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 elec- tron 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 tad- pole 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 devel- oped 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 papil- lae 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).