pISSN 2287-5123·eISSN 2287-4445 http://dx.doi.org/10.9729/AM.2013.43.4.146 Ultrastructure of Capillaria hepatica (Syn. Calodium hepatica) Isolated from the Liver of Mouse Infected with Artificially Embryonated Eggs Collected from House Rats (Rattus norvegicus) Byoung-Hoon Min1,3, Haeng-Sook Lee1, Soo-Jin Kim2,*, Kyoung-Hwan Joo1,* 1Department of Parasitology and the Institute of Travel Medicine, Korea University College of Medicine, Seoul 136-705, Korea 2Department of Life Science, College of Natural Science, Hallym University, Chuncheon 200-702, Korea 3Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul 110-799, Korea Capillaria hepatica (syn. Calodium hepatica) is a parasite found mainly in rodent liver. But, it has also been found in a wide variety of mammals, including humans. This worm is unique as it is the only nematode parasite that is embedded in the liver parenchyma *Correspondence to: of the host even during the adult stage of the life cycle. They produce eggs that elicit Kim SJ, a marked granulomatous reaction that eventually destroys the worms. Fibrosis and Tel: +82-33-248-2091 lymphoplasmacytic inflammatory infiltration are often observed around adult nematodes Fax: +82-33-256-3420 embedded in the liver parenchyma of the host. For this reason, complete isolation of this E-mail: [email protected] slender worm and observation of the intact ultrastructure is very difficult. In this study, 10 intact whole worms (C. hepatica) were isolated from the liver of 3-week-old mouse after Joo KH, inoculation of artificially embryonated eggs collected from house rats (Rattus norvegicus). Tel: +82-2-920-6161 Their external structure of was observed with light and scanning electron microscopy. The Fax: +82-2-924-4905 length of the isolated female and male C. hepatica was approximately 69.60 mm and 36.92 E-mail: [email protected] mm, respectively. More detailed ultrastructure, including bacillary band, eggs and vulva in Received November 19, 2013 female and spicule and spicule sheath in male C. hepatica was also described. Revised December 13, 2013 Accepted December 23, 2013 Key Words: Capillaria hepatica, Genital organ, Spicule, Scanning electron microscopy INTRODUCTION hepatica and infection rates of up to 100% have been reported (Farhang-Azad, 1977; Singleton et al., 1991; Ceruti et al., Capillaria hepatica (syn. Calodium hepatica; Bancroft, 1893) 2001; Claveria et al., 2005). In addition to the principal hosts, is a parasitic nematode living in the liver of rodents and other rodents, other mammals can be infected through ingestion of mammals such as rabbits, dogs, cats, cattle, and humans water or food contaminated by infectious embryonated eggs. (Singleton et al., 1991; Roberts et al., 1996; Anderson, 2000; C. hepatica does not require an intermediate host in their Nabi et al., 2007). About 30 cases of human infections have life cycle and thus death of the host is only way to release the been reported from Asia, Africa, Europe, North and South embedded eggs. When the embryonated eggs are ingested America, and Oceania including United States of America, by a new host, they develop into the adult worm in the Japan, and Korea (McQuown, 1950; Choe et al., 1993; liver of the host within approximately 20 days. The natural Kohatsu et al., 1995; Fuehrer et al., 2011). lifespan of the adult female worm is approximately 60 days Rat species of the genus Rattus are main primary host of C. (Spratt & Singleton, 2001; Kim et al., 2007) and that of male This work was supported by a Korea University grant. CC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyrights © 2013 by Korean Society of Microscopy 146 Applied Microscopy 2013;43(4):146-154 Ultrastructure of Capillaria hepatica Genital Organ worm is approximately 40 days (Luttermoser, 1938; Wright, slide glass and incubated for 4 to 5 hours in phosphate buffer 1961). To date, complete isolation of the adult worm and solution containing type IV collagenase and antibiotics (pH observation of intact ultrastructure has been very difficult, 7.4) at 37oC. The homogenized livers were washed with because the slender and hair-like worms are embedded in normal saline solution including antibiotics at 37oC and the liver parenchyma of the host during the adult stage. For released worms were isolated using a pair of tweezers. these reasons, reports on the morphological study of external structure of C. hepatica are limited. Microscopic Observation In this study, 10 intact whole worms were isolated from the Isolated C. hepatica was fixed with 10% buffered neutral liver of 3-week-old mouse after inoculation of artificially formalin, dehydrated, and then mounted on the slide glass for embryonated eggs collected from house rats (Rattus norvegicus). light microscope observation. External structures of the worm The external and fine structures of genital organ were examined were observed with a light microscope and the length and with light and scanning electron microscopy (SEM). width of the isolated worm was measured with MetaMorph software (Universal Imaging Co., West Chester, PA, USA). For MATERIALS AND METHODS H&E staining, liver was fixed in 10% neutral formalin and embedded in paraffin according to standard protocol. Four μm-thick liver section was deparaffinized and then stained Experimental Animals and Collection of Capillaria with H&E. For SEM observation, the isolated C. hepatica were hepatica Eggs fixed with 1% paraformaldehyde-1% glutaraldehyde (pH 7.4) House rats (R. norvegicus) were taken from an urban market- and 2% osmium tetroxide. Fixed samples were dehydrated in place in Seoul, Korea. The liver of house rats infected with C. a graded ethanol series and treated with isoamyl acetate. Pre- hepatica was homogenized with a sterilized slide glass. Then, treated samples were dried with a critical point dryer (HCP- the eggs were collected with a micropipette. Collected eggs 2; Hitachi, Tokyo, Japan) and the surface was coated with were embryonated by incubating at 37oC for 5 weeks in 0.5% gold in a vacuum using an ion coater (IB-III; Eiko, Tokyo, formalin solution. Individual 8-week-old male BALB/c mouse Japan). Prepared samples were observed with a SEM (S-2500; was inoculated with 1,000 embryonated eggs (n=10, Samtako, Hitachi). Osan, Korea) through oral zonde and mice were bred for 3-weeks. RESULTS Isolation of Capillaria hepatica When the extracted liver harboring C. hepatica was examined Whole worms were isolated from the mouse liver after under a light microscope, several white clusters consisting of C. inoculation of the embryonated eggs using enzyme digestion hepatica and eggs were easily observed and the hepatomegaly methods. Briefly, liver tissue was homogenized with a sterilized around the worms was noticeable. Clusters, measured 2 to 4 Fig. 1. Light micrograph of mouse liver at 3 weeks after embryonated Capillaria hepatica egg infection. (A) C. hepatica was embedded in the liver parenchyma surrounded by fibroblasts and macrophages. (B) The intestine (I), uterus (U), ovary (O), and two bacillary bands (Ba) were observed. Scale bars=100 μm. 147 Min BH et al. mm in diameter, were scattered on the surface of the mouse The length of the isolated female and male C. hepatica was liver. Numerous fibroblasts and macrophages were observed approximately 69.60 mm and 36.92 mm, respectively (Fig. 2). around the clusters in histological sections of infected mouse Female C. hepatica had a thin and pointed anterior end (Fig. liver (Fig. 1A and B). 3A). Many oval shaped eggs (54 μm long and 31 μm wide) Fig. 2. Whole Capillaria hepatica isolated from the liver of mouse. Ten mice were experimentally infected with embryonated eggs of C. hepatica and livers were extracted at 3 weeks after infection. (A, B) Male worm. (C, D) Female worm. Scale bars=5 mm. Fig. 3. Light micrograph of female Capillaria hepatica. (A) Anterior region. (B) Mid region of the worm containing unembryonated eggs in uterus. Vulva region of the female worm showing postvulval uterine sac (egg sac) projected from the worm (C) and posterior region (D). Scale bars=100 μm. 148 Ultrastructure of Capillaria hepatica Genital Organ were observed in the uterus (Fig. 3B) and a pore like vulva the anterior region (Fig. 3D). was opened to outside in the posterior ventral region of the The shape of anterior region of the male worm was similar worm (Fig. 3C). The posterior end of female worms had a to that of the female and had a thin and pointed end (Fig. bluntly conical structure and was about 2.5 times wider than 4A). A funnel-shaped spicular sheath was protruded from Fig. 4. Light micrograph of male Capillaria hepatica. (A) Anterior region. (B~E) Posterior region and genital organ. Spicule (arrowheads) and spicular sheath (arrows). Scale bars=100 μm. 149 Min BH et al. the posterior end (Fig. 4B). A projected spicule (246.49 μm was mostly thin (less than 10 μm) (Fig. 5A), and the width of long and 10.59 μm wide) was observed at the end of the the anterior and posterior regions was approximately 24.68 male worm. Several interesting patterns were observed in μm and 71.23 μm, respectively in the female C. hepatica. The the morphological characteristics of the spicule and spicular anterior region has transverse striations at the interval of sheath. First, a spicule protruded about 40 μm from the about 0.7 μm. The posterior end was tapered to a rounded posterior end (Fig. 4C). Second, a spicule of about 318 μm in end with an excretory pore (Fig.