Nematode capilaridae in the tongue of Cerdocyon thous of free life in Brazil Nematoide capilaridae na língua de Cerdocyon thous de vida livre no Brasil

Studies of helminths in road-killed wild animals are still uncommon but may provide promising results since they can identify the parasites in juvenile and adult hosts and meet the recommendations of current discussions on bioethics that prioritize alternative methods for the use of animals. This study evaluated three Cerdocyon thous individuals that were donated after dying from being run over. Two of them had small nematode adults in the epithelial and connective tissues of the tongue. The diagnosis was based on the presence of eggs, observed in histological sections, and morphological characteristics of the nematodes in the tongue. Morphologically, this nematode has a body with transverse grooves, simple mouth opening and no lips, esophagus and stichosome with stichocytes and bacillary bands along the body, which is characteristic morphology of the family Capilariidae and genus Capillaria. The presence of this nematode in the tongue of C. thous is an extremely important fact that contributes to what is known about the biodiversity of zoonotic parasites in wild canid populations. However, an explanation for these findings remains unclear because, until now, this has not been observed in the biological cycle of the species.


Introduction
Studies involving the helminthofauna of wild animals are of great importance for the conservation of species and for public health, since some of these parasites can injure wild populations and have zoonotic potential (LAFFERTY, 1997).
Some helminths that parasitize wild mammals belong to the family Capillaridae.The taxonomy of this family is still being revised and the group contains many species in the genus Capillaria (GUARDONE et al., 2013;FUEHRER, 2014), of which Capillaria hepatica Bancroft, 1893 is the most commonly cited in the literature as a parasite of various domestic and wild animals (FREITAS & LENT, 1936;ARAÚJO, 1967;SILVEIRA et al., 1975;GALVÃO, 1981;CHIEFFI et al., 1981;MANDORINO & REBOUÇAS, 1991;VICENTE et al., 1997;RUAS et al., 2003).Capillaria comprises a complex group of parasites, which has resulted in the proposal of the genera Calodium, Eucoleus, Paracapillaria, Pearsonema and Aonchotheca (GUARDONE et al., 2013;FUEHRER, 2014).Within this complex, three species are of zoonotic importance: Paracapillaria philippinensis (Syn.Capillaria philippinensis), Eucoleus aerophila (Syn.Capillaria aerophila) and Calodium hepaticum (Syn.Capillaria hepatica, Trichocephalus hepaticus Bancroft, 1893 andHepaticola hepatica Hall 1916.Due to anthropic impacts, wild animals frequently come out of natural habitats in search of food and suffer injuries.Roads, starting at the construction phase, impact native fauna in several ways, producing direct and indirect effects on populations, such as mortality from being run over, loss of habitat, dispersion of exotic species and intensification of the presence of humans (ASCENSÃO & MIRA, 2006).The study of helminths in road-killed animals is still uncommon; however, studies that use this methodology are promising since they can identify these parasites in adults and young individuals and meet the recommendations of current discussions on bioethics that prioritize alternative methods for the use of animals (RICHINI-PEREIRA et al., 2006).In Brazil, among the wild carnivores, Cerdocyon thous is one of the most commonly road-killed species (MALHEIROS, 2004;PRADA, 2004;PINOWSKI, 2005).
Based on the few studies about the presence of capillariids in wild animals, this study describes the first record in Brazil of a Capillariidae nematode parasitizing the tongue of C. thous, in the municipality of Paragominas in Pará State, in addition to collecting and analyzing retrospective data on the fauna of helminths of C. thous in Brazil.

Materials and Methods
We evaluated the tongues of three animals (C.thous) that were donated, after being run over by the company HYDRO Paragominas -bauxite mine, to the Laboratório de Pesquisa Morfológica Animal (LaPMA) -Instituto da Saúde e Produção Animal at the Universidade Federal Rural da Amazônia (UFRA), campus Belém (under authorization of SEMA-PA Nº 455/2009 and 522/2009), animals used for morphological studies.
To extract the tongues, used in morphological studies by LaPMA, an incision in the skin and muscles of the mentonian region was made.The tongue was folded ventrally after cutting the lingual frenulum and then cut caudally at the root to harvest the material.The material was fixed in a 10% formaldehyde solution for the histological analysis and in an AFA solution (93 parts 70% ethyl alcohol, 5 parts formaldehyde, and 2 parts glacial acetic acid) for the morphological analysis.Samples of the nematodes were separated from the lingual tissue, dehydrated in an ethanol series, clarified with lactophenol, placed on a microscope slide under a coverslip as a temporary mount, observed using a light microscope, and photographed with a LEICA DM2500 microscope with an image capturing system.This work was conducted in the Laboratório de Histologia e Embriologia Animal -Instituto da Saúde e Produção Animal.For scanning electron microscopy, fragments of the tongue with nematodes were fixed in 10% formaldehyde, post-fixed in 1% osmium tetroxide, critical point dried with CO 2 , coated with gold-palladium and analyzed with a VEGA 3 LMU/TESCAN scanning electron microscope.This was done in the Laboratório de Microscopia Eletrônica de Varredura -Instituto da Saúde e Produção Animal at the Universidade Federal Rural da Amazônia (UFRA), campus Belém.To avoid confusion, only names of genera of helminths are abbreviated and not those of hosts.

Results
Of the three animals evaluated, two had six small adult nematodes in the epithelial and conjunctive tissues of the tongue (Figure 1A and 2A-C).The six nematodes formed galleries within the epithelium (Figure 1D-E and 2B) and their dispersal had a multifocal character.The diagnosis was based on the presence of eggs (FERRER & CASTELLÀ, 1996), observed in histological sections, and morphological characteristics of the nematodes in the tongues, which were analyzed using scanning electron microscopy (body surface) and light microscopy During the histological analysis of the C. thous tongues, adult female nematodes were observed in transverse sections.These were inserted in the basal layers of stratified epithelial tissue, with eggs both inside the parasite as well as egg isolates and clusters within galleries developed by the nematodes (Figure 1D-E).The eggs were barrel shaped with two polar opercula with plugs on the ends, which are diagnostic characteristics of the genus Capillaria (Figure 1B-C).The size and the number of nematodes inserted in the tegument of the coating of the tongue did not cause inflammatory reactions and did not affect the morphology of the lingual papillae (Figure 1D).
A light microscopy analysis of a fragment of a nematode, clarified with Amman's lactophenol, was conducted on the anterior region of the helminth because the rest of the body was strongly inserted in the tissue and it was not possible to completely remove the parasite (Figure 3A-B).Morphologically, the nematodes have a body with transverse grooves, a simple mouth opening and no lips, stichosome esophagus formed by stichocytes and bacillary bands (Figure 2D) on the surface of the cuticle along the body, which are also morphological characteristics of Capillaria (Figure 3A-B).

Discussion
In studies of the nematofauna of C. thous, published between 1915 and 2017, adult and juvenile nematodes in 13 superfamilies (Ancylostomatoidea, Ascaridoidea, Dioctophymatoidea, Filarioidea, Metastrongyloidea, Oxyuroidea, Physalopteroidea, Rictularoidea, Rhabditoidea, Strongyloidea, Spiruroidea, Thichinelloidea, Trichostrongloidea) were recorded in 13 localities in Brazil (Table 1).Of these, 15 families, 18 genera and 24 species of nematodes were identified.Some taxa were not identified to the specific level because the authors only conducted coprological exams.In the cases described as morphotypes, the authors provided morphological characteristics of the eggs (when possible) and/or measurements of the parasite; due to the lack of data, these were classified only as phylum Nematoda (Table 2).
The nematodes found in the tongue of Cerdocyon thous, collected in the municipality of Paragominas, Pará State, in eastern Amazon, have similar characteristics to those of other species of the family Capillaridae Railliet, 1915, and genus Capillaria Zeder, 1800.The diagnosis of infection by Capillaria in this study is based on the presence of eggs with bipolar plugs, as well as the morphological characteristics of the nematodes found in the tongue.Capillariidae, one of the five families belonging to the superfamily Trichinellidea, comprise more than 300 known species        of nematodes that parasitize all classes of vertebrates around the world (ANDERSON, 2000;GIBSON, 2012;STAPF et al., 2013).
In the last 100 years, three taxonomic inventories of parasites in mammals were conducted, resulting in 13 superfamilies reported for the nematofauna of C. thous in Brazil (VICENTE et al., 1997;VIEIRA et al., 2008;PINTO et al., 2011).Among the nematodes that comprise the nematofauna of C. thous, the members of the superfamily Ancylostomatoidea were the most representative in terms of number of species, such as Uncinaria carinii Travassos, 1915, Ancylostoma buckleyi Le Roux & Biocca, 1957, Ancylostoma caninum Ercolani, 1859, Ancylostoma braziliense Faria, 1910, and Ancylostoma sp.In addition to nematode adults, two egg morphotypes were found, as well as individuals that were not identified to the specific taxonomic level; although, they had features of this superfamily.
In Brazil, superfamily Trichinelloidea is represented by two families, (Trichuridae and Capillariidae), three genera (Trichuris, Capillaria, Pearsonema) and the following four species: Trichuris vulpis Froelich, 1789, a parasite of the cecum of the large intestine in host from Mato Grosso do Sul and Paraíba sates (VIEIRA et al., 2008;LIMA 2009;PINTO et al., 2011;CURI et al., 2010;LIMA et al., 2013); Pearsonema linsi Freitas & Lent, 1935 and Pearsonema feliscati Diesing, 1851, parasites of the urinary bladder in host from Rio de Janeiro state (VIEIRA et al., 2008); and C. hepatica, a parasite of the liver in host from Rio Grande do Sul state (RUAS et al., 2003).In addition, Capillaria sp.occurs in different states of Brazil: Rio Grande do Sul (RUAS, 2005;RUAS et al., 2008), Paraná (SNAK et al., 2017) and Minas Gerais (CURI et al., 2010).This is the first report of a Capillaria infection in the tongue of C. thous in Brazil, but the occurrence of capillariids in the tongue of mammals has already been reported for other countries.Copland (1975) reports that C. papuensis parasitizes the tongue of Sus scrofa papuensis in Papua New Guinea.Scapino & Murphy (1978) describe the infection of the mucous membranes of the oral cavity of Procyon lotor, and suggests that the parasite is possibly Capillaria procyonis.Snyder (1988Snyder ( , 1989) ) reports the occurrence of eggs of C. procyonis in the tongue of Procyon lotor; both accounts were for Illinois, in the United States.Capillaria garfiai is reported to parasitize the tongue of wild Sus scrofa in Spain (FERRER & CASTELLÀ, 1996).
Löwenstein & Kutzer (1989) reported C. garfiai in wild boar in Austria, found a prevalence of 69% based on histopathological studies, and demonstrated that parasitism by C. garfiai in the tongue of this animal produces small pathological changes in the lingual tissue, which is different from what is reported in this article, since the nematodes did not cause injury.
In Brazil, parasitism by capillariids includes different species of parasites and hosts, and C. hepatica is the most commonly cited in the literature as a parasite of wild animals, such as those found in Sciurus aestuans (FREITAS & LENT, 1936), Rattus norvegicus (ARAUJO, 1967;GALVÃO, 1981).Rattus rattus (CHIEFFI et al., 1981), dogs and cats (SILVEIRA et al., 1975), Tayassu tajacu (MANDORINO & REBOUÇAS, 1991), Chrysocyon brachyurus (VICENTE et al., 1997) and Lycalopex gymnocercus (RUAS et al., 2003).Eggs of C. hepatica were found in insects (SOLOMON & HANDLEY, 1971;FARHANG-AZAD, 1977).Lima et al. (2008) reported the occurrence of eggs of Capillaria spp. in the filet of Pseudupeneus maculatus Bloch, 1793, a fish sold in the metropolitan region of Recife, Pernambuco state, and noted the zoonotic potential of the genus and the need for more hygienic and sanitary control of the quality of the product offered to the consumer.
For C. thous, Ruas et al. (2003) reported the first case of parasitism of C. hepatica in the state of Rio Grande do Sul.In the state of Pará, Moreira et al. (2013) describes parasitism by C. hepaticum in synanthropic rodents R. norvegicus and R. rattus.Although Moravec (1982) included C. hepatica in the genus Calodium (Calodium hepaticum), this name is rarely used, and many authors place it in Capillaria.
The site of infection of the capillariids reported in this study is possibly an erratic occurrence in the cycle of this parasite, and the hosts acquired the infection due to certain habits, such as licking the genitals and anus of other animals in the same pack.The life cycle of capillariids is monoxenic and the infection occurs by ingestion of infective eggs after death (e.g., due to cannibalism or predation) and decomposition of the host (FARHANG-AZAD & SCHLITTER, 1978;KATARANOVSKI et al., 2010).
The presence of capillariids in C. thous and other wild canids is possibly related to their diet, which includes wild and urban rodents, and is often influenced by habitat loss and the ease of obtaining food close to urban centers.Ruas et al. (2003) describes capillariids as parasites of the hepatic parenchyma and urinary bladder of multiple hosts, which are most commonly found in rodents, especially rats.Pedó et al. (2006) describe C. thous as a versatile predator and that small rodent species are the most important food item in its diet, with a 52.8% relative frequency, including at least seven species from distinct habits, such as open areas (Cavia aperea Erxleben, 1777), semi-aquatic (Holochilus brasiliensis Desmarest, 1819), cursorial in forest and open areas (Akodon sp.), semi-arboreal (Oligoryzomys sp.), arboreal (Phyllomys dasythrix Hensel, 1872), semi-fossorial in forest and open areas (Oxymycterus sp.) and peridomiciliary (Mus musculus Linnaeus, 1758).Birds, insects and fruits, as well as consuming the most abundant food resources in each season, have also been reported (BISBAL & OJASTI, 1980;BERTA 1987;MOTTA-JUNIOR et al., 1994).

Conclusions
The presence of eggs and nematodes of Capillaria sp. in the tongue of C. thous is extremely important, not only as a public health issue, but to the knowledge of the biodiversity of zoonotic parasites present in wild populations of canids.However, an explanation of why the eggs and nematodes were in the epithelial and connective tissues of the tongue remains unclear since, until now, this has not been observed in the biological cycle of the species.Parasitism of C. thous by capillariids in Pará state requires a more detailed investigation into the circumstances of this finding, in order to obtain adults for morphological and molecular analyses that can elucidate the epidemiological implication of this parasitism and the potential zoonotic factor of this species, as well as to determine the wild rodents that can serve as reservoirs for the parasite.

Figure 3 .
Figure 3. Photomicrographs of Capillaria sp.parasitizing the tongue of C. thous in Pará State.(A) Overview off anterior region in lateral position, simple oral opening with no lips and initial portion of stichosome esophagus (esf ); (B) Lateral view of the middle region of Capillaria sp.showing the presence of stichocytes (st).Scale bars: A= 50 µm, B= 50 µm.

Figure 2 .,
Figure 2. Scanning electron microscopy of Capillaria sp.parasitizing the tongue of C. thous in Pará State.(A) Overview of the parasite inserted between the lingual papillae.(B) Detail of Capillaria sp.inserted in the interstitial space formed by keratinized, stratified, flat epithelial tissue.(C) Detail of the posterior region of the nematode strongly inserted in the keratinized, stratified, flat epithelial tissue of the tongue.(D) Detail of the bacillary bands present along the body of the nematode.Scale bars: A= 100 µm, B= 10 µm, C= 50 µm, D= 5 µm.

Table 2 .
Morphological description of egg morphotypes found in Cerdocyon thous, in Brazil, according to different studies conducted between 1915 and 2017.