Helminth parasites of Galictis cuja ( Carnivora , Mustelidae ) , from localities in the Atlantic forest of Brazil

The current study aimed to investigate the helminth parasites of a population of Galictis cuja (Carnivora, Mustelidae) that occur in Atlantic Forest in the Southeastern region of Brazil. We necropsied 18 specimens of G. cuja, collected between January 2009 and May 2014, ran over victims on BR-040 highway, between the municipalities of Duque de Caxias, state of Rio de Janeiro and Juiz de Fora, state of Minas Gerais, localities inserted in Atlantic rainforest Biome. A total of six species of helminths were identified: Dioctophyme renale, Molineus elegans, Physaloptera sp., Strongyloides sp., Platynosomum illiciens, and Pachysentis gethi. Molineus elegans, Physaloptera sp. and P. illiciens were recorded for the first time in this host. Data provided in the current study when compared to the previous reports of parasitism by helminths in G. cuja in Brazil demonstrate that this study is the most representative with this host species.


Introduction
The lesser grison, Galictis cuja (Molina, 1872) (Carnivora, Mustelidae), is the smallest representative of the family Mustelidae.It is restricted to South America, with distribution from northeastern Brazil to southern Peru, western Bolivia, central and southern Chile, Paraguay, Uruguay, Argentina, and southeastern Brazil, at elevations from sea level to 4200 m, and habitats from Atlantic Forest, and cold steppe in Patagonia to exotic forest plantations in Chile (YENSEN & TARIFA, 2003;PREVOSTI & TRAVAINI, 2005;NABTE et al., 2009;ZÚÑIGA et al., 2009;ROCHA-MENDES et al., 2010;BORNHOLDT et al., 2013;POO-MUÑOZ et al., 2014).According to the IUCN (2016), this species is not included in the main endangered categories, and its conservation status is of "least concern".
We consider that the studies about helminth parasites of G. cuja in Brazil have been more representative.The most recent studies on helminths in mammals in Brazil have so far recorded five species of helminths in this host, in several localities in this country (VIEIRA et al., 2008;PINTO et al., 2011).Therefore, the current study aimed to investigate the helminth parasites of a population of G. cuja living in a stretch of Atlantic Forest in the southeastern region of Brazil, and elaborate a checklist of helminth parasites of this host species in South America.
After being collected, the hosts were kept frozen in a freezer at -18 °C until necropsy.The organs were dissected in plastic trays, and washed in saline solution 0.9%.The helminth collected were fixed in AFA (70° GL ethanol, 93 parts; 37% formalin, 5 parts; glacial acetic acid, 2 parts) for 48 hrs, and then stored in 70° GL ethanol.
For microscopic identification the nematodes were clarified in Amann's lactophenol (1: 1: 2: 1 -phenol: lactic acid: glycerin: distilled water) and mounted in temporary slides.Trematodes and acanthocephalans were stained with Mayer's carmalum (HUMASON, 1979) and mounted in Canada balsam for examination as whole mounts.The helminth species were identified in Olympus BX 41 light microscope.
The nematodes were identified at generic level according to Anderson et al. (2009).Sistematic classification of nematodes is according to Hodda (2011).The specimens of the genus Molineus Cameron, 1923 (Strongylida, Molineoidea) were identified according to the bursal rays distribution (ANDERSON et al., 2009) and by the specific morphological descriptions made by Travassos (1921Travassos ( , 1937)).Trematodes were identified at generic level according to Bray et al. (2008), and at specific level according to Rodrigues (1963).Acanthocephalans were identified according to Machado (1950) and Schmidt (1972).The ecological terms and quantitative parameters of infrapopulations were according to Bush et al. (1997).
Voucher specimens of helminths are deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro, Brazil (Table 1).The current study was performed under authorization from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis -IBAMA/SISBIO (Request No 30727-4).

Results
Among the specimens of Galictis cuja examined in the current study, the total prevalence of parasitism was 94.4% (17 infected hosts out of the sample of 18 hosts analyzed).The helminth richness was six species, of which four were nematodes: Dioctophyme renale  1 and 2).
Until the current study, five species of helminth parasites of G. cuja in Brazil were known, and other six species of helminth in this host from Paraguay and Argentina was reported (Table 2).

Discussion
The genus Molineus (Strongylida, Molineoidea) was proposed by Cameron (1923), and its type species is Molineus felineus Cameron, 1923, which is a parasite of the small intestine of Puma (Herpailurus) yagouaroundi (Carnivora, Felidae) in South America.Currently, this genus has 28 nominal species, of which seven have been reported in Neotropical primates and 21 in carnivorous mammals throughout the world, except in Australia (DURETTE-DESSET et al., 2000, 2001).In carnivorous mammals in Brazil, seven species of this genus have been reported: Molineus barbaris Cameron, 1936, which was found to be a parasite in Eira barbara (Linnaeus, 1758)  1938;VICENTE et al. 1997;VIEIRA et al., 2008;LIMA et al., 2013).In Brazil, Molineus elegans also is reported in this type host Saimiri sciureus (Linnaeus, 1758) (Primates, Cebidae) of the state of Pará (CORRÊA et al., 2016).Until the current study, no reports of nematodes of the genus Molineus parasitizing G. cuja existed.Therefore, the current report of M. elegans in G. cuja in Brazil is the first report of this genus of nematodes in this host.Currently, the genus Physaloptera Rudolphi, 1819 (Spirurida, Physalopteroidea), has approximately 104 nominal species, which occur in amphibians, reptiles, birds and mammals throughout the world (PEREIRA et al., 2012).According to Vieira et al. (2008) and Pinto et al. (2011), six species of this genus have been reported in wild carnivorous mammals in Brazil.Physaloptera anomala Molin, 1860, was found in Panthera onca (Linnaeus, 1758) (Felidae) at an unspecified locality (VICENTE et al., 1997;VIEIRA et al., 2008); P. digitata Schneider, 1866, was reported parasitizing Leopardus wiedii (Schinz, 1821) (Felidae) in the state of Pará (NORONHA et al., 2002;VIEIRA et al., 2008) and Puma concolor (Linnaeus, 1771) (Felidae) in the states of Rio de Janeiro and São Paulo (VICENTE et al., 1997;VIEIRA et al., 2008); P. maxillaris Molin, 1860, was reported as a parasite of Conepatus chinga (Molina, 1782) (Mephitidae) in the states of Pernambuco and Rio Grande do Sul (VICENTE et al., 1997;VIEIRA et al., 2008); P. praeputialis Linstow, 1889.was reported in Cerdocyon thous (Linnaeus, 1766) (Canidae) in the state of Paraná (VIEIRA et al., 2008) and in C. brachyurus in the state of Minas Gerais (VICENTE et al., 1997;VIEIRA et al., 2008); P. semilanceolata Molin, 1860, was found parasitizing N. nasua in the states of Mato Grosso do Sul and Pará (VICENTE et al., 1997;VIEIRA et al., 2008); and P. terdentata Molin, 1860, was found in P. concolor in the state of Rio de Janeiro (VICENTE et al., 1997;VIEIRA et al., 2008).Unidentified species of Physaloptera was reported in C. thous in the state of Mato Grosso do Sul, in E. barbara in the state of Amazonas, in L. wiedii in the state of Pará (VIEIRA et al., 2008), in Leopardus pardalis (Linnaeus, 1758) (Felidae) in the states of Mato Grosso do Sul and Pará (TRAVASSOS & FREITAS, 1943;VICENTE et al., 1997;VIEIRA et al., 2008) and in N. nasua in the states of Mato Grosso, Mato Grosso do Sul and Rio de Janeiro (TRAVASSOS & FREITAS, 1943;VICENTE et al., 1997;VIEIRA et al., 2008).In the current study, we found only female specimens of this nematode, therefore, it was not possible to identify the species.The occurrence of an unidentified species of Physaloptera parasitizing C. cuja in the current study is the first record of this nematode in this host species.
The monotypic genus Dioctophyme Collet-Megret, 1802 (Enoplida, Dioctophymatoidea) is composed by D. renale (Goeze, 1782), which is a parasite of the kidneys or peritoneum of mammals, especially of the families Mustelidae and Canidae (VICENTE et al., 1997;ANDERSON et al., 2009).In Brazil, this species has been reported in several wild carnivorous hosts, including specimens of G. cuja that originated from the state of Rio de Janeiro (VIEIRA et al., 2008).
Species of Strongyloides Grassi, 1879 (Rhabditida, Rhabiditoidea) are parasites of several groups of domestic and wild vertebrates throughout the world (ANDERSON et al., 2009).Nevertheless, reports of this genus in wild carnivorous mammals in Brazil are rare and the occurrences have been limited to unidentified species of Strongyloides parasitizing C. thous and Galictis vittata (Schreber, 1776), both in the state of Rio de Janeiro (VIEIRA et al., 2008), and G. cuja in the state of Rio Grande do Sul (MULLER et al., 2009;PINTO et al., 2011).Species of Strongyloides have not been previously recorded in G. cuja in Atlantic forest localities in the states of Rio de Janeiro and Minas Gerais, and therefore this is the first report of this nematode in this host species in these states.The genus Pachysentis Meyer, 1931 (Oligacanthorhynchida, Oligacanthorhynchidae), was proposed to accommodate Pachysentis canicola Meyer, 1931, a parasite of domestic dogs in South America (PETROCHENKO, 1971).Currently, this genus contains 10 nominal species that parasitize domestic and wild mammals throughout the world (PETROCHENKO, 1971;AMIN, 2013).Pachysentis gethi (MACHADO, 1950) was originally described by Machado (1950) and was placed in the genus Prosthenorchis Travassos, 1915 (Oligacanthorhynchida, Oligacanthorhynchidae), from descriptions of specimens collected from E. barbara in the states of Rio de Janeiro and Pará.Vieira et al. (2008) searched for information on helminth parasites in wild carnivorous mammals in Brazil that was stored in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC) and reported the same data as in the original description made by Machado (1950), along with two new records of this species, parasitizing G. cuja and G. vittata, both in the state of Rio de Janeiro.
Platynosomum illiciens (Braun, 1901) (Trematoda, Dicrocoeliidae) described as an intestinal parasite of Falco sp.(Aves, Falconiformes) (TRAVASSOS, 1944).This species was reported by Travassos (1944) and Yamaguti (1958) as an exclusive parasite of the liver of some bird species in Brazil.However, Rodrigues (1963) observed large polymorphs in P. semifuscum Looss, 1907, which is a parasite of European birds; in P. proxilliciens (Canavan, 1937), which is a parasite of Asian birds; in P. fastosum Kossack, 1910, which is a parasite of several species of mammals in Asia and North and South America; and in P. brauni (Freitas & Lent, 1937), which is a parasite of Brazilian birds.This author therefore considered all these species to be junior synonyms of P. illiciens.Pinto et al. (2016) also considered that the morphometric differences observed in these species of Platynosomum were insufficient for specific differentiation, and stated that P. illiciens is a species with wide geographic distribution and host range, parasitizing both birds and mammals.In carnivore mammals from Brazil, P. illiciens has been previously reported as a parasite of G. vittata in the state of Rio de Janeiro (TRAVASSOS, 1944;TRAVASSOS et al., 1969;VIEIRA et al., 2008), of P. yagouaroundi in the state of Pernambuco (BARBOSA & PONTUAL, 1949;RODRIGUES, 1963;VIEIRA et al., 2008).Therefore, the current study is the first report of P. illiciens parasitizing G. cuja.
The quantitative data from this study could not be compared with other previous studies from helminth from G. cuja (Table 1), since these previous studies do not show a number of compatible hosts with the present study, or do not provide quantitative data from parasitism.Some data of mean intensity and mean abundance of certain helminth infrapopulations observed herein, showed a large standard deviation (Table 1), suggesting heterogeneity in size of these infrapopulations.When the 95% confidence intervals of the mean intensity and abundance of these data were analyzed found that the quantitative data of P. gethi (Confidence intervals of mean intensity 0.959, 4.241; and of mean abundance -0.344, 1.744), P. illiciens (Confidence interval of mean abundance -0.439, 3.439), D. renale (Confidence intervals of mean intensity -0.953, 2.047; and of mean abundance -0.098, 0.698), and Strongyloides sp. were not statistically significant (Confidence interval of mean abundance -3.143, 17.743) (Table 1), which indicate that the number of studied hosts could not estimate the totality of the helminth infrapopulations, and that a higher sampling hosts should be analyzed for a more consistent study of the population ecology of G. cuja helminth at the studied area.
The most recent report of parasites in this host was in the study by Vieira et al. (2012), who proposed a new species of nematode, Crenosoma brasiliense Vieira, Muniz-Pereira, Souza Lima, Moraes Neto, Gonçalves & Luque, 2012 (Strongylida, Metastrongyloidea), which was collected in the lungs of one specimen of G. cuja in the municipality of Juiz de Fora, state of Minas Gerais.
Comparison of the data provided in the current study with the previous reports of helminth in G. cuja in Brazil (Table 2) demonstrates that the current study is the most representative report relating to this host species.Here, we studied a considerably sized sample of hosts (18 specimens) and recorded three new occurrences of helminths in G. cuja in Brazil, which is equivalent to 60% of what was previously known about this fauna.

Table 1 .
Species of helminths collected in Galictis cuja of the current study, site of infection, and ecological data of each helminth.

Table 2 .
Checklist of helminth parasites of Galictis cuja from South America.