Detected in the Body Cavity of Garrulus Glandarius Brandtii from Republic of Korea

pISSN 1598-298X / eISSN 2384-0749 J Vet Clin 36(3) : 133-138 (2019) http://dx.doi.org/10.17555/jvc.2019.06.36.3.133

Description of Diplotriaena manipoli (Nematoda: Diplotriaenoidea) Detected in the Body Cavity of Garrulus glandarius brandtii from Republic of Korea

Eui-Ju Hong, Si-Yun Ryu, Joon-Seok Chae*, Hyeon-Cheol Kim**, Jinho Park***, Jeong-Gon Cho***, Kyoung-Seong Choi****, Do-Hyeon Yu***** and Bae-Keun Park1 College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea *Laboratory of Veterinary Internal Medicine, BK21 PLUS Program for Creative Veterinary Science Research and College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea **College of Veterinary Medicine, Kangwon National University, Chuncheon 24341, Korea ***College of Veterinary Medicine, Chonbuk National University, Jeonju 54896, Korea ****College of Ecology and Environmental Science, Kyungpook National University, Sangju 37224, Korea *****College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea

(Received: November 12, 2018 / Accepted: June 03, 2019)

Abstract : The present study was performed to identify the nematodes recovered from the Eurasian jay, Garrulus glandarius brandtii, from Daejeon Metropolitan City, the Republic of Korea. Total five nematode worms were detected in the body cavities of two out of the twenty birds necropsied, and they were identified using morphological features, light and scanning electron microscope (SEM), and molecular (18S rRNA analysis) methods. The nematodes were all female Diplotriaena manipoli and had numerous eggs at different developmental stages in the uterus. The nematodes were long and slender measuring about 123-145 mm. The eight submedian cephalic papillae were arranged into four large, outer papillae and four small, inner-circle papillae. A pair of amphidal pores were located at the lateral portion of the mouth. The manubrium apex of trident was rounded and three branches of trident were bluntly rounded at the posterior ends. Using 18S rRNA partial sequence arrangements, DNA analysis of nematode worms was also carried out, and they were identified to be close to the Serratospiculum tendo based on a phylogenic tree analysis. To our knowledge, this is the first report on the molecular characterization and SEM study of D. manipoli. Key words : Diplotriaena manipoli, Garrulus glandarius brandtii, abdominal cavity, Korea.

Introduction ing results owing to the periodicity exhibited by microfilar- iae. However, at necropsy, the detection of worms in the air The genus Diplotriaena belonging to the family Diplotri- sacs or thoracic and abdominal cavities has diagnostic value aenidae was established by Henry and Ozoux (9). The world- (11). wide occurrence of these worms has been reported. The Eurasian jay, Garrulus glandarius, is currently classified prevalence of Diplotriaena may be related to the migratory into a total of 33 subspecies (22). In Republic of Korea habits of the birds, thus lacking intermediate host speciﬁcity (ROK), Garrulus glandarius brandtii is found as a resident (26). Past reports have described the occurrence of Diplotri- bird. In this study, we describe the morphology and molecu- aena spp. in different parts of the world, such as Europe, lar characteristics of female D. manipoli found in G. glandar- Russia, Australia, Asia, America and Africa. About 77 spe- ius brandtii from Daejeon, ROK. cies of this genus have been reported in the literature (2,3,5,8,13,15,23,25,28). Case All species of the genus Diplotriaena are nematodes that parasitize the air sacs, lungs, and body cavity of birds. From April 2013 to June 2018, a total of twenty dead bod- Diplotriaena is pathogenic in birds, and causes pneumonia, ies of G. glandarius brandtii were received as donation from lung consolidation, central nervous system disturbance, diar- the Daejeon Wildlife Rescue Center. They were all dissected rhea, marked weight loss, loss of appetite and sudden death in the laboratory of College of Veterinary Medicine, Chun- caused by larval and adult migrations. Also, they are well gnam National University in Daejeon, ROK. Around two to known to often cause subcutaneous emphysema, pneumonia three nematodes each were found in the body cavity of two and/or air sacculitis, including fatal cases (2,4,11,22,32). of those deceased birds (Fig 1). The worms were tentatively Diagnosis in alive birds, using blood smears, has given vary- identified by observation under the light microscope (LM), and later were precisely classified using the scanning electron microscope (SEM) and molecular methods. For the LM 1Corresponding author. study, the worms were placed in the lacto-phenol solution E-mail : [email protected] (glycerin 20 ml, lactic acid 10 ml, phenol 10 ml, D.W. 10 ml)

133 134 Eui-Ju Hong et al.

Fig 1. Gross finding of D. manipoli (arrows) in body cavity of G. glandarius bandtii. Scale bar = 1 cm. for 24 hrs. For the SEM study, the parasites were washed five times with 0.2 M cacodylate buffer (pH 7.3) and fixed in 2.5% glutaraldehyde, followed by fixing in 1% osmium o tetroxide at 4 C. The specimens were then dehydrated in a Fig 2. Light microscophical view of female D. manipoli. (A) A graded ethyl alcohol series, dried by using a CO2 critical pair of trident (circle). Scale bar = 50 µm. (B) Diagram of tri- point dryer, coated with gold, and examined under SEM (S- dent. (C) A trident. Apex is rounded (square). Scale bar = 20 4800, Hitachi) at 15 kV. µm. (D) Lateral view of tail part. Scale bar = 20 µm. Genomic DNA was extracted from the worms by using a DNeasy® blood and tissue kit (Qiagen, Alameda, CA) according to the manufacturer’s instructions. Using 18S ribo- les, CA) using EmeraldAmp GT PCR master mix (Takara, somal RNA (18S rRNA) gene sequences of Setaria digitata, Shiga, Japan) with a 1 µl aliquot of template DNA. The PCR common nematoda primers were designed using the online products were next visualized via electrophoresis on 1.2% tool (Primer3Plus, http://www.bioinformatics.nl/cgi-bin/prim- agarose gel, and then purified using QIAquick PCR purifica- er3plus/primer3plus.cgi). The oligonucleotide sequences of tion kit (Qiagen, Alameda, CA). PCR amplicons were directly primers employed to detect 18S rRNA gene (DNA) in the sequenced using ABI Prism Big Dye Terminator v3.0 ready worms were 5'-GTCTTGTACCGGCGACGTAT-3' (forward) reaction kits in cycle sequencing (Applied Biosystems), with and 5'-TTTTCTCGAAACGGCTCAGT-3' (reverse). The the same primers as those used in PCR. The sequencing data primer set were designed to amplify a fragment of around revealed that the amplicons were around 1349 bp. Further- 1397 bp, which varies in the different sequences aligned. more, the sequence data were aligned by Clustawl Omega Under standard conditions of denaturation at 95oC for 30 sec, program (Clustawl O 1.2.1). Phylogenetic tree was con- annealing at 60oC for 30 sec, and extension at 72oC for 1 min, structed based on sequence analysis using the neighbor-join- polymerase chain reaction (PCR) was performed in a MyCy- ing (NJ) method and blast tree (http://www.ncbi.nlm.nih.gov/ cler Personal Thermal Cycler (Bio-Rad Laboratories, Hercu- BLAST). NJ method used were based on a guide tree as

Fig 3. The developmental stages of eggs from D. manipoli uterus. Scale bar = 20 µm. (A) Eggs in the early stage and embrionated egg (circle) which is fully developed. (B) The early stage eggs. (C) Unembrionated egg. (D) The early tadpole stage egg. (E) The tadpole stage egg. (F) The fully developed embrionated egg. Description of Diplotriaena manipoli (Nematoda: Diplotriaenoidea) 135 parameters for pair wise and multiple alignment (10). The nematodes were long and slender measuring in 123- 145 mm (Fig 1). Body was elongated and slightly tapering with rounded ends (Fig 1, 2A-2D). Cuticle was smooth with fine transverse striation (Fig 4C-4E). The worms were all female and had numerous eggs detected in their uterus, with the eggs being in different developmental stages. The size of eggs from the uterus varied based on their developmental stages and were measured as being in the range of 68.5-40.0 µm × 53.0-22.5 µm (Fig 3). The mean size of the fully developed eggs was 64.45 µm × 50.96 µm (n = 20). The oral opening was a small dorso-ventral slit without lips sur- rounded by a large rectangular plate whose sides were slightly pressed inward by large circular plates surrounding the openings of tridents. In SEM study, the eight submedian cephalic papillae were arranged into four large, outer papillae and four small, inner-circle papillae (Fig 4A). A pair of oval amphidal opening was located at the lateral portion of the mouth. Two amphidal openings connected with the manubria of tridents were present on the sides of esophagus. In LM study, the manubrium apex of trident was rounded Fig 4. SEM finding of female D. manipoli. (A) Apical view of (Fig 2B, 2C). Three branches of trident were bluntly rounded anterior end; ampid (arrow); Mouth opening (square); subme- at the posterior ends and had no cleft (Fig 2A-2C). The max- dian cephalic papillae (circle). Scale bar = 20 µm. (B) Apico- µ imum size of the longest trident measured was 94.44 m× ventral view; incisura (arrow). Scale bar = 10 µm. (C) Vulva and 9.45 µm. The maximum size of shortest trident measured excretory pore (circle). Scale bar = 10 µm. (D) Ventro-lateral was 72.22 µm × 9.10 µm. Vulva and excretory pore opened view of tail; anus (circle). Scale bar = 40 µm. (E) Anus. Scale ventrally at 577.77 µm (n = 5) posterior incisura portion from bar = 10 µm. (F) Cutting view of midbody; two uterus (U) con- the anterior extremity (Fig 4B, 4C). In the cutting view of the sist of stratified squamous epithelium; intestine (I) consist of SEM study, two uteruses consisting of stratified squamous simple columnar epithelium; neural cord (arrow). Scale bar = epithelium, the intestinal simple columnar epithelium, and a 200 µm. neural cord were present (Fig 4F). The body cuticle had smooth striations (Fig 4C, 4E) and the anus was noted to be similarity to that of Oxyspirura petrowi (97%), Cyrnea simple (Fig 4D, 4E). Tails had no appendages and were mea- seurati (97), Setaria digitata (97%), Cyrnea mansion (97%), sured to be 128.24 µm-135.38 µm (Fig 2B and 4D). Serratospiculum tendo (97%), Cyrnea leptoptera (96%), Spi- Using the ITS1 region of 18S rRNA in Diplotriaena spec- rocerca sp. (96%), Spirocerca lupi (96%), and Streptophara- imen from wild bird (G. glandarius brandtii), its genomic gus sp. (96%), as shown in Fig 5. Although the DNA DNA was analyzed by sequencing. We defined partial ITS1 sequences of Diplotriaena specimens were not completely sequences of the Diplotriaena specimens containing identical matching with above sequences of several nematode worms, sequences that belonged to the same clade. BLAST revealed their identification was supported by the fact that they the ITS1 region of their 18S rRNA sequences shared high belonged to the same clade as Diplotriaena, in the phyloge-

Fig 5. Phylogenetic tree (Neighbor Joining) based on sequence analysis. 136 Eui-Ju Hong et al.

Table 1. Sequence of Diplotriaena specimens (Nematoda: Diplotriaenoidea) CGTTCCGCCTTATCACTTTCGATGGTAGTTTATGTGCCTACCATGGTTGTAACGGGTAACGGAGAATAAGGGTTCGACT CCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCTCGGAATG AGGAGGTAGTGACGAAAAATAACGAGACCGTTCTCTTTGAGGCCGGTTATCGGAATGGGTACAATTTAAACCCGTTAA CGAGAATCTATGAGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCTCAAAGTGTATATCGTTATTGCTGC GGTTAAAAAGCTCGTAGTTGGATCTTCGTCTTAGGACCTGGTCCGTCCATTGGACGAGAACTGGGAACCTAGACTTCA TTGGCCAGTTTTCCCTATGTTACCTTAATTGGTTGCATAGGGTGGCGGGCAAGTTTACCTTGAAAAAATTAGAGTGCTC AATGCGGGCTAATGCCTGAATATTCGTGCATGGAATAATGAAATAGGATCTCGGTTCTATTTTGTTGGTTTTCTGATCTG AGATAATGGTTAAGAGGGACGGACGGGGGGCATTCGTATCGCTGCGTGAGAGGTGAAATTCTTGGACCGTAGCGAGA CGCCCGACTGCGAAAGCATTTGCCAAGAATGTCTTCATTAATCAAGAACGAAAGTCAGAGGTTCGAAGGCGATCAGA TACCGCCCTAGTTCTGACCGTAAACGATACCAACTAGCGTTCCGTTGGCGGTAAATACGCCTTGACGGGCAGCTTCCC GGAAACGAAAGTGTTTCGGTTCCGGGGGAAGTATGGTTGCAAAGCTGAAACTTAAAGAAATTGACGGAAGGGCACC ACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTCACCTGGCCCGGACACCGTGAGGATTGACA GATTGAGAGCTCTTTCATGATTCGGTGGTTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTTATTC CGATAACGAGCGAGACTCTAGCCTACTAAATAGTTACTGGATAAGATCACAAACGTCCAGATAACTTCTTAGAGGGAC AAGCGGTATTTTAGCCGCATGAAGTTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCAGGGCTGCACGCGCGCT ACACTGGAGGAATCAGCGTGCTGTAACCATTGCCGAAAGGTATTGGTAACCCCTTGAAAATCCTCCGTGATCGGGATC GGGAATTGCAATTATTTCCCTTGAACGAGGAATTCCTAGTAAGTGTGAGTCATCAGCTCACGTTGATTACGTCCCTGCC CTTGTCACCCGCCCTGTTGCCC netic tree based on the NJ method and the ITS1 rRNA gene Dewi and Zhang (7) reported that Diplotriaena anthreptis that was used. When the sequence of the Diplotriaena speci- was found in Amthreptes malacensis from Kangean Island, men was compared with genes from other nematodes, it was Indonesia. Diplotriaena lagopusi and D. andersoni were found to have closest similarity to S. tendo (AY702704.1). found in Lagopus leucurus (21). Diplotriaena bargusinica was found in different hosts, such as Chrysomus ruficapillus Discussion (27) and Turdus (Turdus) eunomus (30). Diplotriaena monticolae was isolated from Monticola solitarius magnus (30), Diplotriaena is a parasite with a heterogenous dual-host Diplotriaena nipponensis from Turdus cardis (31), Diplotri- life cycle. The eggs containing the first stage larvae are aena thomasi from Zonotrichia albicollis (23), Diplotriaena released from infected birds through feces and are ingested utae from Perisoreus Canadensis (29), and Diplotriaena pas- by the locust (order Orthoptera) which is their intermediate seri from Passer domesticus (17). host. The definitive host (tit) feed on the infected locust con- Five species, namely, D. bargusinica from Tordus euno- taining the third stage larvae. The larvae invade the intestine mus, D. manipoli from Garrulus glandarius japonicus, D. and then migrate to liver through the bile duct, where molt- monticolae from Monticola solitarius magnus, D. nipponen- ing of 3rd stage cuticle occurs, and is followed by their turn- sis from Turdus cardis cardis, and D. henryi from Poecile ing into fourth stage larvae. The fourth stage larva migrates varius have been reported in Japan (30,31). In ROK, Kim to heart through portal vena and then to aerial sac through and Rim (12) were the first to report the isolation of D. blood, where worms become adult male and female. After manipoli from G. glandarius brandtii, similar to the findings mating, the released ova travel through aerial sac to reach from this study. upper respiratory tract; from there the swallowed ova go to Taxonomy of the Diplotriaena spp. is based on morpho- digestive tract and is released from the tit by defecation. logical characteristics such as, body size, texture and apex Since the adult stage occurs in the aerial sac, it causes respi- format of the trident, number of caudal papillae, shape of the ration difficulty (19). This genus consists of the large nema- spicules, and size of eggs (26). Dewi and Zhang (7) dis- tode parasites that are usually found in the air sac, lung and/ cussed that D. anthreptis is similar to Diplotriaena ozouxi, or body cavity of birds (1,16). Anderson (1) reported that Diplotriaena obtuse, D. bargusinica, Diplotriaena isabellina they were also found in body cavities, but that finding was and Diplotriaena delta, all of which belong to this genus. considered erroneous and was believed to have occurred due However, D. anthreptis differs from other similar species in to postmortem migration. In our study, a total of five nema- the structure of the tridents, and in that the apex of the manu- tode worms were detected in the body cavity of two birds. bria is concave (versus not concave in others), and the mid- The final or definitive hosts of the Diplotriaena species dle branch of the trident has a concaved posterior end (versus include a variety of passerine species (1,16). Diplotriaena flat in others). D. utae have trident with flattened apex and henryi was found in different hosts, such as Athene noctua, varying numbers of rounded elevations (29); D. bargusinica Asio otus (Strigiformes), Actenoides princes, Coracias garru- have tapered apex (27); D. lagopusi have tridigitate apex; D. lous, Merops apiaster (Coraciiformes), Alauda arvensis, andersoni have rounded apex (21); Diplotriaena couturier Anthus spinoletta, Galerida cristata, Lanius excubitor, have two grooved apex and lack elevation (2); Diplotriaena Lanius cristatus, Laniu. minor, Parus major, Turdus philome- ezzati have a sharply pointed apex (25). Seibert (23) illus- los, Turdus pilaris, Turdus ruficollis, Turdus viscivorus, Tur- trated that, D. manipoli have no cleft in their trident and dus sp., Zosterops erythropleura, Parus major, Sturnus Diplotriaena bifidus have cleft in one arm of their trident, in vulgaris, Garrulus glandarius and Corvus cornix (14,24). his report on the criteria for identification of Diplotriaena. Description of Diplotriaena manipoli (Nematoda: Diplotriaenoidea) 137

The size of female trident was 83-102 µm in D. anthreptis There are few SEM studies of Diplotriaena. D. anthreptis (7), 120-246 µm in D. lagopusi, 80 µm in D. andersoni (21), have a pair of lateral amphid on the inner circle; two oval 150-177 µm in D. henryi (18), 110-130 µm in D. bargusi- openings connected with manubria of tridents are present on nica (27), up to 240 µm in D. monticolae (30), 84-105 µm in both sides of the esophagus; two lateral branches of trident D. manipoli (30), 130-135 µm in D. nipponensis (31), 100 µm are bluntly rounded at the posterior ends, middle one has a in Diplotriaena nagpurensis (8), and 54 µm in D. thomasi concaved posterior end; the esophagus divided into short (23). posterior parts; and cuticle with smooth striation (7). We In our study, the trident was distinguished from the other could not find the difference of those descriptions in our nematodes: the apex of the manubria was rounded and the study. trident had no cleft and the length of trident was measured as As a nuclear ribosomal DNA, 18S rRNA gene was used to 94.44-72.22 µm. study inter- and intra-specific relationships studies because it The female in our study (123-145 mm) was longer than is highly repetitive and contains variable regions flanked by that reported for D. bargusinica (48.73-60.55 mm) (27), D. more conserved regions (6), and is used for diagnostic pur- andersoni (65-75 mm), D. lagopusi (60-80 mm) (22), D. poses (20). Although the genetic divergence of Diplotriaena anthreptis (83-102 mm) (7), D. monticolae (44-52 mm) (30), genus suggests their nonspecific relationship with other nem- D. nipponensis 83-114 mm (31) and similar to D. manipoli atode worms, the sequence of Diplotriaena specimens can be (88-126 mm) (30), D. henryi (94-134 mm) (18), D. nagpu- separated from those worms such as O. petrowi (LC316613.1, rensis (135.6 mm) (8), D. thomasi (112 mm) (23), D. passeri KF110800.1, KF110799.1), C. seurati (EU004816.1), S. dig- (158-176 mm) (17). itata (DQ094175.1), C. mansion (AY702701.1), C. leptop- D. anthreptis can be distinguished from D. ozouxi by their tera (EU004815.1), Spirocerca sp. (AY751498.1), S. lupi two alae on the right spicule (versus one ala in others). D. (AY751497.1), and Streptopharagus sp. (HM067977.1). anthreptis is also different from D. bargusinica by the size Although the sequence of Diplotriaena specimens did not difference in their spicules. D. anthreptis have a longer left have a high similarity with the sequence of S. tendo spicule (910 µm versus 700 µm), two alae in their right spic- (AY702704.1) compared to above sequences of several nem- ule (versus one in others), a difference in the spicule ratio atode worms, both sequences were shown in a monophyletic (1:1.6 versus 1:1.1-1.3) (7). D. anthreptis can be separated tree (27), as shown in Fig 5. To our knowledge, this is the from D. isabellina by presence of two rather than one alae in first report on the molecular characterization and SEM study the right spicule, and their spicule ratio (1:1.6 versus 1:1.1- of D. manipoli. 1.2). Finally, D. anthreptis differs from D. obtusa in having a shorter left spicule (910 µm versus 1,064-1,232 mm) and a Acknowledgements different spicule ratio (1:1.6 versus 1:1.8) (7). Yamaguti reported that D. manipoli had spiral spicule 750-880 µm and This research was supported by Basic Science Research straight spicule 112-128 µm, D. monticolae had spiral spic- Program through the National Research Foundation of Korea ule 810-850 µm and straight one 600-720 µm D. bargusi- (NRF) funded by the Ministry of Education (Grant No. nica had spiral spicule 440-540 µm and straight one 530- 2017RIDIAIB06031728). 600 µm (30). In D. lagopusi, the right spicule with two twists is 572-688 (627) µm long; left spicule with slight sword-like References curve toward tip, but mostly straight, is 731-890 µm long (21). In D. andersoni, the right spicule is 700 µm long, with 1. Anderson RC. Nematode parasites of vertebrates: their two twists, tip spatulate; left spicule is 1.015-1.7 mm long, development and transmission Wallingford: CABI Publishing; arcuate, and slender (21). In D. manipoli, the spiral spicule is 2000. Available from: http://www.cabi.org/CABeBooks/default. 0.75-0.88 mm long and the other slightly arcuate one 1.12- aspx?site=107&page=45&LoadModule=PDFHier&BookID=2 1.28 mm long (30). Unfortunately, we could not detect any 5. 2. Anderson RC. Preliminary revision of the genus Diploiriatna male nematode in our study. Henry and Ozoux, 1909 Diplotriaenidae: Diplotriaeninae. With respect to dimensions of eggs, D. manipoli were 51- Parasitologia 1959; 1: 195-307. µ µ µ 58 m × 36-40 m, D. lagopusi 29-52 (average 50) m × 27- 3. Bain O, Mawson PM. On some oviparous filarial nematodes 37 (average 31) µm, D. andersoni 61-69 (average 67) µm× mainly from Australian birds. Rec S Aust Mus 1981; 198: 45-53 (average 49) µm, D. anthreptis 36-42 µm×29-31µm, 265-284. D. ozouxi 50 µm×40µm, D. bargusinica 40-52 × 25-37 µm, 4. Bockheim G, Congdon S. The Sturnidae husbandry manual D. nipponensis 39-45 µm, D. nagpurensis 41-60 µm × 35-41 and resource guide. Disney Animal Kingdom, Lake Buena µm (7,8,21,27,30,31). In our study, the eggs were very differ- Vista. 2001. ent depending on various developmental stages in uterus and 5. Borji H, Razmyar J. Detection of Diplotriaena spp. from the fully developed eggs were measured as 64.45 × 50.96 µm the body cavity of Myna (Acridotheres tristis) in Mashhad, (n = 20). Iran. Sci Parasitol 2011; 12: 223-225. 6. Choudhury A, Rosas Valdez R, Johnson RC, Hoffmann B, Finally, even though the egg size in our specimen is some- Perez-Ponce de Leon G. The phylogenetic position of what larger than that of D. manipoli reported by Yamaguti Allocreadiidae (Trematoda: Digenea) from partial sequences (30), all other characteristics, such as the length of body and of the 18S and 28S ribosomal RNA genes. J Parasitol 2007; trident, morphology of trident and final host in our speci- 93: 192-196. men, agreed well with that reported for D. manipoli. 7. Dewi K, Zhang L. Two new species of spiruroid nematodes 138 Eui-Ju Hong et al.

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