First record and morphological contributions of Procyrnea leptoptera (Rudolphi, 1819) (Nematoda: Habronematidae) in the kestrel Falco tinnunculus Linnaeus (Falconiformes: Falconidae) from Egypt

The kestrel Falco tinnunculus, Linnaeus, 1758 is widely distributed in Europe, Asia, Africa, and East coast of North America. It is a predator species that belongs to family Falconidae. Nematode species of the genus Procyrnea infects the proventriculus and gizzard of the birds and may cause serious health hazards including inflammation, erosions, and ulcers for the infected gastric mucosa, general weakness and may lead to death. The present study aimed to improve the limited information regarding the endoparasites of Falco tinnunculus in Egypt. Nine males and twenty-one female nematodes were identified as Procyrnea leptoptera, Rudolphi 1819, Chabaud 1958, isolated from the proventriculus of common kestrel; Falco tinnunculus gathered from El-Faiyum governorate, Cairo, Egypt, as a new geographical record. The isolated species has been studied morphologically by light and scanning electron microscopy. It was revealed that it is characterized by two lateral pseudolabia with dorsal and ventral labia. The pseudolabium is divided into 2 lobes at the internal side, a number of medium-size teeth (4–6) in the buccal cavity on the interior border of pseudolapia, in addition to the presence of transverse striations. Also, the histopathological effects of the isolated nematode on the liver of the infected host revealed severe damage in the bird's tissues and morphological alterations including infiltration of inflammatory cells, congestion, focal necrosis, and degeneration of hepatic cells. Raptors like Falco tinnunculus are infected with parasites via prey ingestion, so they are serving as intermediate hosts for many species of parasites. Due to the scarcity of studies for the helminth parasites of raptors in Egypt, further studies are needed to get additional precise data on the helminthic infection of raptors and their histopathological effects.


Study area and sampling
Samples of the kestrel Falco tinnunculus were hunted at El-Faiyum Governorate in the middle Egypt 29.31° N 30.8° E by specialist hunters. El-Fayoum is a representative example of the oases of the great North African Sahara Desert in its form, origin, geologic formation, culture, and ecosystem. It is located less than 100 km from Cairo city, Egypt. It is one of the most beautiful regions in Egypt and has rich heritage of flora, fauna, and archeology.
Eight adults of common kestrel, Falco tinnunculus (three males and five females), ranged in weight from 180 to 260 gm. were collected at June 2020 ( Fig. 1a). They were anesthetized then slaughtered and examined at the biological laboratory, Department of Biological and Geological sciences, Faculty of Education, Ain-Shams University, Cairo, Egypt. Nematodes were isolated from the underlining of proventriculus and gizzard (Fig. 1b). Specimens were washed in physiological saline solution 0.7% NaCl several times under a stereomicroscope and then fixed and preserved in hot 70% ethanol. Some specimens were prepared for light microscopy, and others were ready for scanning electron microscopy.

Light microscopy
The collected nematodes were fixed in hot 70% ethanol, cleared, mounted on slides with lactophenol, and observed under a light microscope. Mounted specimens were photographed using an Olympus C X 31 microscope and an Olympus digital camera E-330-ADU1X Japan. The morphometric studies were based on ten specimens (three males and seven adult females). Measurements were made by 0.01 mm microscope objective micrometer glass scale (Japan) and a calibrated lens. Measurements (range, followed by mean in parentheses) are given in millimeters (mm), unless otherwise stated. The mounted slides were drawn diagrammatically using an Axioplan Zeiss light microscope (Carl Zeiss, Germany) that was outfitted with a camera lucida. One male and one female voucher specimen were deposited in the helminthes collection in the Biological Department, Faculty of Education, Ain Shams University, Cairo, Egypt.

Scanning electron microscopy
The nematode parasites were deposited for 2 h in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (PH 7.2) as a fixative and post-fixed in osmium tetroxide (OsO4) for 2 h, then washed in sodium cacodylate buffer, dehydrated in an ascending ethanol series and critical-pointdried with CO2, coated with gold (Nel-S, 2008). The specimens were examined and photographed by scanning electron microscope (Jeol. JSM-5400) at the Atomic Energy Agency, Cairo, Egypt.

Histopathological preparation
Small pieces of liver were collected from infected and uninfected birds, then fixed in alcoholic Bouin's fluid for 24 h, and then preserved in 70% ethyl alcohol. The fixed tissues were dehydrated in ascending series of ethanol, cleared in terpineol, and embedded in the paraplast wax (melting point 58-60). Sections of 5µ thickness were deparaplastinized, hydrated to water, stained with hematoxylin and eosin, and cleared in xylene and mounted in DPX (Bancroft & Gamble, 2002). Finally, the slides were examined and photographed using an Olympus C X 31 microscope and an Olympus digital camera E-330-ADU1X Japan to determine the histopathological effects of Procyrnea leptoptera on the liver tissues (Additional file 1).

Results
Helminthological examinations of the common kestrel Falco tinnunculus Linnaeus (Falconidae) (Linnaeus) (Mullidae) from El-Fayium Governorate in the middle Egypt revealed the presence of nematodes identified as Procyrnea leptoptera. Thirty nematodes (9 males and 21 females) of medium size were recovered from five out of eight examined hosts. The morphological description of this nematode species is presented herein.

Description
Body is small to medium size, sexually dimorphic, females are longer than males, and their length ratio is 1.19-1.45:1. The cuticle is marked by distinctive transverse striations on the whole body (Fig. 4a). The posterior end of male is coiled while it is broad with pointed tip in female (Fig. 3g, h). The cephalic region is consisting of a short buccal capsule which laterally compressed and labial region that is continuous with body contour. Labial region is consisting of two lateral pseudolabia with dorsal and ventral labia; each has two sub-median lobes ( Fig. 4c and d). The pseudolabium is the widest and divided into 2 lobes at the internal side. As revealed by scanning electron microscope, there are a number of medium-size teeth (4-6) in the buccal cavity on interior border of pseudolabia and two amphids were located near the distal base of the later (Fig. 4d). Among two prominent cephalic papillae, one is large and the other is small are located on the cephalic region on both sides (Fig. 4d). Cervical papillae were present at the level of nerve ring (Fig. 4a). Two lateral alae were present extending just posterior to the excretory pore (Fig. 4b). Esophagus is long divided into short anterior muscular part and long posterior glandular part (Fig. 2a). Many types of annulations were observed. The first type is on the dorsal side of the worm characterized by not deep transverse striations with regular pattern (Fig. 4e). The second type is characterized by deeply corrugated annules separated by an equal distance and divided internally by a longitudinal striation located at the middle body of the worm (Fig. 4f ). Another type of annulations was observed on the dorso-lateral side of the posterior region of body worm that is represented by a series of imbricated tegumental annulations in a regular pattern with more closed distances (Fig. 4i). The caudal alae are present narrow and covered with slight transverse striations (Fig. 3i). The nerve ring is situated approximately at the middle of muscular esophagus (Fig. 2a). Excretory pore is located just posterior to the nerve ring.  (Fig. 3i). Tail measures 0.14-0.19 (0.17); it bears a number of papillae: four pairs precloacal of pedunculated papillae and   (Fig. 2e). Histopathological lesions were noticed in the liver of Falco tinnunculus infected with P. leptoptera nematode as compared with the uninfected ones. These lesions are demonstrated as accumulation of inflammatory cells, congestion, and dilation of the central vein with erosion of its lining endothelial cells. Some specimens showed infiltration of inflammatory cells. Also, it was seen parenchymal lesions in the hepatic lobules accompanied by the loss of their regular pattern. Focal necrosis and degeneration of hepatic cells were also observed (Fig. 5).

Discussion
The present study is the first record of the nematode Procyrnea leptoptera Chabaud 1958, (Habronematidae) from the kestrel Falco tinnunculus (Falconidae) in Egypt. Chabaud (1958) established the genus Procyrnea as a subgenus of Cyrnea. (Chabaud) in 1975 raised it to generic level. Sixty valid species of Procyrnea have been described from avian hosts, especially Falconiformes and Strigiformes. Also, this genus is rarely recorded from Piciformes, Passeriformes, Tinamiformes, and Rheiformes (Bagnato et al., 2018).
However, the present nematode species is morphologically similar with Procyrnea leptoptera in the presence of two symmetrical lateral alae, left spicule with pointed tip, without barbs and absence of median precloacal papillae (Barus et al., 1978). Moreover, our specimens show very close metrical data to that of Procyrnea leptoptera regarding the length of male (7.23-7.58 vs 7.1 mm), the length of left spicule (0.53-0.65 vs 0.64 mm) and the length of gubernaculum (0.026-0.032 vs 0.03 mm). Procyrnea leptoptera as well as the material described here are characterized by the presence of four pairs of preanal papillae and two pairs of postanal papillae.
However, P. leptoptera has a group of eight sessile papillae near tail end that was not observed in the present specimens. Regarding to the female of the present species, it is approximately similar to that of P. leptoptera in the total length (8.62-11.9 vs. 11.7-14.0), the length of muscular esophagus (0.39-0.91 (0.65) vs. 0.36-0.38 mm), the length of glandular esophagus (2.7-2.82 vs. 2.55-2.71 mm), the position of nerve ring and excretory pore in relation to anterior extremity (0.19-0.35 and 0.25-0.38, respectively, vs. 0.23-0.25 mm and 0.30-0.32 mm) and the egg size (39-52 X 24-32 µm vs. 0.032-0.041 X 0.019-0.024 mm). Therefore, based on the present data this is the first record of the nematode Procyrnea leptoptera in Egypt.
The histopathological effects of Procyrnea leptoptera on the liver of Falco tinnunculus showed the appearance of some lesions such as accumulation and infiltration of inflammatory cells, congestion, and dilation of the central vein with erosion of its lining endothelial cells, necrosis, and degeneration of hepatic cells. The circulating antigen toxins and immune complex produced as a result of parasitic infection are responsible for the degenerative changes and necrosis that were observed in the present study (Peter & Robert, 1989). These lesions are coincident with the results of (Yen-Li et al., 2017) who found vasodilation, necrotic cell, and slight chronic inflammation in the infected liver of eclectus parrots (Eclectus roratus) with Filarial nematode.
Moreover, the present lesions are very similar to the finding of (Brener et al., 2006) in their study of the effect of nematode Heterakis gallinarum on the liver of turkey,