INTRODUCTION
Manatees are aquatic mammals that are considered vulnerable to extinction according to IUCN (2014), and endangered based on the Brazilian Red List (Mma, 2014). In Brazil, the presence of these animals has been reported on the coast and in estuary areas of all states from Amapá to Sergipe, with some discontinuous areas along the coast (Luna et al., Reference Luna, Lima, Araújo and Passavante2008; Lima et al., Reference Lima, Paludo, Soavinski, Silva and Oliveira2011; Alves et al., Reference Alves, Schwamborn, Borges, Marmontel, Costa, Schettini and Araújo2013).
For a long time, the indiscriminate hunting of animals was considered the major threat to manatee conservation in Brazil (Domning, Reference Domning1982; Lima et al., Reference Lima, Paludo, Soavinski, Silva and Oliveira2011). Moreover, other factors such as accidental death in fishing nets (Meirelles, Reference Meirelles2008), the silting of estuaries, environmental degradation (Nishida et al., Reference Nishida, Nordi and Alves2008; Icmbio, 2011), indiscriminate use of motorized boats (Borges et al., Reference Borges, Vergara-Parente, Alvite, Marcondes and Lima2007), contamination of water resources (Anzolin et al., Reference Anzolin, Sarguis, Diaz, Soares, Serrano, Borges, Souto, Taniguchi, Montone, Bainy and Carvalho2012) and the stranding of manatee calves (Parente et al., Reference Parente, Vergara-Parente and Lima2004; Meirelles, Reference Meirelles2008) contributed to the reduction of the manatee population.
It is well known that the presence of certain pathogens such as bacteria (Vergara-Parente et al., Reference Vergara-Parente, Sidrim, Teixeira, Marcondes and Rocha2003), viruses (Ghim et al., Reference Ghim, Joh, Mignucci-Giannoni, Rivera-Guzmán, Falcón-Matos, Alsina-Guerrero, Rodríguez-Villanueva, Jenson and Bossart2014) and parasites (Borges et al., Reference Borges, Alves, Faustino and Marmontel2011; Bando et al., Reference Bando, Larkin, Wright and Greiner2014) may interfere in the health of these animals and in many cases have been associated with their mortality (Buergelt et al., Reference Buergelt, Bonde, Beck and O'Shea1984; Beck & Forrester, Reference Beck and Forrester1988). So far, about 25 species of helminth parasites have been reported as infecting manatees (Mignucci-Giannoni et al., Reference Mignucci-Giannoni, Beck, Montoya-Ospina and Williams1999a; Lieven et al., Reference Lieven, Uni, Ueda, Barbuto and Bain2011; Bando et al., Reference Bando, Larkin, Wright and Greiner2014), however, clinical signs are associated with the parasitic infection in only a few of these cases (Beck & Forrester, Reference Beck and Forrester1988; Bossart, Reference Bossart, Dierauf and Gulland2001).
From 1991 to 2003, a large copromicroscopic survey using different techniques (i.e. direct methods, flotation and sedimentation) was conducted in Brazil, which showed that all manatees studied were negative for the presence of helminths (Borges et al., Reference Borges, Vergara-Parente, Lima, Brito, Alves and Solamac2004). On the other hand, a research study carried out in the state of Ceará in 2009, detected Pulmonicola cochleotrema in the upper respiratory tract of free-living specimens (Carvalho et al., Reference Carvalho, Meirelles, Motta, Maia, Campello and Bevilaqua2009).
Therefore, in order to bridge the gap on the knowledge of helminths fauna in sirenian species, this study was to assess the frequency of infection by Pulmonicola cochleotrema in Antillean manatees (Trichechus manatus manatus), in the North-eastern region of Brazil.
MATERIALS AND METHODS
Between the years of 1989 and 2014, 88 manatees were studied, 39 specimens representing stranded animals found dead, rescued between the states of Sergipe (11°25′32S 37°19′18W) and Rio Grande do Norte (04°49′55S 37°15′09W). The carcasses were necropsied (Vergara-Parente, Reference Vergara-Parente and Vergara-Parente2005) and careful examinations of the respiratory and digestive systems were performed (Marigo & Andrade, Reference Marigo, Andrade and Vergara-Parente2005).
In addition, considering the actions planned for the strategy of manatee conservation in Brazil (ICMBIO, 2011), 49 live specimens were monitored; some of them during their rehabilitation process and maintenance in pools (N = 23), some during the rehabilitation period in captivity, built in the natural environment (N = 5), and others after reintroduction (N = 21).
All animals in captivity and those reintroduced were physically examined and faecal samples were collected for laboratory analyses. The age of animals was estimated based on data previously published on Florida and Antillean manatees (Marmontel, Reference Marmontel1993; Borges et al., Reference Borges, Freire, Attademo, Serrano, Anzolin, Carvalho and Vergara-Parente2012). In the physical examination, three reintroduced animals eliminated parasites from their nostrils during breathing.
All faecal samples and helminths collected were fixed and preserved in a solution containing alcohol, formalin, glacial acetic acid and distilled water. Afterwards, the faecal material was analysed through sedimentation and flotation techniques (Bando et al., Reference Bando, Larkin, Wright and Greiner2014) and helminths were clarified in lactophenol, stained and mounted on microscope slides for morphological identification (Amato, Reference Amato1985). Parasites were measured and identified using previous descriptions (Blair, Reference Blair1981, Reference Blair, Jones, Bray and Gibson2005; Carvalho et al., Reference Carvalho, Meirelles, Motta, Maia, Campello and Bevilaqua2009).
In order to investigate the interaction between infection by P. cochleotrema and gender (male or female) and/or age (calf, young or adult) the log-linear method was used (Quinn & Keough, Reference Quinn and Keough2002). All analyses were performed by the glm() function of the MASS package (Venables & Ripley, Reference Venables and Ripley2002) of Software R (R Core Team, 2014).
All procedures herein performed were approved by the Sistema de Autorização e Informação em Biodiversidade (SISBIO) (licence number: 33.819–1) and by the Ethics Committee of Animal Experimentation (ECAE) of the Universidade Federal Rural de Pernambuco (licence number: 23082.011095/2013).
RESULTS
Out of 88 animals analysed, 7.95% (7/88) scored positive for the presence of P. cochleotrema in the nostrils (Fig. 1), trachea and/or bronchi. The trematodes herein found presented oval bodies with 6.6 mm in length and 4.83 mm in width. Morphologically they were concave ventrally and convex dorsally. In addition, they presented an oral sucker subterminal with transversal opening, branched intestinal caecum and multilobated and irregular ovaries. Testicles were rounded, multilobulated, and located in the posterior third of the body. All trematodes were identified as P. cochleotrema (Digenea: Opisthotrematidae).
Pulmonicola cochleotrema (Digenea: Opisthotrematidae) found in the nostril of Antillean manatee.
Although a higher frequency of infection was observed in females (71.42%; 5/7), this gender did not present any correlation with the occurrence of the parasite (gl = 1; F = 0; P = 1). Conversely, age was correlated with the occurrence of infection by P. cochleotrema (gl = 2; F = 4.6193; P < 0.001), because all positive animals were adults. On the other hand, no correlation between the positivity and gender/age was observed.
The frequencies of infection in dead and live animals were 10.25% (4/39) and 6.12% (3/49), respectively. Positive results were obtained only in free-living animals, including native and reintroduced species. No parasites were detected in animals maintained in captivity.
In three reintroduced specimens (6.12%; 3/49), respiratory changes (i.e. atypical noises and nasal mucous secretion) were observed. In addition, clinical signs suggestive of bronchitis and bronchopneumonia were reported. Some specimens eliminated parasites from the nostrils during breathing. All clinical signs herein reported are compatible with infection by P. cochleotrema.
All analyses of faeces scored negative for the presence of eggs, larvae and/or oocysts.
DISCUSSION
The frequency of infection by P. cochleotrema observed in the present research study (i.e. 7.95%) was lower than that reported in a previous study in the USA, where a positivity of 38% was detected (Beck & Forrester, Reference Beck and Forrester1988), and in Puerto Rico (Mignucci-Giannoni et al., Reference Mignucci-Giannoni, Beck, Montoya-Ospina and Williams1999b) where 26% of manatees were parasitized by this trematode. Similarly, a study conducted in the state of Ceará, Brazil, reported a positivity of 26.7% (Carvalho et al., Reference Carvalho, Meirelles, Motta, Maia, Campello and Bevilaqua2009).
The findings of this study are relevant and expand the knowledge on the occurrence area (states of Paraíba and Sergipe) of P. cochleotrema in Antillean manatees in Brazil.
Special attention has been given to the factors that contributed to the occurrence of P. cochleotrema in manatees in the last years. Until 2013, no infection had been reported in Antillean manatees (free-living animals or in captivity) in the states of Sergipe, Alagoas, Pernambuco, Paraíba and Rio Grande do Norte. In addition, during a large copromicroscopic survey conducted from 1991 to 2003, and during the examination of manatee carcasses (Borges et al., Reference Borges, Vergara-Parente, Lima, Brito, Alves and Solamac2004), no trematode species were detected.
In order to better understand the epidemiological factors associated with the infection by P. cochleotrema in sirenian species, as well as the host–parasite relationship, it is pivotal to know the biology of this trematode. Indeed, until now, information about its life cycle in manatees, as well as factors influencing its survival in environmental conditions and routes of infection remain unknown (Carvalho et al., Reference Carvalho, Meirelles, Motta, Maia, Campello and Bevilaqua2009).
In fact, it is believed that P. cochleotrema can use molluscs or crustaceans as intermediate hosts (Beck & Forrester, Reference Beck and Forrester1988). These invertebrates are found attached to algae and seagrasses, which are important sources of food for manatees (Borges et al., Reference Borges, Araújo, Anzolin and Miranda2008). Therefore, it is likely that the infection occurs after ingestion of these (Blair, Reference Blair1981; Beck & Forrester, Reference Beck and Forrester1988; Bando et al., Reference Bando, Larkin, Wright and Greiner2014). In addition, it has been proved in other studies that environmental factors such as salinity and water temperature can influence the occurrence of P. cochleotrema (Beck & Forrester, Reference Beck and Forrester1988; Carvalho et al., Reference Carvalho, Meirelles, Motta, Maia, Campello and Bevilaqua2009).
The infection by P. cochleotrema was observed only in native and reintroduced species. Therefore, it is likely that intrinsic factors related to the condition of free-living animals may play an important role in the susceptibility to infection of the Antillean manatees herein studied. Moreover, it has been demonstrated that manatees living in the Florida Peninsula were more exposed to the infection due to their displacements and consequent diversity of food resources ingested by them (Bando et al., Reference Bando, Larkin, Wright and Greiner2014). In Brazil, this sirenian species may also move over a large area, which makes them vulnerable to infection by these parasites (Lima et al., Reference Lima, Alvite, Reid and Júnior2012; Normande et al., Reference Normande, Luna, Malhado, Borges, Junior, Attademo and Ladle2014).
Most likely, the lack of infections by this trematode in animals maintained in captivity, especially in pools with physical and chemical water treatment, occurs due to the absence of intermediate hosts.
Only adult animals were found infected with P. cochleotrema. This finding is similar to another study previously reported, in which only older animals were affected, probably because these animals use a large area of habitat during their life-time (Beck & Forrester, Reference Beck and Forrester1988).
The presence of atypical noises and nasal mucous secretion in animals naturally infected by P. cochleotrema has already been reported (Beck & Forrester, Reference Beck and Forrester1988; Mignucci-Giannoni et al., Reference Mignucci-Giannoni, Williams, Toyos-Gonzáles, Pérez-Padilla, Rodríguez-Lópes, Veja-Guerra and Ventura-González1999b). In fact, clinical signs related to the infection by this trematode are seldom recorded (Bossart, Reference Bossart, Dierauf and Gulland2001), but when they occur the animal may present chronic rhinitis, pulmonary oedema, pneumonia, and in some cases these may lead to death (Beck & Forrester, Reference Beck and Forrester1988).
The absence of helminth eggs and larvae in the faeces herein analysed is similar to the findings previously reported by Borges et al. (Reference Borges, Vergara-Parente, Lima, Brito, Alves and Solamac2004). Therefore, it is important to adopt new tools with higher sensitivity in order to improve diagnoses of parasite infections.
In conclusion, the displacement ability of Antillean manatees along the Brazilian coast might be an important factor for the spreading of P. cochleotrema. In addition, it is important to note that this is the first report of manatee infection by this trematode in the states of Paraiba and Sergipe from the North-eastern region of Brazil.
ACKNOWLEDGEMENTS
The authors kindly thank all the staff of the Fundação Mamíferos Aquáticos and of the Centro Mamíferos Aquáticos/ICMBio, as well as of Projeto Viva o Peixe-Boi Marinho, who were important during the rescue, rehabilitation and reintroduction activities of the manatees used in this study. This paper employed data generated by the Regional Program for Stranding and Abnormal Activity Monitoring in the Sergipe-Alagoas Basin carried out by the Fundação Mamíferos Aquáticos, Instituto de Tecnologia e Pesquisa and Petrobras, as a mitigating measure of the Federal Environmental Licensing conducted by the Brazilian environmental Agency IBAMA.
FINANCIAL SUPPORT
The authors would like to thank CAPES for their financial support.
