Oesophagostomum asperum infection in a domestic goat

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1 Note Oesophagostomum asperum infetion in a domesti goat in Yamaguhi, Japan Patrie MKOULOUOU l.*, Mihiko MSUD 2, Kaori HRDONO \ etsuya YNID 1 and Hiroshi SO 1 1 Laboratory of Parasitology, Joint Faulty of Veterinary Mediine, Yamaguhi University 2 NOSl Y amaguhi-seibu Veterinary Clini Center, Hohkan Branh, Shimonoseki (*Present address: L'Institut de Reherhe en Eologie ropiale (IRE), Le Centre National de la Reherhe Sientifique et ehnologique (CENRES), abon) BSRC Infetion of goats and sheep with nodular worms (Oesophagostomum spp.) is ommon worldwide. lthough oproulture allows for generi identifiation based on the morphology of third-stage larvae, further speifi differentiation requires onsiderable expertise. In the present study, oproultured larvae from a young goat with diarrhea were morphologially and genetially haraterized as 0. asperum. he PCR tehnique used here is appliable to the identifiation of the ausative Oesophagostomum sp (p). in symptomati goats and sheep enountered in routine veterinary work. Keywords : Oesophagostomum asperum, goat, Japan, internal transribed spaer (IS), rdn. Nodular worms (Oesophagostomum spp.) in the large intestine are one of the most widely distributed and prevalent gastrointestinal nematodes in mammals worldwide, suh as ruminants, pigs, and non-human primates [1]. dditionally, human infetion with 0. bifurum is endemi in the northern parts of hana and ogo [3, 16, 20]. Ostensibly, oesophagostomiasis aused by these parasites has onsiderable eonomi impats upon the produtivity of domesti animals worldwide and is of publi health importane in the endemi areas on the frian ontinent [7, 13]. Oesophagostomum asperum, 0. olumbianum, and 0. venulosum are major speies found in goats and sheep [1]. lthough aurate identifiation and differentiation of the speies are essential for studying their epidemiology and ontrolling the disease, it is diffiult to identify the speies based solely on the morphology of eggs and larval stages [ 4, 9, 14]. Consequently, Oesophagostomum spp. are usually identified and differentiated based on the morphologial features of adult worms olleted at neropsy. Currently, DN tehnology is a feasible hoie for the reliable speifi identifiation of parasites [2, 5, 6, 9, 14]. DN sequenings of the seond internal transribed spaer (IS-2) of the ribosomal RN gene (rdn) allow differentiation of six Oesophagostomum spp. found in livestok [17]. Usability of the first internal transribed spaer (IS-1) as a reliable geneti marker for Oesophagostomum spp. has also been demonstrated [8, 11, 171 In the present study, we have employed the rdn sequening tehnology to identify the possible ause of diarrhea in a young goat. In mid-september 2013, a farmer in a rural area of Shimonoseki, Japan, onsulted the NOSl Veterinary Clini Center on diarrhea of a nine-month-old female goat. From mirosopi examination of diarrheal fees, abundant numbers of oidial ooysts and nematode eggs were deteted (Fig. 1). o ollet third-stage larvae and identify the nematode speies, oproulture using the petri-dish feal ulture method with an unglazed tile was onduted. On the 7th day, third-stage larvae with strongyliform esophagi were olleted from the oproulture. hey showed typial -16- Jpn.]. Vet. Parasitol. Vol. 13. No

2 Patrie MKOULOUOU. Mihiko MSUD. Kaori HRDONO. etsuya YNID and Hiroshi SO 501Jm Fig. 1. Oesophagostomum egg found in a feal sample from a young goat. morphology of sheathed Oesophagostomum larvae, i.e. triangular intestinal ells. a gradually tapered and pointed tail. and prominent transverse striations on the sheath throughout most of its length (Fig. 2). Larvae exluding the sheath (n=9) were (average 657) 11m in length and (19) 11m in width. Larvae inluding the sheath were (711) 11m in length and (22) 11m in width. Other morphologial features were as follows: bual avity, 19 11m in depth; strongyliform esophagus (123) 11m in length; nerve ring loated (93) 11m from the anterior end; triangular intestinal ells alternatively positioned, 28 to 32 in number; onial tails (58) 11m in length; and the tail part of the sheath, (113) 11m in length. Parasite DN was extrated separately from two oproultured larvae using an Illustra tissue & ells genomiprep Mini Spin Kit (E Healthare UK. Bukinghamshire. UK) aording to the manufaturer's instrutions. PCR amplifiation of the sequene ontaining partial 18S rdn, IS-1, 5.8S rdn, IS-2, and partial 28S rdn was performed using a primer ombination of NSF1419/20 (5'-CCCCC-3') and NC2 (5'-CCCCCC-3') [15]. he following PCR yling protool was used: 3 min at 94oC, then 35 yles at 94t for 30 se, 63t for 45 se. and 72 oc for 90 se. followed by a final extension at 72t for 10 min. Subsequent proedures were performed in a similar way to our previous work [10. 11]. fter purifiation and sequening of a PCR produt of 1,192 bp in length, a omplete IS sequene was obtained as 1001Jm Fig. 2. stylized drawing of a oproultured Oesophagostomum asperum larva. follows: 367-bp long IS-1, 151-bp long 5.8S rdn, and 250-bp long IS-2 (DDBJ/ EMBL/enBank aession no. B971665). Searhes with the basi loal alignment searh tool (BLS) against the DDBJ/ EMBL/enBank databases speified our IS sequene to be absolutely idential to that of 0. asperum from goats in China (aession no. JX188460) or almost idential to those of other 0. asperum isolates from goats in China at >99% identities (able 1), followed by those of 0. venulosum (HQ283349) and other Oesophagostomum spp. at <98% identities. he phylogeneti relationships of our isolate with these Oesophagostomum spp. based on the IS- 1 and IS-2 nuleotide sequenes were assessed by the phyml method desribed previously [12]. he onstruted phylogeneti trees based on either IS-1 ( 40 sequenes of six Oesophagostomum spp. and four 2 (51 sequenes of eight Oesophagostomum spp. and four isolates of Chabertia ershowi as an outgroup) demonstrated the monophyly of 0. asperum with the losest speies. 0. venulosum (data not shown). as having been shown by Yu et a!. [17] as simple trees. Jpn. J. Vet. Parasitol. Vol. 13. No isolates of Chabertia ershowi as an outgroup) or IS- -17-

3 OesoPhagostomum asperum infetion in a domesti goat in Yamaguhi, Japan o the best of our knowledge, oesophagostomiasis of goats and sheep is asribed to 0. olumbianum and 0. venulosum, with little referene to 0. asperum, in popular textbooks of 'Veterinary Parasitology' used urrently or in the past in Japan. In China, 0. asperum and 0. olumbianum are onsidered as predominant speies in sheep and goats [17, 18]. In the present study, we identified 0. asperum infetion in a symptomati goat kept in a rural area of Yamaguhi, Japan, suggesting that we should onsider a possible infetion of this speies in goats and sheep in our ountry as well. In line with this notion, routine veterinary work should determine the prevalene of 0. asperum and other Oesophagostomum spp. in small ruminants distributed throughout Japan. s shown in able 1, there are urrently at least 10 nuleotide variants of 0. asperum IS sequenes. Yu et al. [ 17] and Zhao et al. [ 19] reorded suh geneti divergene of the parasite olleted from ashmere goats in Shaanxi Provine, China. lthough the sequene obtained in the present study is idential to one of them (JX188460), little is known about the signifiane of suh relatively high intraspeifi geneti variation of IS as well as mitohondrial ox-1 nuleotide sequenes of Oesophagostomum spp. suh as 0. asperum [17, 19] and 0. dentatum [8, 9]. We also experiened relatively high nuleotide variation in the IS and ox-1 nuleotide sequene of 0. stephanostomum olleted from western lowland gorillas within a limited area in abon [11] (d. B B821030). he signifiane of suh intraspeifi geneti divergenes for reognition of parasite epidemiology or their usability as a marker of transmission dynamis should be pursued in future works. REFERENCES 1. nderson, R. C Nematode Parasites of Vertebrates; their Development and ransmission. CB International, Oxon, W alingford, UK, 578 pp. 2. Cutillas, C., uevara-martinez, D., Oliveros, R., rias, P., and uevara, D. C Charaterization of porine and ovine Oesophagostomum spp. by isoenzymati patterns and restrition-fragmentlength polymorphisms (RFLPs). ta rap. 73 : de ruijter, ]. M., asser, R. B., Polderman,. M., sigri, V., and Dijkshoorn, L High resolution DN fingerprinting by FLP to study the geneti variation among Oesophagostomum bijurum (Nematoda) from human and non-human primates from hana. Parasitology 130 : Dobson, R. ]., Barnes, E. H., Birlijin, S. D., and ill, ]. H he survival of Ostertagia iruminta able 1. Nuleotitide hanges found in the IS regions of Oesophagostomum asperum * IS1 IS2 enotype Sequenes deposited in the DDBJ /EMBL/enBank databases * * JN (OeSYM2); JN (OeSYF2); JN (OeSYF3); JX (OSZM1); JX (OSZM2); JX (OSYM1); JX (OSYM4); JX (OSSMl); JX (OSSM2); JX (OSSM3); JX (OSSM4) JX (OSYFl) JX (OSYF2); B (YM) JX (OSYM2); JX (OSYM3) JX (OSSF2) JN (OeSYMl) JN (OeSYF1) JX (OSSF1) - JX (OSZF1) JN (OeSYM3) * single sequene of the parasite in Japan and 21 retrieved sequenes from the DDBJ/EMBL/enBank databases of the parasite olleted in Shaanxi. China. he site of nuleotide variation is expressed for eah IS region from the 5'-terminus. " " means the same nuleotide of the uppermost line, and " - " means a gap. **he sequnes ontain IS1 (367-bp) and IS2 (250-bp), separated by 5.8S rdn (151-bp) Jpn.]. Vet. Parasitol. Vol. 13. No

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