Globocephalus urosubulatus (Alessandrini, 1909) (Nematoda: Ancylostomatidae) in Brazil: a morphological revisitation

Helminthiasis occurs frequently in wild or domestic pigs in the most varied environments or production systems; however, the literature includes few taxonomic studies for the purpose of expanding this knowledge about the parasitic diversity in these animals. Thus, in order to expand this knowledge regarding parasitic diversity in these animals, the present study reports the occurrence and redescribes Globocephalus urosubulatus infecting domestic pigs in the state of Pará, Brazil, using microscopy. Four hundred and ten specimens of Sus scrofa intestinal nematodes were collected extensively in the municipality of Moju, northeastern mesoregion of Pará and observed under light and scanning electron microscopy. Based on morphological characteristics, the nematodes were identified as G. urosubulatus , and new morphological data were added by light microscopy and scanning electron microscopy for the diagnosis of this parasite.


Introduction
Helminthiasis frequently occurs in wild or domestic pigs in the most varied environments or production systems around the world, but has generally received less attention when compared to parasitological studies in ruminants (Thamsborg et al., 2017). For Hale & Stewart (1979) and Roepstorff et al. (2011), helminth infections in these animals are not always apparent and persist at subclinical levels for extended periods, often leading to death for the animals.
Parasitism in pigs can affect performance in terms of efficient feeding, growth rate, general health and condemnation after slaughter (Weng et al., 2005). There are few studies that detail or inventory the parasitological fauna of wild and domestic species, especially in taxonomic assessments and that trace differences and similarities between different groups of parasites. Therefore, the accumulation of information over time will enable validation of the use of helminths in monitoring the health of ecosystems and pig breeding systems (Bongers & Ferris, 1999;Brandão et al., 2009).
Despite the Brazilian literature involving several studies on the parasitic fauna of pigs, these data are scarce for the Amazon region, mainly regarding helminth diversity in pigs raised extensively and not submitted to the hygienicsanitary inspection process. Among the nematodes present as fauna parasites in pigs in Brazil, the most genera most often recorded genera are Ascaris, Ascarops, Globocephalus, Hyostrongylus, Metastrongylus, Oesophagostomum, Physocephalus, Stephanurus, Strongyloides, Trichostrongylus and Trichuris (D'Alencar et al., 2006;Pinto et al., 2007;Brito et al., 2012;Anjos et al., 2015;Mattos et al., 2020).
The genus Globocephalus is one of the least known and is represented in Brazilian territory by two species G. urosubulatus (Alessandrini, 1909) and G. marsupialis Freitas & Lent, 1936(Vicente et al., 1997. Only G. urosubulatus has been recorded as parasitizing pigs in different rearing systems in Brazil (Freitas & Costa, 1967;Francis, 1978;Carneiro et al., 1979Carneiro et al., , 1980Pinto et al., 2007). Thus, the aim of the present study was to report the occurrence and redescribe Globocephalus urosubulatus infecting domestic pigs in the state of Pará, Brazil, using microscopic tools.

Survey data
The nematodes were recovered from the small intestine of a single pig slaughtered at a market in the municipality of Moju (01º53'02" S; 48º46'08" W), mesoregion of northeastern Pará, with slaughter done without the sanitary control. The intestine was placed in a basket and washed with water, and macro-residues were separated with the aid of a steel granulometric sieve (9.50mm opening). What passed through the sieve was then fixed in 70% ethanol, stored at room temperature according to (Ahn et al., 2015;Kuzmin et al., 2019;Morais et al., 2020) and transported in a 30L bag to the Laboratório de Parasitologia Animal, Instituto da Saúde e Produção Animal, Universidade Federal Rural da Amazônia. Aliquots of the material were separated into petri dishes, observed with the aid of a stereomicroscope (LEICA-ES2) and the parasites collected.

Light microscopy
For morphological and morphometric analysis twenty-five nematodes (15 females, 10 males) were dehydrated in an ethanol series, clarified with Aman's Lactophenol (20%) and observed using a light microscope, and LEICA DM2500 camera with an imaging capture system. Measurements are shown in micrometers as the mean followed by the range, or as otherwise indicated. Taxonomic classification of nematodes was in accordance with Cameron (1924), Freitas & Lent (1936), Vicente et al. (1997) and Anderson et al. (2009).

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Globocephalus urosubulatus in pigs in Brazil Scanning electron microscopy For scanning electron microscopy (SEM), twenty nematodes (10 females and 10 males) were post-fixed in 1% osmium tetroxide, dehydrated to the critical point for CO 2 , metalized with gold-palladium, and analyzed with the VEGA 3 LMU/TESCAN scanning electron microscope at the Laboratório de Microscopia Eletrônica de Varredura, Instituto de Saúde e Produção Animal da Universidade Federal Rural da Amazônia.

Results
A total of 410 [females= 264, males= 146] nematodes were recovered from Sus scrofa. All specimens collected showed characteristics compatible with the genus Globocephalus (Nematoda: Ancylostomatidae). The morphological and morphometric characteristics of the nematodes recovered from domestic pigs are presented below and in Table 1.  Freitas & Lent (1936) a Measurements in micrometers unless indicated and the parameter number of buds is given in amplitude; b Abbreviations: L: length, W: width; c Distance between the vulva and the posterior body end.

Ancylostomatidae Looss, 1905
Globocephalus Molin, 1861 Globocephalus urosubulatus (Alessandrini, 1909) (Based on light microscopy and scanning electron microscopy examination: Figures 1-3) Medium size nematode. Females larger than males, the morphology of the anterior region being similar in both sexes (Figures 1a, b, 3a). Thick cuticle, with thin transverse streaks along the body. Long, rounded cephalic end, circular mouth opening, surrounded by a delicate cuticular ring (Figure 1c, 3b). Subglobular buccal capsule, without cutting plates or teeth on its margin, a pair of large sub-ventral teeth near the base of the capsule (Figure 1d). A rib with small spines reaching the top of the inner surface of the mouth capsule on its back (Figure 3c). Four external cephalic papillae and four internal papillae and a pair of lateral amphids. Well-developed claviform muscular esophagus, opening to the intestine through a strong valve. Excretory pore and spoon-shaped deirids located near the nerve ring (Figures 1a, b, 3a).
There are few taxonomic studies for the genus Globocephalus. Even the original descriptive articles have limitations in their morphological and morphometric data, which makes it necessary to update these data again (Lane, 1922;Cameron, 1924;Freitas & Lent, 1936), as in studies by Nanev et al. (2007) for G. urosubulatus collected from wild boars in Bulgaria and Ahn et al. (2015) to provide morphological features, and measurements of various parts of the adult nematodes by both light and scanning electron microscopies from G. samoensis in wild boars from South Korea.
This is the first study to describe ultrastructural aspects of G. urosubulatus in Brazil, in the world most studies for this species were based on ligth microscopy, but in this study the use of SEM made it possible to observe the mouth opening, distribution of cephalic papillae, deirids, vulva and a panorama of the male's tail, in addition to the presence of a pair of papillae present in the female's tail. According to Cesaroni et al. (2017) nematodes are small and usually have small external morphological characters, although the use of SEM has been used in taxonomic and systematic publications for helminths, its use for large groups such as nematodes this technique is still limited, even if it allows for observation of details that light microscopy hardly detects.
In this study we present new morphological and morphometric data and the first record of G. urosubulatus recovered from pigs in the State of Pará. Different authors have reported the presence of this parasite in the same host in Brazil: Amazonas (Freitas & Costa, 1967), Goiânia and Distrito Federal (Carneiro et al., 1979(Carneiro et al., , 1980, Rio de Janeiro (Francis, 1978) and Bahia (Pinto et al., 2007).
Species of Globocephalus are taxonomically characterized by the morphology and morphometry of the oral capsule, teeth, position of the vulva and the distribution of rays in the male copulatory bursa, as well as the morphology of the dorsal ray (Freitas & Lent, 1936;Ahn et al., 2015). For Nanev et al. (2007) characteristics such as body size, esophagus, nerve ring, length of specimens and gubernaculum used independently as taxonomic criteria for species identification are not reliable.
Based on these morphological features, the nematodes were identified as Globocephalus urosubulatus. In Brazil, only Francis (1978) presented morphometric data for this species recovered from the intestine of pigs in the state of Rio de Janeiro, the morphometry being very similar to this study, with only differences in the location of the vulva in females (3-5mm), while that for the specimens found in Rio de Janeiro, the location was closer to the cephalic region (1.73-2.91mm), which can also be observed in the study by Nanev et al. (2007) for female specimens of G. urosubulatus (2.20−2.40mm) in pigs in Bulgaria. However, the use of young females can justify such variation, which in this work we have resolved by presenting the morphometry of ovigerous females and juvenile females separately.
Globocephalus urosubulatus differs from G. marsupialis, in addition to the host group, in morphology and morphometry, especially the gubernaculum (G. urosubulatus = 60-88µm × G. marsupialis = 38-48µm) and spicules (G. urosubulatus = 337-527µm × G. marsupialis = 338-378µm), in addition to the size of the female esophagus and tail. For Freitas & Lent (1936), although the genus Globocephalus presents a small number of species, it presents difficulties regarding the literature described for the genus, especially due to the impossibility of studying the species, since Molin drawings have weak taxonomic arguments, which would hardly support the species distinction. Morphological and morphometric data for G. urosubulatus and G. marsupialis are presented in Table 1.
Morphologically the G. urosubulatus eggs observed in the womb presented an elliptical or round shape, shell single, thin, smooth and transparent and morulated embryo. Even though the description of the eggs of this species is presented, we do not believe that it is a strong character for differentiation or diagnosis of this parasite, since egg morphology is very similar to that of other species within the Ancylostomatidae. So far, no coprological diagnosis of G. urosubulatus has been made in Brazil, with most of the positive specimens obtained from necropsies (see Freitas & Costa, 1967;Francis, 1978;Carneiro et al. 1979Carneiro et al. , 1980Vicente et al., 1997;Pinto et al., 2007). However, the records of occurrence and prevalence of G. urosubulatus by coprological examinations are also few in the literature: Permin et al. (1999) registered in Ghana (2.7% for eggs and 20% for adults after necroscopic examination); Dadas et al. (2016) in Mumbai demonstrated low prevalence (0.74%) of parasitism. Nwoha & Ekwurike (2011) registered in Abia State (Nigeria) 68% of the eggs of Globocephalus sp. in fecal samples from pigs of different ages. Stojanov et al. (2018) registered a prevalence of 30% of Globocephalu sp. in pigs in Serbia. Pinto et al. (2007) emphasize the importance of nematodes rarely reported in the parasite of pigs literature, especially in Brazil, and they use G. urosubulatus as an example, emphasizing that the conditions of the reproduction environment, especially the free-living ones, favor the maintenance of the parasites in the cycle. Nejsum et al. (2012), states that in addition to the close similarity in the morphology of eggs of different species present and the same host, cases of cross-infection would not be detected by standard coprology methods, and mentions that studies based only on coprological exams, for example, may be the reason that the zoonotic potential of certain nematodes such as T. suis has been ignored so far.

Conclusion
This study describes the occurrence of Globocephalus urosubulatus adding data in light microscopy and scanning electron microscope, in addition to expanding the knowledge about this species and contributing to a better understanding of the diversity of nematodes in pigs in Brazil.