while water levels are dropping fast. Our aim was to better understand the role of depth variation in this migratory process. We analyzed the sole tracking data on wild manatees (n=10 males), 30 years of Landsat images, a 14-year hydrograph and a 3-D bathymetric model. Migratory routes contained shallower segments, here called 'migratory bottlenecks', which dried out at the end of most lowering-water seasons, blocking the passage to the refuge. Manatees began migrating just in time to traverse the bottlenecks furthest away, suggesting they fine-tuned their departure so as to maximize time within the foraging home range without compromising safety. They apparently achieved this by estimating depth at the bottlenecks. Moreover, a bottleneck was created in >15 years, illustrating the environment's dynamism and the challenge this imposes upon manatees. Our results are probably generalizable to most of the species' range. We contend manatees possess an updatable cognitive map of their environment and are behaviorally plastic. Current dam-building plans, if implemented, would create more bottlenecks and make flooding less predictable, increasing manatee mortality from unsuccessful migrations. It would also partition the species into small populations, each prone to short-term extinction. The natural outcome would be the second species-level collapse. Economic growth should not come at the expense of the extinction of the iconic manatee.

Em uma região particular da Amazônia ocidental, peixes-boi amazônicos (Trichechus inunguis) vivem em um ambiente que se torna inóspito para eles durante a água-baixa anual. Para fugir dele, realizam uma migração perigosa para o refúgio enquanto o nível da água desce rapidamente. Nosso objetivo foi compreender melhor o papel da variação da profundidade neste processo migratório. Analisamos os únicos dados de rastreamento de peixes-boi selvagens (n=10 machos), 30 anos de imagens Landsat, 14 anos de hidrógrafa e um modelo batimétrico 3-D. As rotas migratórias possuíam trechos mais rasos, denominados gargalos migratórios, que secaram no final da maioria das vazantes, bloqueando o acesso ao refúgio. Os peixes-boi começaram a migração em tempo justo para atravessar os gargalos mais distantes, sugerindo que a sintonizaram para maximizar o período se alimentando sem comprometer a segurança. Para tal, parecem ter estimado a profundidade nos gargalos. Adicionalmente, um gargalo foi criado em 15 anos, ilustrando o dinamismo do ambiente e o desafio que isto impõe aos peixes-boi. Esses resultados provavelmente valem para boa parte da área de distribuição da espécie. Argumentamos que peixes-boi possuem um mapa cognitivo atualizável do ambiente e são comportamentalmente plásticos. Os planos de construção de barragens hidrelétricas, se concretizados, criariam mais gargalos e regimes de inundação menos previsíveis, dificultando a migração e consequentemente aumentando a mortalidade de peixes-boi. Também particionariam a espécie em populações pequenas, vulneráveis à extinção no curto-prazo. O desfecho seria o segundo colapso da espécie. O crescimento econômico não deve vir às custas da extinção do icônico peixe-boi.

Variáveis hematológicas e bioquímicas de peixe-boi da Amazônia (Trichechus inungui) jovens / Hematological and biochemical variables of young Amazonian manatee (Trichechus inungui)

Peixes-boi da Amazônia (Trichechus inungui) jovens são frequentemente encontrados e resgatados de rios, no entanto para avaliar e monitorar o estado de saúde desses animais é necessário conhecer os valores de referência das variáveis hematológicas e bioquímicas nessa faixa etária. O trabalho objetivou determinar os valores hematológicos e bioquímicos de peixes-boi da amazônia jovens e saudáveis, mantidos em cativeiros. Foram coletadas 20 amostras sanguíneas de peixes-boi para a realização do hemograma e bioquímica. Os valores médios e desvio padrão das variáveis hematológicas foram: volume globular, 37±4%; eritrócitos, 4,4±0,35x106/µL; hemoglobina, 12,5±1,6g/dL; volume corpuscular médio (VCM), 85,33±4,28fL; hemoglobina corpuscular media (HCM), 28,29±1,39pg; e concentração de hemoglobina corpuscular media (CHCM), 33,13±0,20%, leucócitos totais, 9.080±1.868x103/µL; segmentados, 5.078±1.044,24x103/µL; linfócitos 3.556±1,063x103/µL; eosinófilos, 288± 215,51x103/µL; e monócitos, 165± 114,11x103/µL. A média e o intervalo (mínimo e máximo) das variáveis bioquímicas foram: creatinina quinase (CK), 112,53 (50,10-295,80) U/L; aspartato aminotransferase (AST), 6,02 (2,80-10,20) U/L; gama glutamiltransferase (GGT), 25,24 (10,80-45,30) U/L; fosfatase alcalina (FA), 98,73 (14,66-198,30) U/L; proteína total, 6,12 (5,38-7,10) g/dL; albumina, 4,23 (3,50-5,00) g/dL; colesterol, 310,13 (144-518) mg/dL; triglicerídeos, 127,98 (57,50-213,50) mg/dL; glicose, 43,63 (24,50-73,80) mg/dL; ureia, 30,21 (14,30-54,25); creatinina, 1,34 (0,93-1,76) mg/dL; cálcio, 10,08 (9,25-11,10) mg/dL; e fósforo inorgânico; 6,50 (4,10-8,80) mg/dL. No presente trabalho foi possível determinar valores hematológicos e bioquímicos de peixes-boi da Amazônia (Trichechus inungui) jovens, mantidos em cativeiro, os quais podem ser utilizados como referência para animais em cativeiro e de vida livre, mantidos em condições ambientais, e de manejo, semelhantes.(AU)

Amazonian manatee (Trichechus inunguis) young are often found and rescued from rivers, however to assess and monitor the health status of the animals is necessary to know the reference values of hematological and biochemical variables in this age group. The study aimed to determine hematological and biochemical values of healthy young manatee amazonian, kept in captivity. Were collected 20 blood samples of manatees in captivity for the hematological and biochemical evaluation. Means and standard deviations of hematological variables were: pack cell volume, 37±4%; red blood cell, 4.4±0.35x106/µL; hemoglobin, 12.5±1.6g/dL; mean corpuscular volume (VCM), 85.33±4.28fL; mean corpuscular hemoglobin (HCM), 28.29±1.39pg; and mean corpuscular hemoglobin concentration (CHCM), 33.13±0.20%. White blood cell, 9,080±1,868x103/µL; neutrofil, 5,078±1,044.24x103/µL; lymphocytes 3,556±1,063x103/µL; eosinophil, 288±215.51x103/µL; and monocyte, 1.65±114.11x103/µL. Means and ranges (minimum and maximum) of values for the analytes were: creatine kinase (CK), 112.53 (50.10-295.80) U/L; aspartate aminotransferase (AST), 6.02 (2.80-10.20) U/L; γ-glutamyltransferase (GGT), 25.24 (10.80-45.30) U/L; alkaline phosphatase (FA), 98.73 (14.66-198.30) U/L; total protein, 6.12 (5.38-7.10) g/dL; albumin, 4.23 (3.50-5.00) g/dL; cholesterol, 310.13 (144-518) mg/dL; triglycerides, 127.98 (57.50-213.50) mg/dL; glucose, 43.63 (24.50-73.80) mg/dL; urea, 30.21 (14.30-54.25); creatinine, 1.34 (0.93-1,76) mg/dL; calcium, 10.08 (9.25-11.10) mg/dL; and inorganic phosphorum, 6.50 (4.10-8.80) mg/dL. In the present study we determined hematological and biochemical values of manatees Amazonian (Trichechus inunguis) young held in captivity, which can be used as a reference for animals in bondage and free life, kept under similar ambient conditions, and management similar.(AU)

Acute necrotizing colitis with pneumatosis intestinalis in an Amazonian manatee calf.

On 25 January 2014, a 1 mo old female Amazonian manatee Trichechus inunguis calf weighing 12 kg was rescued by air transport in Guajará, Brazil, and transferred to Mamirauá Institute's Community-based Amazonian Manatee Rehabilitation Center. The calf presented piercing/cutting lesions on the back, neck, and head, in addition to dehydration and intermittent involuntary buoyancy. X-ray analysis revealed a large amount of gases in the gastrointestinal tract. Daily procedures included wound cleaning and dressing, clinical and laboratory monitoring, treatment for intestinal tympanism, and artificial feeding. Adaptation to the nursing formula included 2 kinds of whole milk. Up to 20 d post-rescue the calf presented appetite, was active, and gained weight progressively. Past this period the calf started losing weight and presented constant involuntary buoyancy and died after 41 d in rehabilitation. The major findings at necropsy were pneumatosis intestinalis in cecum and colon, pulmonary edema, and hepatomegaly. The microscopic examination revealed pyogranulomatous and necrohemohrragic colitis with multinucleated giant cells, acute multifocal lymphadenitis with lymphoid depletion in cortical and paramedullary regions of mesenteric lymph nodes, and diffuse severe acinar atrophy of the pancreas. Anaerobic cultures of fragments of cecum and colon revealed colonies genotyped as Clostridium perfringens type A. We speculate that compromised immunity, thermoregulatory failure, and intolerance to artificial diet may have been contributing factors to the infection, leading to enterotoxemia and death.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P<0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli.

Acoustical and anatomical determination of sound production and transmission in West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees.

West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees are vocal mammals, with most sounds produced for communication between mothers and calves. While their hearing and vocalizations have been well studied, the actual mechanism of sound production is unknown. Acoustical recordings and anatomical examination were used to determine the source of sound generation. Recordings were performed on live captive manatees from Puerto Rico, Cuba and Colombia (T. manatus) and from Peru (T. inunguis) to determine focal points of sound production. The manatees were recorded using two directional hydrophones placed on the throat and nasal region and an Edirol-R44 digital recorder. The average sound intensity level was analyzed to evaluate the sound source with a T test: paired two sample for means. Anatomical examinations were conducted on six T. manatus carcasses from Florida and Puerto Rico. During necropsies, the larynx, trachea, and nasal areas were dissected, with particular focus on identifying musculature and soft tissues capable of vibrating or constricting the airway. From the recordings we found that the acoustical intensity was significant (P < 0.0001) for both the individuals and the pooled manatees in the ventral throat region compared to the nasal region. From the dissection we found two raised areas of tissue in the lateral walls of the manatee's laryngeal lumen that are consistent with mammalian vocal folds. They oppose each other and may be able to regulate airflow between them when they are adducted or abducted by muscular control of arytenoid cartilages. Acoustic and anatomical evidence taken together suggest vocal folds as the mechanism for sound production in manatees.

The mammary glands of the Amazonian manatee, Trichechus inunguis (Mammalia: Sirenia): morphological characteristics and microscopic anatomy.

The mammaries from carcasses of two female Amazonian manatees were examined. Trichechus inunguis possesses two axillary mammaries beneath the pectoral fins, one on each side of the body. Each papilla mammae has a small hole on its apex--the ostium papillare. The mammaries are covered by a stratified squamous keratinized epithelium. The epithelium of the mammary ducts became thinner more deeply in the tissue and varied from stratified to simple cuboidal. There was no evidence of glandular activity or secretion into the ducts of the mammary glands.

Conocimientos, conservación y avistamiento del manatí amazónico (Trichechus inunguis), según los pobladores de la cuenca del río Ucayali (Loreto, Perú) / Knowledge of the inhabitants from the Ucayali river basin (Peru) about the Amazonian manatee (Trichechus inunguis)

Identificar conocimientos que poseen los pobladores de la cuenca del río Ucayali sobre la biología, conservación y problemática, asi como avistamientos previos del manatí amazónico Trichechus inunguis. Materiales y métodos: Se encuestaron 421 personas de diferentes edades, sexos y ocupaciones, en diferentes localidades de la cuenca del río Ucayali. Resultados: El 88.8% reconoció a la especie, y el 80.1% conocen al manatí con el nombre de vaca marina. El 65.1% declaran haber consumido su carne de manera poco frecuente y oportunista. La principal razón que justifican para capturarlos fue su buen sabor (46.4%). Así mismo, 51.9% de los encuestados afirmó saber que su caza es ilegal y el 24% refirió haberlo avistado en el área de estudio. Asimismo, 96.7% manifestaron interés para que esta especie animal se conserve. Conclusiones: Los conocimientos de los pobladores encuestados son insuficientes sobre la situación poblacional, las amenazas y la legislación actual que rige la caza del manatí. Actualmente, con poca frecuencia, se siguen avistando ejemplares en vida silvestre dentro del área de estudio, pero también se sigue dando su captura. Se recomienda incluir la educación ambiental en estas comunidades como principal herramienta para la conservación del manatí amazónico...

To collect information about the knowledge of the inhabitants from the Ucayali River basin about the amazonian manateeÆs, Trichechus inunguis, biology, conservation and problematic as well as previous sightings. Materials and methods: 421 people were surveyed without bias of age, sex or occupation, in different localities of the Ucayali River basin. Results: 88.8% of the interviewed recognized species, most of them referring to it as ôsea cowõ (80.1%). The principal use attributed to this animal is the consumption of its meat (65.1%). Also 51.9% of the surveyed are aware that hunting is illegal. According to the surveyed, the consumption of this species is rare and opportunistic, being the good taste of its meat (46.4%) the main reason for its capture. There is a great interest from behalf the population in the conservation of this animal (96.7%). As for the sightings, 24.0% were within the study area. Conclusions: There is a lack of knowledge from the surveyed on the current population, threats and the laws regulating the hunting of the amazonian manatee. Currently, with low frequency, sightings are still occurring within the study area, but also their capture. Environmental education is the main tool required for the conservation of the amazonian manatee in these communities...

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.