Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Environmental characterization of home range of Antillean manatees (Trichechus manatus) released in northeastern Brazil.

The Antillean manatee occurs discontinuously from the state of Amapá to the state of Alagoas on the coast of Brazil. There is also evidence of reintroduced manatees using the coasts of Sergipe and Bahia, with a preference for calm shallow waters. This study characterized the home range areas of six rehabilitated manatees released in northeastern Brazil. The activities were conducted in the states of Paraíba, Sergipe, and Bahia. Type of environment, substrate, depth, aquatic vegetation, physicochemical variables of the water, presence of solid waste, human settlements, and watercraft were considered to characterize the areas. The results showed a manatee preference for sheltered areas. Resources were available in larger quantities in the dry season, and a reduction in the availability of food items was fund over the years. High overlap was found in the multivariate space of the individuals in terms of the characteristics of the habitats. The estuary of the Paraíba River and the coastal area of Cabedelo Beach in Paraíba showed the greatest amount of solid waste, human settlements, and watercraft. Released manatees exhibited a preference for sites shallower than two meters, with food resources and fresh water availability.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

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.

Use of the FLOTAC technique as a new coproparasitological diagnostic method in aquatic mammals and comparison with traditional methods.

The inadequate choice of a diagnostic method or the option for techniques that have low sensitivity and specificity may limit the diagnosis of parasitic agents that affect aquatic mammals. The aim of this study was to evaluate the performance of the FLOTAC technique and compare it with three traditional methods (Willis, sedimentation and centrifugation- flotation) used in the diagnosis of gastrointestinal parasites in aquatic mammals. For this, 129 fecal samples from 12 species were collected. Each sample was submitted to laboratory processing using the Willis, Hoffman techniques, Faust method and FLOTAC. Sensitivity, specificity, real prevalence, estimated prevalence, positive predictive value, negative predictive value, correct classification (accuracy) and incorrect classification were evaluated to compare the different diagnostic methods. The highest frequency of positive samples occurred using FLOTAC (46.51%), compared to Hoffman (23.25%), Faust (10.07%) and Willis techniques (6.97%). In the samples analyzed, the occurrence of Strongylidae eggs and Eimeriidae oocysts was frequently observed. The FLOTAC technique proved to be the most appropriate technique and due to its efficacy, is strongly recommended for coproparasitological evaluations in aquatic mammals.

Repeated downsweep vocalizations of the Araguaian river dolphin, Inia araguaiaensis.

Araguaian botos (Inia araguaiaensis) are known to produce pulsed as well as tonal sounds. This study documents the first evidence for repetitive sequences of downsweep whistles in botos that appear to be shared between individuals, and the context of their occurrence is investigated. Boat surveys were conducted along the Tocantins River located in the Eastern Amazon over a period of 42 days between 2012 and 2018. Eighty-two groups of Araguaian botos were observed, and 43 h of sound recordings were acquired. 632 downsweep whistles were recorded in 10 encounters. Four of these encounters contained downsweep bouts (21 bouts with ≥2 whistles) with short inter-call intervals (bout criterion 50 s) and up to 161 whistles. A statistical relationship was not found between downsweep occurrence and any of the contextual parameters that were investigated, including socializing, travelling, feeding, group size, presence of calves, and socio-sexual displays. The rarity of these signals makes them unlikely candidates for individual or group identification. It is more likely that they are associated with very specific contexts, such as nursing or mating, both of which were rarely observed in this study. Further studies are required to investigate context specificity and elucidate the function of these signals.

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.

Retrospective pathological survey of pulmonary disease in free-ranging Amazon river dolphin Inia geoffrensis and tucuxi Sotalia fluviatilis.

The Amazon river dolphin Inia geoffrensis and tucuxi Sotalia fluviatilis are classified as Data Deficient species. Despite very limited knowledge on health and disease aspects of these species, the main threats to their conservation include incidental mortality in fishing gear, population fragmentation, habitat loss and environmental pollution. It is also suggested that underlying diseases may contribute to their mortality rates. Herein, we retrospectively describe gross and microscopic pulmonary lesions in free-ranging I. geoffrensis (n = 24) and S. fluviatilis (n = 28) found dead. Nearly 85% of the examined animals presented some kind of primary lung disease, wherein the main etiological diagnoses were verminous pneumonia by Halocercus brasiliensis (25%), bacterial pneumonia (25%) and a single case of meconium aspiration syndrome (1.9%). An etiology was not determined in 36.5% (19/52) of animals. These results indicate a high incidence of pulmonary pathology in these species, raising concerns about population impacts and potential zoonotic implications in some instances. These data may provide a scientific basis for future medical and conservation efforts focused on Amazonian dolphins.

Cryptosporidium spp. and Giardia sp. in aquatic mammals in northern and northeastern Brazil.

Cryptosporidium and Giardia are protozoans that can infect humans and wild and domestic animals. Due to the growing importance of diseases caused by protozoan parasites in aquatic species, we aimed to evaluate the frequency of infection by Cryptosporidium spp. and Giardia sp. in aquatic and marine mammals in the northern and northeastern regions of Brazil. We collected 553 fecal samples from 15 species of wild-ranging and captive aquatic mammals in northern and northeastern Brazil. All samples were analyzed by the Kinyoun technique for identification of Cryptosporidium spp. oocysts. Giardia sp. cysts were identified by means of the centrifugal-flotation technique in zinc sulfate solution. Subsequently, all samples were submitted for direct immunofluorescence testing. The overall frequency of infection was 15.55% (86/553) for Cryptosporidium spp. and 9.04% (50/553) for Giardia sp. The presence of Cryptosporidium spp. was detected in samples from 5 species: neotropical river otter Lontra longicaudis (15.28%), giant otter Pteronura brasiliensis (41.66%), Guiana dolphin Sotalia guianensis (9.67%), Amazonian manatee Trichechus inunguis (16.03%), and Antillean manatee T. manatus (13.79%). Giardia sp. was identified in L. longicaudis (9.23%), P. brasiliensis (29.16%), pygmy sperm whale Kogia breviceps (100%), dwarf sperm whale K. sima (25%), S. guianensis (9.67%), T. inunguis (3.81%), and T. manatus (10.34%). This is the first report of Cryptosporidium spp. in L. longicaudis, P. brasiliensis, and S. guianensis, while the occurrence of Giardia sp., in addition to the 2 otter species, was also identified in manatees, thus extending the number of hosts susceptible to these parasitic agents.

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.

Occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil.

Infections by Cryptosporidium spp. in aquatic mammals is a major concern due to the possibility of the waterborne transmission of oocysts. The aim of the present study was to report the occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil. Fecal samples were collected and processed using Kinyoun's method. Positive samples were also submitted to the direct immunofluorescence test. The results revealed the presence of Cryptosporidium spp. oocysts in 12.5% (17/136) of the material obtained from the Antillean manatees and in 4.3% (05/115) of the samples from the Amazonian manatees. Cryptosporidium spp. infection was more prevalent in captive animals than in free-ranging specimens.

The sustainable development goals in two sustainable development reserves in central amazon: achievements and challenges.

The 2030 Agenda was set in 2015 by the United Nations, with 17 Sustainable Development Goals. The Amazonian riverine people are recognized as traditional communities that have their own culture and use the local natural resources of their territories in an ancestral and traditional way. The Sustainable Development Reserve is a Brazilian protected area category which aims to ensure the protection of the natural environment while allowing the residence and the use of these lands by traditional populations. This article reports and discusses the achievements and challenges of the Sustainable Development Goals in two sustainable development reserves in Central Amazonia. The goals were evaluated in the Mamirauá and Amanã Sustainable Development Reserves, due to the large research programs developed in those areas along the past 20 years. The 17 Sustainable Development Goals have a clear connection with the mission of these sustainable development reserves in Central Amazon. Despite the many achievements conquered over the years, there are many challenges yet to overcome; and while striving to achieve the goals from the 2030 Agenda, new challenges will emerge. The current main challenges to reach the Sustainable Development Goals in the Mamirauá and Amanã Sustainable Development Reserves, in Central Amazon, are connecting to the reality of rural areas.

The evolutionary history of manatees told by their mitogenomes.

The manatee family encompasses three extant congeneric species: Trichechus senegalensis (African manatee), T. inunguis (Amazonian manatee), and T. manatus (West Indian manatee). The fossil record for manatees is scant, and few phylogenetic studies have focused on their evolutionary history. We use full mitogenomes of all extant manatee species to infer the divergence dates and biogeographical histories of these species and the effect of natural selection on their mitogenomes. The complete mitochondrial genomes of T. inunguis (16,851 bp), T. senegalensis (16,882 bp), and T. manatus (16,882 bp), comprise 13 protein-coding genes, 2 ribosomal RNA genes (rRNA - 12S and 16S), and 22 transfer RNA genes (tRNA), and (D-loop/CR). Our analyses show that the first split within Trichechus occurred during the Late Miocene (posterior mean 6.56 Ma and 95% HPD 3.81-10.66 Ma), followed by a diversification event in the Plio-Pleistocene (posterior mean 1.34 Ma, 95% HPD 0.1-4.23) in the clade composed by T. inunguis and T. manatus; T. senegalensis is the sister group of this clade with higher support values (pp > 0.90). The branch-site test identified positive selection on T. inunguis in the 181st position of the ND4 amino acid gene (LRT = 6.06, p = 0.0069, BEB posterior probability = 0.96). The ND4 gene encodes one subunit of the NADH dehydrogenase complex, part of the oxidative phosphorylation machinery. In conclusion, our results provide novel insight into the evolutionary history of the Trichechidae during the Late Miocene, which was influenced by geological events, such as Amazon Basin formation.

Highly divergent herpesviruses in threatened river dolphins from Brazil.

River dolphins are a highly threatened polyphyletic group comprised of four odontocete families: Iniidae, Pontoporiidae, Lipotidae, and Platanistidae, the first two endemic to South America. To address the knowledge gap regarding infectious agents in this cetacean group, we surveyed the presence of herpesviruses by PCR in skin and/or blood samples of live-captured Amazon (Inia geoffrensis, n = 25) and Bolivian (Inia boliviensis, n = 22) river dolphins of the Amazon basin and in selected tissue samples of franciscanas (Pontoporia blainvillei, n = 27) stranded or bycaught in southeastern Brazil. Additionally, available franciscana tissue samples were examined by histopathology. Herpesvirus DNA was amplified in 13 Bolivian river dolphins (59.1%, 95% CI 38.5-79.6%) and 14 franciscanas (51.9%, 95% CI 33.0-70.7%). All Amazon river dolphins were herpesvirus-negative. Two different herpesviruses were found in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and/or skin samples of all positive individuals and a novel alphaherpesvirus in the skin of one animal. A new gammaherpesvirus was found in several franciscana samples-the first herpesvirus recorded in Pontoporiidae. Intranuclear inclusion bodies consistent with herpesvirus were observed in the lymph node of one franciscana. The high divergence among the obtained herpesviruses and those previously described can be explained by viral-host coevolution, and by the fact that these populations are fairly isolated.

One Health Implications of Antimicrobial Resistance in Bacteria from Amazon River Dolphins.

Studies on the microbiota of freshwater cetaceans are scarce and may provide important data on animal and environmental health. This study aimed to evaluate the antimicrobial susceptibility of Gram-negative bacteria recovered from two populations of free-ranging Amazon river dolphins (Inia geoffrensis). Twenty-one animals were captured and released, 13 from Negro River and 8 from Tapajós River, Brazil. Swab samples were obtained from the oral cavity, blowhole, genital opening and rectum and were cultured on MacConkey agar. Isolates were biochemically identified, and antimicrobial susceptibility was assessed by disk diffusion method. Overall, 132 isolates were recovered, of which 71 were recovered from animals from Negro River and 61 from Tapajós River. The most commonly recovered bacterial species were Enterobacter cloacae, Morganella morganii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed (P < 0.001). The results indicate that free-ranging Amazon river dolphins host resistant bacteria, contributing for their maintenance in the environment. This study highlights the importance of the One Health approach to monitor the emergence of antimicrobial resistance. Summary Gram-negative bacteria recovered from 21 free-ranging Amazon river dolphins (Inia geoffrensis) from the Negro River and the Tapajós River populations were evaluated for their antimicrobial susceptibility. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed. Thus, free-ranging Amazon river dolphins, never treated with antimicrobials, host resistant bacteria, contributing for their maintenance in the environment and highlighting the importance of the One Health approach to monitor the emergence of antimicrobial resistance.

On the brink of isolation: Population estimates of the Araguaian river dolphin in a human-impacted region in Brazil.

Populations of freshwater dolphins are declining in response to increased human pressure, including habitat degradation, overfishing, bycatch, poaching and obstruction of free-flowing river corridors by dams. At least three river dolphin species occur in South America: the Amazonian river dolphin, or boto (Inia geoffrensis), the Bolivian river dolphin (Inia boliviensis) and the tucuxi (Sotalia fluviatilis). A fourth species, the Araguaian boto (Inia araguaiaensis), been proposed for the Tocantins-Araguaia, a large river basin in northern Brazil. Here we show that the Araguaian boto population in the Tocantins River is relatively small (N = 1083, CV = 0.52). During a survey to estimate density and abundance, 138 groups (198 individuals) of botos were observed along a ~600 km stretch of the Tocantins River in five different habitats (river margin, river channel, channel, island margin, and a dam reservoir). Overall, lower densities of the Araguaian boto were registered downstream of the Tucuruí dam, the world's fifth largest hydropower dam. Density was 68% lower in the river margin habitat downstream (0.23 ind./km2, CV = 0.92) than upstream (0.72 ind./km2, CV = 0.53). In addition, density within the Tucuruí reservoir decreases from upstream areas towards the dam. Geographic post-stratification of data into sub-regions (downstream, reservoir, upstream) in relation to the Tucuruí dam helped to reduce CV by ~70%, which illustrates the high variability in the encounter rate in these areas. Our findings suggest that the Araguaian boto population has been impacted by the construction of the Tucuruí dam. The construction of other dams proposed for the Tocantins-Araguaia basin should be planned strategically to minimize overlapping with the Araguaian boto distribution. Coordinated conservation actions are imperative to prevent the Araguaian boto from reaching extinction or near-extinction as some of their Asian counterparts such as the Yangtze, Ganges, and Indus river dolphins.

The newly described Araguaian river dolphins, Inia araguaiaensis (Cetartiodactyla, Iniidae), produce a diverse repertoire of acoustic signals.

The recent discovery of the Araguaian river dolphin (Inia araguaiaensis) highlights how little we know about the diversity and biology of river dolphins. In this study, we described the acoustic repertoire of this newly discovered species in concert with their behaviour. We analysed frequency contours of 727 signals (sampled at 10 ms temporal resolution). These contours were analyzed using an adaptive resonance theory neural network combined with dynamic time-warping (ARTwarp). Using a critical similarity value of 96%, frequency contours were categorized into 237 sound-types. The most common types were emitted when calves were present suggesting a key role in mother-calf communication. Our findings show that the acoustic repertoire of river dolphins is far from simple. Furthermore, the calls described here are similar in acoustic structure to those produced by social delphinids, such as orcas and pilot whales. Uncovering the context in which these signals are produced may help understand the social structure of this species and contribute to our understanding of the evolution of acoustic communication in whales.

Morphology of mucosa-associated lymphoid tissue in odontocetes.

This study describes the mucosa-associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment.

Morphological analysis of lymph nodes in Odontocetes from north and northeast coast of Brazil.

The morphology and location of lymph nodes from seven species of Odontocetes, of both sexes and different age groups, were described. All animals were derived from stranding events along the North and Northeastern coasts of Brazil. After the identification of lymph nodes in situ, tissue samples were analyzed for light and electron microscopy. Vascular volume density (VVD) and vascular length density (VLD) were evaluated in the mesenteric lymph nodes. Lymph nodes occurred as solitary nodules or in groups, varying in shape and size. In addition to using the nomenclature recommended by Nomina Anatomica Veterinaria, new nomenclatures were suggested based on the lymph nodes topography. Lymph nodes were covered by a highly vascularized and innervated capsule of dense connective tissue, below which muscle fibers were observed, inconsistently, in all studied species. There was no difference in VLD among different age groups. However, VVD was higher in adults. Lymph nodes parenchyma was divided into an outer cortex, containing lymph nodules and germinal centers; a paracortical region, transition zone with dense lymphoid tissue; and an inner medulla, composed of small irregular cords of lymphatic tissue, blood vessels, and diffuse lymphoid tissue. Abundant collagen fibers were observed around arteries and arterioles. Germinal centers were more evident and developed in calves and young animals, being more discrete and sparse in adults. The morphology of lymph nodes in Odontocetes was typical of that observed in other terrestrial mammals. However, new groups of lymph nodes were described for seven species occurring in the Brazilian coast.