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Three Echinostome Species from Wild Birds in the Republic of Korea

Three echinostome species, i.e., Patagifer bilobus, Petasiger neocomense, and Saakotrema metatestis, are newly recorded in the trematode fauna of the Republic of Korea. They were recovered from 3 species of migratory birds (Platalea minor, Podiceps cristatus, and Egretta garzetta), which were donated by the Wildlife Center of Chungbuk (WCC) and the Conservation Genome Resource Bank for Korean Wildlife (CGRB). Only 1 P. bilobus specimen was recovered from the intestine of a black-faced spoonbill (P. minor), and characterized by the bilobed head crown with a deep dorsal incision and 54 collar spines. Twenty P. neocomense were recovered from the intestine of a great crested grebe (P. cristatus), and they had a well-developed head crown with 19 spines and 2 testes obliquely located at the posterior middle of the body. Total 70 S. metatestis were collected from the bursa of Fabricius of 1 little egret (E. garzetta). It is characterized by stout tegumental spines covered in the entire leaf-shaped body, posterior extension of the uterus, presence of the uroproct and a well-developed head crown with 12 pairs of collar spines on each side. By the present study, these 3 echinostome species are newly added to the trematode fauna in Korea.


MATERIALS AND METHODS
Parasite specimens were recovered from the visceral organs of their hosts. Bird carcasses were donated by the Wildlife Center of Chungbuk (WCC) and the Conservation Genome Resource Bank for Korean Wildlife (CGRB). A black-faced spoonbill (Platalea minor), coded as CGRB12252, was collected from Yeocha-ri, Hwado-myeon, Ganghwa-gun, Incheon Metropolitan City, Korea in May 2010. A great crested grebe (Podiceps cristatus) was collected from a factory district in Cheongju-si, Chungcheongbuk-do, Korea in February 2011. The host animal had a fractured clavicle and hypothermia. Veterinarians of the WCC tried to save the bird but it eventually died. A little egret (Egretta garzetta, WCC no. 20120224) with severe fractures was found in Yeongdong-gun, Chungcheongbuk-do, Korea, in September 2013 and was necropsied immediately after death. Carcasses of 2 additional birds were donated to our laboratory for parasitological examinations and stored at -20˚C.
Visceral organs of the hosts were dissected and washed with tap water. Parasitic worms were recovered by filtration through sieves (mesh size: 250 µm to 1 mm). Collected worms were flattened with a coverslip pressure and fixed with 10% neutral buffered formalin. The specimens were stained with Semichon's acetocarmine and were dehydrated through a graded alcohol series. After clearing with xylene, morphological characteristics of each specimen were observed with a light microscope with a reticle.
Examined specimens were deposited at the National Institute of Biological Resources (NIBR), Incheon Metropolitan City, Korea and the Parasite Resource Bank (PRB), Cheongju City, Chungcheongbuk-do, Korea.
Description (n= 1): A ribbon-shaped body with blunt end. The longitudinal muscle was well developed. Tegumental spines were absent. The body length was 12.3 mm and the width was 1.6 mm at the level of the uterus and 1.3 mm at the ventral sucker. The ratio between the forebody and body length was 12.2%. The head collar was large and well developed, bilobed, fan shaped, and with a deep incision. The collar was 937 µm in length and 1,415 µm in width. A total of 54 collar spines were present. Lateral spines were similar in size to the angle spines. The oral sucker was small, measured 283× 370 µm. The pharynx was small, oval in shaped and located immediately behind the oral sucker, measured 253× 224 µm. The esophagus was 341 µm, with an intestinal bifurcation anterior to the cirrus sac. The cecum ran through the lateral side of the body to the posterior extremity. The cirrus sac was elongate-oval in shape, measured 468× 263 µm and was located anteriodorsally to the ventral sucker. The ventral sucker was large, well developed, and measured 1,190× 995 µm. The uterus was filled with numerous eggs, loops intercaecal, and located between the ventral sucker and ovary. Eggs in the uterine loops (n= 23) measured 95-105 (96± 18)× 52.5-57.5 (54± 1) µm. The ovary was oval in shape, displaced by the uterine loops, and measured 175× 335 µm. Mehlis' gland was oval in shape, located behind the ovary, and measured 458× 292 µm. The vitelline follicles were located laterally on the body, between the ventral sucker and the posterior extremity and overlapped the cecum. The excretory vesicle and the pore were obscured. There were 2 testes, tandem: the anterior testis measured 1,035 × 332 µm and the posterior testis measured 1,308× 332 µm ( Fig. 1A-C).

DISCUSSION
The present study revealed 3 unrecorded echinostome species in Korea. Since the first report of Echinostoma revolutum by Issiki [3], more than 14 echinostome species have been identified in Korea. Park [4] recovered Echinostoma hortense from rats, while Yamaguti [5] identified Acanthoparyphium marilae in Melanitta nigra, as well as A. tyosenense in M. nigra america and M. fusca stejnegeri as new species in Korea. E. cinetorchis and Euparyphium murinum were also recovered from wild rats by Seo et al. [6]. Chu et al. [7] reported the presence of E. revolutum, E. gotoi, E. miyagawai, and Echinoparyphium koizumi in wild birds. Echinochasmus perfoliatus was found from cats in Daegu-si [8]. Eom et al. [9] found E. miyagawai and E. japonicus in domestic ducks in Korea. However, E. miyagawai was considered as synonym of E. revolutum by Yamaguti [2,17] and Bykhovskaya-Pavlovskaya [18], but this species was re-validated upon closer examination of various characteristics, including the morphology and life cycle [19,20]. Lee et al. [11] found E. revolutum and Echinoparyphium recurvatum from house rats in Yangyang-gun, Gangwon-do and considered E. koizumi that reported by Chu et al. [7] as a synonym of E. recurvatum.
The worms recovered from Podiceps cristatus had 19 collar spines, including obvious longer angle spines, oblique to symmetrical testes, an elongate-oval cirrus sac and a tubular cirrus. These morphological characters were in line with those of P. neocomense, including size variation [27]. P. neocomense was first recovered from P. cristatus in Lake Neuchàtel by Furhmann [28]. In Korea, the host of P. neocomense is also P. cristatus. P. neocomense resembles P. nitidus morphologically but can be easily distinguished by the shape of the cirrus and the cirrus sac, as well as by geography (Eurasia vs North America) [27].
P. bilobus is the type-species of the genus Patagifer and was recovered from the Threskiornithidae birds in Europe, Asia, Africa, Australia, and America [29]. P. bilobus was the only known species found in spoonbills (genus Platalea) [29]. In this study, we recovered only 1 damaged specimen, which had similar characteristics to P. bilobus; a ribbon-like body, non-confluent vitelline fields, 4 pairs of angle spines on the ventral lappet and a deep dorsal incision on the head collar [29]. Our specimen was missing some of the tegumental spines and a section of the collar spines; however, we could confirm traces of these spines and identified the specimen as P. bilobus based on the morphology and host ranges. P. minor was added to the list as a new host of P. bilobus and was the first reported case of an echinostome fluke from the Threskiornithidae birds in Korea.
S. metatestis was first described by Saakova in 1952 and named Opisthotrema metatesti. However, in 1956, Skrjabin and Baschkirova [30] identified it as an echinostome species based on its echinostomid characteristics. They created a new genus, Saakotrema, for the fluke of Saakova and assigned it to the genus Saakotrema in the family Echinostomatidae [30]. The specimens collected in this study were identical morphologically (leaf-shaped body, posterior extension of the uterus, presence of the uroproct) and in size to S. metatestis (Table 2). S. metatestis has been recovered from piscivorous birds in Europe and Asia [31], including China [32]. This is, so far, the first report of S. metatestis in Far-East Asia.
Among echinostomes reported in Korea, 7 species (A. tyosenense, E. japonicus, E. perfoliatus, E. cinetorchis, E. hortense, E. revolutum, and E. recurvatum) were known as zoonotic parasites infecting humans [33]. There was no evidence that the 3 present species could cause human infections. However, in the case of A. tyosenense, it was primarily considered only as a parasite of birds [5], but 60 years later it was revealed to be a zoonotic parasite [34]. Thus, further monitoring is required in aspects of public health.
The Korean peninsula bridges China and Japan, so we can assume that helminth fauna overlaps the 2 countries. However, the investigation of parasitic fauna in Korea is not thorough enough. In China [32] and Japan, some faunistic revisions are available, but not in Korea. In this study, we found 3 unrecorded echinostome flukes from migratory birds in Korea and presented new area records for them. It is likely that many more unrecorded parasites are present in Korea, underlining the needs for additional studies.