Introduction

Dioctophymatidae are composed of nematode species belonging to three genera that parasitize vertebrates: Eustrongylides Jägerskiöld, 1909 and Hystrichis Molin, 1861, which are parasites of the esophagus, proventriculus and/or gizzard of aquatic birds; and Dioctophyma Goeze, 1782, which are parasites of the kidneys of wild and domestic carnivores. All the species include oligochaetes as intermediate hosts (^{ANDERSON, 2000}).

^{Hendricks et al. (1969)} established five species of Hystrichis: Hystrichis coronatus Molin, 1861 in the gizzard of Mergus merganser Linnaeus, 1758 (Anseriformes: Anatidae); Hystrichis pachicephalus Molin, 1861 in the gizzard of Cygnus olor Gmelin, 1789 (Anseriformes: Anatidae); Hystrichis corvi Hendricks, 1969, in the proventricular glands of Corvus brachyrhynchos Brehm, 1822 (Passeriformes: Corvidae); Hystrichis tricolor Dujardin, 1845 in the esophagus of Anas tadorna Linnaeus, 1758 (Anseriformes: Anatidae); and Hystrichis acanthocephalicus Molin, 1861 in the proventricular glands of Phimosus infuscatus Lichtenstein, 1823 (Pelecaniformes: Threskiornithidae), host that is object of this study.

Phimosus infuscatus known as the bare-faced ibis, inhabits shallow-water regions - flooded fields, wetlands and rice fields - where it finds the main items of its diet: aquatic arthropods, crustaceans, mollusks, seeds and leaves (^{BELTON, 2003}). These birds are widely distributed throughout South America and can be observed in all seasons of the year (^{SICK, 1997}).

The helminths reported in P. infuscatus in the Neotropical region include the cestode Chimaerula bonai Georgiev, 2000 (^{GEORGIEV & VAUCHER, 2000}), the nematodes Eustrongylides sp. and H. acanthocephalicus and the trematode Stomylotrema sp. (^{TRAVASSOS & FREITAS, 1942}). The objective of the present study was to describe the histopathological alterations to the proventriculus of Phimosus infuscatus caused by Hystrichis acanthocephalicus and to report new morphometric data on this parasite.

Materials and Methods

The birds examined were collected in the municipalities of Pelotas (31° 46’ 19” S; 52º 20’ 33” W), Capão do Leão (31° 46' 3” S; 52° 26' 55” W) and Rio Grande (31° 51' 12.1” S; 52° 18' 48.0” W), in Rio Grande do Sul State, Brazil. We examined thirty specimens of P. infuscatus (16 males and 14 females), nine of which were donated by the Núcleo de Reabilitação da Fauna Silvestre e Centro de Triagem de Animais Silvestres da Universidade Federal de Pelotas (NURFS-CETAS/UFPel), where they had died. The others were shot using hunting guns between December 2015 and February 2016, with authorization from the Instituto Chico Mendes de Conservação da Biodiversidade – Sistema de Autorização e Informação em Biodiversidade/ICMBIO-SISBIO no. 507541); and from the CEEA/UFPel ethics committee for animal use, under no. 21300013.

The necropsies on the birds and helminth collection and preparation followed the recommendations of ^{Amato & Amato (2010)}.

The proventriculus of each bird was subjected to enzymatic digestion in order to collect parasites. This was done in a solution of 1% hydrochloric acid and 10% pepsin at 37 °C for 45 minutes. After collection of parasites, the specimens were fixed in AFA (solution of ethanol, formaldehyde and acetic acid) and stored in alcohol 70%. The nematodes were clarified using Amman's lactophenol and were observed under an Olympus CX21 microscope in order to identify them and obtain morphometric data. They were then identified morphologically (^{HENDRICKS et al., 1969}). The ecological terms following the recommendations of ^{Bush et al. (1997)}.Voucher specimens (male and female) were deposited in the Coleção de Helmintos do Laboratório de Parasitologia de Animais Silvestres do Instituto de Biologia, Universidade Federal de Pelotas (CHLAPASIL/UFPel), under the number 636.

For histopathological analysis, two proventriculus were fixed in 10% buffered formalin. Each proventriculus was processed in accordance with the recommendations of ^{Caputo et al. (2009)}.

Results

Hystrichis acanthocephalicus was found in the proventriculus of 25 birds. A total of 90 specimens (58 females and 32 males) of H. acanthocephalicus was collected, with prevalence of 83%, average abundance of 3.0 and mean intensity of infection of 3.6 nematodes per host.

The morphometric description was based on five male specimens and five female specimens (adults) and 25 eggs. The females were slightly longer than the males (Table 1), with mean lengths of 37.9 mm and 32.5 mm, respectively. The spines are of uniform size at different points in the anterior end (Figure 1a). The eggs presented morphology specific to the species (Figure 1d and Figure 1e), with a symmetrical format covered by a thick, rough shell marked by a irregularly shaped ridges; morphologically characteristic of Dioctophymatidae. A single spicule was seen in each male. The copulatory bursa also showed uniform measurements among the different specimens (Figure 1b).

Figure 1 (a) Anterior end; (b) Posterior end of male showing the copulatory bursa (arrow) and the spicule (arrow tip); (c) Proximal end of spicule; (d) Posterior end of female showing the egg (arrow tip) in the vulvar opening (arrow); (e) Eggs. 

In the histological analysis, we observed parasite structures in the proventriculus of P. infuscatus. Parasites were found with the anterior end inserted in the mucous glands, directed towards the lumen of the proventriculus. The intermediate and posterior regions were spread along the submucosa, muscle and adventitia layers (Figure 2a). The regions of parasite structures showed infiltration of heterophils, hemorrhage and hemosiderin (Figure 2b). The intermediate and posterior regions were mainly in the muscle and adventitia layers. In these regions, adult females with a pregnant uterus (Figure 2f) and calcified parasites (Figure 2c and Figure 2d) were found. The parasites were delimited by a conspicuous hypereosinophilic halo (cellular remains), a thick layer of multinucleated giant cells and a fibrous connective tissue capsule (Figure 2e). In addition, bordering these regions, a conspicuous inflammatory response with the presence of mononuclear cells and heterophils and hemosiderin (Figure 2b and Figure 2d) was observed. Some areas had necrotic crypts with cellular remains in the lumen and inflammatory responses of different degrees of severity (Figure 2g).

Figure 2 (a) Parasite encapsulated in the proventriculus, extending from the mucosa to the adventitia; (b) Hemosiderin around the anterior end (arrow); (c) Parasite bounded by necrosis (*), inflammatory multinucleated cells (arrow) and fibrosis (arrow tip); (d) Viable parasite (*) bounded by hypereosinophilic halo, surrounded by infiltrated inflammatory of multinucleated giant cells, fibrosis and abundant inflammatory infiltrate of mononucleosis; necrotic parasite (+); (e) External cuticle of the parasite (arrow); necrotic tissue surrounding the parasite (*) and multinucleated giant-cell layer (arrow tip); (f) Adult parasite in adventitia layer; gravid uterus with eggs (arrow); remains of parasite undergoing degradation process and necrosis (*); (g) Normal crypt (N); parasite inside crypt (*); crypt undergoing degeneration process with remains of cells in the lumen (+). 

Discussion

In Brazil, there is only one record of H. acanthocephalicus in the proventricular glands of P. infuscatus (^{VICENTE et al., 1995}), but neither the number of birds examined nor the geographical location were reported. In the present study, the hosts were caught systematically and central geographical coordinates relative to the sampling points were supplied, including for the hosts that were donated by NURFS.

In the morphological description of H. acanthocephalicus in P. infuscatus, only the length and width measurements of the males and females were reported: 2 mm and 35 mm, and 35 mm and 3 mm (^{MOLIN, 1861}), respectively. In the present study, we found differences in length and width, both among the males and among the females, thus agreeing with what was described by ^{Molin (1861)}. The males were shorter and wider than the females (32.5 mm in length and 1.720 mm in width). The females, in turn, were slightly longer and narrower (37.9 mm in length and 1.646 mm in width) than those originally described, and in relation to the males. The esophagus and cuticular spines of the females were also of different sizes, compared with those of the males. Measurements for the copulatory bursa and spicules of P. infuscatus were reported here for the first time. Hence, it was only possible to make morphometric comparisons with other species of Hystrichis.

Hystrichis acanthocephalicus is smaller than H. corvi: the females measure 90.70 mm x 2.11 mm and the males, 40.79 mm x 0.98 mm. However, the copulatory bursa of H. corvi is smaller, with a diameter of 138 µm (^{HENDRICKS et al., 1969}), compared with 253 µm for H. acanthocephalicus (Figure 1b and Figure 1c). Reports of the female length and width are available for H. tricolor and H. coronatus: 35 mm x 5 mm, and 27 mm x 3 mm, respectively. For H. pachicephalus, only the length of the female is known: 30 mm (^{MOLIN, 1861}). Thus, there is a small difference in relation to the measurements of H. acanthocephalicus.

Hystrichis acanthocephalicus and H. tricolor can be differentiated through the morphology of the eggs (^{HENDRICKS et al., 1969}). Hystrichis acanthocephalicus has a shell marked by a network of irregularly shaped ridges and the eggs measure 0.078 μm in length x 0.0432 μm in width. Hystrichis tricolor has eggs of regular shape, measuring 85 μm by 50 μm (^{AL-LABBAN et al., 2013}). The irregular shape of H. acanthocephalicus eggs, along with the disposition of cuticular spines, was essential for species identification in the present study. The morphometric data generated were important for species differentiation.

Hystrichis spp. have been reported in many hosts and geographical regions with varied prevalence, mean abundance and intensity: Hystrichis tricolor was reported in Anas platyrhynchos, in Poland (^{KAVETSKA et al., 2012}) with P = 0.19%; and larvae of H. tricolor were reported in Bufo viridis, in Uzbekistan (^{VASHETKO & SIDDIKOV, 1999}), with A = 0.004 and MI = 0.4, and. B. viridis is probably a paratenic host. ^{Al-Labban et al. (2013)} reported H. tricolor with P = 10%, in ducks (Anatidae), in Iraq. ^{Youssefi et al. (2010)} provided the first report of Hystrichis sp. in the digestive tract of otters (Lutra lutra), in Iran. In a comparative study between Gallinula chloropus cachinnans and Porphyrula martinica (Rallidae), in Florida, ^{Kinsella et al. (1973)} found H. tricolor with prevalence of 10% for Gallinula chloropus cachinnans and 2% for Porphyrula martinica (Rallidae), in the proventriculus of both birds. ^{Canaris et al. (2010)}, in Mexico, reported H. tricolor with P = 58% and A = 5.08 in Limnodromus scolopaceus (Scolopacidae). The results from these studies, except ^{Canaris et al. (2010)}, show distinctly lower rates than those in P. infuscatus (P = 83%). We attribute this difference in prevalence to the climatic conditions of the places where the studies were conducted. The higher prevalence in Mexico and Brazil, which are both in the Neotropical region, probably occurred because of better environmental conditions for the intermediate hosts throughout the year. In the Palearctic and Nearctic regions, the intermediate hosts can complete the life cycle in the summer.

The pathological effects of H. acanthocephalicus have not yet been described. However, the histopathological changes caused by H. tricolor in the proventriculus of ducks were reported by ^{Al-Labban et al. (2013)}, who observed that the cells were distributed irregularly, with infiltration of inflammatory cells. In addition to the inflammatory response in P. infuscatus of this study, parasites were found encapsulated and calcified. Similar inflammatory reactions were also reported by ^{Clark (1978)} in relation to Tetrameres tarapungae Clark 1978, in which the anterior end is also characterized by spines and the organ selected is the proventriculus.

Histrichosis is rare, but can cause large impacts on bird populations, as has been reported in Russia, Ukraine, and Georgia (^{KARMANOVA, 1986}). Our new information on the histopathology of H. acanthocephalicus, in association with morphometric and prevalence data, among other information, will make it possible to recognize outbreaks in the Neotropical region. Finally, the parasitological indices of H. acanthocephalicus in P. infuscatus reported here were the first records for the Neotropical region.