Parasitology International 65 (2016) 58–61 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Case report Visceral pentastomiasis caused by Armillifer armillatus in a captive striped hyena (Hyaena hyaena) in Chiang Mai Night Safari, Thailand Sakorn Dechkajorn a, Raksiri Nomsiri a, Kittikorn Boonsri b, Duanghatai Sripakdee b,c, Kabkaew L. Sukontason d, Anchalee Wannasan d, Sasisophin Chailangkarn b,e, Saruda Tiwananthagorn b,e,⁎ a Chiang Mai Night Safari, Chiang Mai 50230, Thailand b Veterinary Diagnostic Laboratory, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand c Veterinary Central Laboratory, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand d Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand e Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand article info abstract Article history: Visceral pentastomiasis (porocephalosis) caused by Armillifer armillatus larvae was incidentally diagnosed in a Received 4 April 2015 female striped hyena (Hyaena hyaena) of unknown age which died unexpectedly in 2013. The hyena had been Received in revised form 7 October 2015 imported from Tanzania 8 years earlier and have been since then in a zoo in Chiang Mai, northern Thailand. Accepted 8 October 2015 Pathological examination revealed visceral nymph migrans of pentastomes throughout the intestine, liver, Available online 14 October 2015 diaphragm, omentum and mesentery, spleen, kidneys, and urinary bladder. Polymerase chain reaction and se- fi Keywords: quencing that targeted the pentastomid-speci c 18S rRNA gene determined 100% identity with reference sequence Visceral pentastomiasis for A. armillatus, suggesting that its ova can infect the hyena to serveasanintermediatehostfortheparasite.Further Striped hyena studies to identify the source of infection, its risk factors, and host range for A. armillatus are important to determine Armillifer armillatus its zoonotic potential and to better prevent and manage the disease to protect animal and human health. Pathological finding © 2015 Elsevier Ireland Ltd. All rights reserved. Scanning electron microscopy 18 s rRNA gene 1. Introduction via host feces and secretions. When rodents or other typical mammalian intermediate hosts ingest those eggs, the larvae hatch and migrate to Visceral pentastomiasis is a parasitic zoonosis caused by the larval their viscera. There, encystation and molting occur several times [3]. stage of several pentastome species. Parasites belonging to the subclass Humans, non-human primates, and domestic animals may become Pentastomida are obligate parasites which commonly infect both accidental intermediate hosts upon ingestion of food or water contam- humans and animals. The most common genera are Armillifer, inated with parasite eggs or by consumption of uncooked contaminated Kiricephalus, Porocephalus, Raillietiella, and Sebekia, with the majority snake meat [2]. found as adults in the lungs of snakes, lizards, and crocodiles [1]. The Pentastomiasis is a rare zoonosis in Southeast Asia; when found it is genus Armillifer consists of eight species with different geographical almost exclusively by infection with A. moniliformis [5]. Human cases distribution: Armillifer aborealis, Armillifer agkistrodontis, Armillifer were reported among aborigines in West and East Malaysia in the armillatus, Armillifer australis, Armillifer grandis, Armillifer mazzai, 1960s, and recently in 2011 [5]. In Thailand, to the present, only six Armillifermoniliformis,andArmillifer yoshidai [2]. Of these, most reported human cases of pentastomiasis have been reported, all caused by infections in humans are caused by Armillifer armillatus [3,4]. Armillifer spp., probably A. moniliformis infection [6]. None in animals The adult worms of Armillifer spp. inhabit the respiratory tract of have been described. Here we report a case of A. armillatus infection in large snakes and other reptiles as definitive hosts. Reproduction is a striped hyena which had lived 8 years in a zoo in Chiang Mai, northern sexual and a large number of fertile eggs are shed into the environment Thailand after its importation from Tanzania. ⁎ Corresponding author at: Department of Veterinary Biosciences and Veterinary Public 2. Case history and diagnosis Health, Faculty of Veterinary Medicine, Chiang Mai University, Mae Hiae, Muang, Chiang Mai 50100, Thailand. In December 2013, a 9-year-old, female, striped hyena (Hyaena E-mail addresses: [email protected] (S. Dechkajorn), hyaena) died unexpectedly in the animal shelter of a “safari” zoo located [email protected] (R. Nomsiri), [email protected] (K. Boonsri), in Chiang Mai, the major city of northern Thailand. The animal had been [email protected] (D. Sripakdee), [email protected] (K.L. Sukontason), [email protected] (A. Wannasan), [email protected] (S. Chailangkarn), imported from Tanzania in 2005, and since then had lived in no other [email protected] (S. Tiwananthagorn). venue in Thailand. Upon discovery of the death, it was found to be http://dx.doi.org/10.1016/j.parint.2015.10.004 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved. S. Dechkajorn et al. / Parasitology International 65 (2016) 58–61 59 emaciated, with many areas of alopecia (Fig. 1A). Necropsy revealed surface of the pseudo-segmented nymph body (Fig. 2A). Its cephalic end moderate autolysis (Fig. 1B). No remarkable lesions were observed in was characterized by four prominent mouth-hooks (Fig. 2B), and an the thoracic cavity. In the abdomen, semitransparent encysted parasites oral aperture arranged vertically on its antero-medial end (Fig. 2C). were obvious in various visceral organs (i.e., diaphragm, omentum and Genomic DNA of two representative nymphs was extracted sepa- mesentery, spleen, intestine, kidneys, and urinary bladder). These rately using QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany). Poly- encysted forms were C-shaped, measuring 9 mm × 6 mm × 3 mm merase chain reaction (PCR) amplification targeted the 18S rRNA gene (Fig. 1B and B-inset). Each cyst contained a segmented nymph stage, of Pentastomida with primers Pent629F and Pent1011R [7] using and was encapsulated by connective tissue. The nymph morphology TaKaRa Ex Taq® DNA Polymerase (Takara Bio, Otsu, Shiga, Japan). PCR indicated pentastomiasis as the etiopathological diagnosis. products were purified and subsequently sent to the Macrogen DNA se- Further histopathological, parasitological, and molecular examina- quencing service (Macrogen, Seoul, Korea). Partial 18S rRNA gene tion of representative specimens of the dissected organs and amplicons (383 bp) were analyzed by nucleotide BLAST program pentastomid nymphs were performed by the Veterinary Diagnostic (National Center for Biotechnology Information, National Library of Laboratory of the Faculty of Veterinary Medicine, and the Department Medicine, Bethesda, USA) [8]. The sequences of both representative of Parasitology of the Faculty of Medicine, both of Chiang Mai University. nymphs were 100% identical to that of A. armillatus (GenBank accession Two nymphs were processed and examined by scanning electron mi- number HM756289). Our 18S nucleotide sequences of A. armillatus are croscopy (SEM). available in the GenBank database under the accession numbers Light microscopic examination detected pentastomid nymphs in KJ877183 and KJ877184. many visceral organs, with surrounding mild inflammatory reactions, including the sub-serosal layers of the spleen, kidneys, and urinary blad- 3. Discussion der. A thin layer of fibrous tissue was observed encircling the encysted nymphs. The submucosal and muscularis layers of the intestine Pentastomiasis is a parasitic zoonotic disease caused by the nymphal displayed catarrhal enteritis with larval infestation. stages of pentastomes, and is being increasingly reported in humans [5, The liver was characterized by multifocal chronic necrotic hepatitis 9–11], non-human primates [12,13], and dogs [7,14]. Worldwide, the with hemosiderosis. In the spleen were found multifocal necrotic genus Armillifer is the most common in human infections, accounting inflammation and lymphoid depletion; worms were present. Both for more than 90% of reported cases [1]. The four pathogenic Armillifer kidneys were infected with pentastomid worms, mainly in the subcap- species are Armillifer armillatus in Africa and the Arabian Peninsula, sular spaces. The lungs and heart were free of parasites. Armillifer agkistrodontis in China, Armillifer grandis in Africa and Examination of the pentastomid nymphs under light microscopy re- Armillifer moniliformis in Southeast Asia [3,5,15]. vealed a pseudo-segmented body with prominent bunches of acidophil- In Thailand, six cases in human pentastomiasis have been reported ic glands surrounding the gut (Fig. 1C). SEM revealed waved folds on the in the northern and northeastern regions of the country, including one in Chiang Mai [6]. This patient lived in San Pa Tong district of the prov- ince, was infected with Armillifer spp., probably A. moniliformis infection, and gave a history of having eaten a viper snake [6]. Prior to this report, only one animal case of visceral pentastomiasis was known in Thailand, caused by A. moniliformis in a domestic dog in Bangkok, the national capital (personal communication, Woraporn Sukhumavasi).