Natural infection by Procyrnea uncinipenis (Nematoda, Habronematidae), a parasite from rheas, an autoctone bird from South America, in emus Dromaius novaehollandiae, a ratite from New Zealand

The present study reports a natural infection of emus, Dromaius novaehollandiae, by the nematode Procyrnea uncinipenis. Five adult emus from a scientific breeding farm at North Fluminense State University located in the city of Campos dos Goytacazes, Rio de Janeiro state, Brazil were necropsied, and their gastrointestinal tract were collected and examined for the presence of parasites from October 2013 to November 2015. Two of the five (40%) emus necropsied were infected with nematodes, and a portion of the nematodes were processed for light microscopy. In addition, two other nematodes (a male and a female) were prepared for scanning electron microscopy. In a female bird, one nematode was collected in the proventriculus and two nematodes in the gizzard and in the male bird four nematodes were collected in the gizzard. The morphological and morphometric analyzes allowed to identify the nematodes as P. uncinipenis, this being the first report of an infection by P. uncinipenis in emus. Therefore, we infer that these emus were naturally infected by nematodes that were considered specific to rheas.

The present study reports a natural infection of emus, D. novaehollandiae, by the nematode P. uncinipenis.

Material and methods
Five adult emus (3 males and 2 females) from a scientific breeding farm regulated by the governmental agency IBAMA under number 18981-2 and approved by Ethics Committee at the Universidade Estadual do Norte Fluminense (North Fluminense State University) located in the city of Campos dos Goytacazes, State of Rio de Janeiro, Brazil were necropsied after natural death, and the gastrointestinal tract were collected and examined for the presence of parasites from October 2013 to November 2015. All applicable institutional, national and international guidelines for the care and use of animals were followed. These birds live in proximity to R. americana from the same breeding. The contents of the proventriculum and gizzard were passed through a sieve with a 75 µm mesh, and the mucosa was observed under a stereomicroscope (Opton TIM-2T, China). The koilin layer was removed and observed for the presence of nematodes. The nematodes that were found were washed in a saline solution (0.09% NaCl). A portion of the nematodes were processed for light microscopy, and two (a male and a female) were prepared for scanning electron microscopy. Scanning electron microscopy. The nematodes were fixed for 2 h in 2.5% glutaraldehyde, 4% freshly prepared paraformaldehyde, and 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2. The nematodes were postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer. The samples were dehydrated in an acetone series, critical-point dried with CO 2 , sputter-coated with gold and examined under a Zeiss 962 scanning electron microscope (SEM) operating at 15 kV. ethical approval. All applicable institutional, national and international guidelines for the care and use of animals were followed.

Results
Two of the five (40%) analyzed emus (one male and one female) were infected with nematodes. In one bird (female), one nematode was collected from the proventriculus and two from the gizzard, under the koilin layer. In another bird (male), four nematodes were collected from under the gizzard koilin layer. No gross pathology was observed.
The morphological and morphometrical analyses identified the nematodes as P. uncinipenis. The nematodes were large and whitish in vivo and had two lateral lips with denticles and two interlabia, four labial papillae and two amphids (Fig. 1a). The esophagus was divided into an anterior muscular portion and a posterior glandular portion. Only one female specimen was measured, which had a total body length approximately 21,724 by 734 wide. Buccal cavity 55 long by 38 wide. Muscular esophagus 357 long by 93 wide, and glandular esophagus 4874 long by 306 wide. Distances from nerve ring, excretory pore and cervical papillae to anterior end were approximately 289, 1368 and 1401, respectively. Anus and vulva with a transverse slit (Fig. 1b,c) opening at 236 and 1484 from posterior end, respectively. Female posterior end with two lateral phasmids and a circular structure at the tip tail (Fig. 1c). Eggs (n = 10) 26 ± 1.31 long, ranging from 24 to 28, by 43 ± 2.36 wide, ranging from 40 to 48.
Two males were measured, and the total body length ranged from 14,855 to 20,861 long by 624 to 625 wide, buccal cavity 47-65 long by 42-60 wide, muscular esophagus 313-516 long by 104-108 wide, glandular esophagus 3305-3442 long by 264-266 wide, nerve ring at 422-432 from anterior end. Excretory pore was not observed in the specimens. Spicules unequal in size and shape, with a proportion of approximately 1:4. Left spicule long and thin, measuring 3224 long, with a more robust spicule head (Fig. 2a). Distal tip ended sharply (Fig. 2b). Spicule head shorter (Fig. 2c). Right spicule short and thick, measuring 741 long, with a curved distal end, similar to a hook, and a dilation at the tip (Figs. 1d, 2d). Gubernaculum well chitinized, "v" shaped ( Fig. 2d)

Discussion
The morphology, observed by light and scanning electron microscopy, and the morphometry of the nematodes collected from D. novaehollandiae are similar to those of the nematode species that infect R. americana in South America, P. uncinipenis, which shows that this exotic bird can host this parasite from rheas, a native bird of the continent. This is the first report of an infection by P. uncinipenis in emus. Other studies that evaluated this species of parasites in emus have not reported the presence of P. uncinipenis 14,15 . There are reports of infection by other nematode species, including Chandlerella quiscali 16 , Baylisascaris spp. 17 , Cyathostoma variegatum 18 , Dromaestrongylus bicuspis, Trichostrongylus tenuis, and Syngamus trachea 19 . This report of P. uncinipenis, a parasite from R. americana, in emus bred in captivity in Brazil together with rheas shows that these birds developed an adaptation to this nematode parasite, which was considered to be host-specific. In rheas, there can be a high intensity infection, with more than 400 nematodes infecting the proventriculus and gizzard, causing widespread necrosis accompanied by a hemorrhagic appearance. The nematodes deeply penetrate the gizzard glands 4 . Although the emus had few specimens of P. uncinipenis in the gizzard (n = 4), infections with a high parasite load can occur and lead to the death of the birds.
The female and male specimens of P. uncinipenis that infected the emus in the present study are smaller than those from R. americana and larger than P. choique from R. pennata (Tables 1 and 2). However, the morphology   www.nature.com/scientificreports/ After analyzing the nematode specimens collected from the gizzard of emus, a bird from Oceania that were introduced in Brazil for commercial and ornamental purposes, along with rheas, a native bird from South America, the present study can infer that the emus were naturally infected by nematodes that were considered specific to rheas.