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Case report: First confirmed case of canine peritoneal larval cestodiasis caused by Mesocestoides vogae (syn. M. corti) in Japan
Veterinary Parasitology 201 (2014) 154–157
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Case report: First confirmed case of canine peritoneal larval cestodiasis caused by Mesocestoides vogae (syn. M. corti) in Japan Takuya Kashiide a , Jun Matsumoto a,∗ , Yoshiki Yamaya b , Aya Uwasawa a , Ai Miyoshi b , Kazuo Yamada c , Toshihiro Watari b , Sadao Nogami a a Laboratory of Medical Zoology, Department of Veterinary Medicine, Faculty of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan b Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, Faculty of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan c Yamada Animal Hospital, Asahi-ku, Yokohama, Kanagawa 241-0822, Japan
a r t i c l e
i n f o
Article history: Received 7 September 2013 Received in revised form 22 January 2014 Accepted 24 January 2014
Keywords: Mesocestoides vogae (syn. M. corti) Tetrathyridium Canine peritoneal larval cestodiasis (CPLC)
a b s t r a c t Canine peritoneal larval cestodiasis (CPLC) is an unusual parasitic disease in dogs that is caused by asexual proliferation of larval Mesocestoides. A 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Japan. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging. In addition, a number of parasites were observed in the ascitic fluid collected by abdominal paracentesis. Each of the whitish colored parasites was less than 1 mm in size. The parasites were morphologically identified as Mesocestoides sp. tetrathyridia. The parasites had four suckers and calcareous corpuscles, but no hooks or rostellum. Mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 DNA amplified from the tetrathyridia were used for molecular identification to species level. DNA sequence analysis showed that the tetrathyridia shared more than 99% identity with M. vogae (syn. M. corti) for each gene. The patient was treated with a standard dose (5 mg/kg) of praziquantel, which was administered subcutaneously twice at an interval of 14 days. This resulted in successful deworming. This is the first case that CPLC was diagnosed in a dog that had never been taken outside of Japan, indicating that M. vogae is distributed in this country. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cestodes of the genus Mesocestoides belong to the order Cyclophyllidea. Unlike other Cyclophyllidea, Mesocestoides require at least two intermediate hosts to complete their life cycles (Loos-Frank, 1991; Padgett and Boyce, 2004). However, the complete lifecycle is not known for any species within this genus. In Japan, Kugi (1983) tried to elucidate complete life cycle of M. paucitesticulus and found
∗ Corresponding author. Tel.: +81 466 84 3964; fax: +81 466 84 3964. E-mail address: [email protected] (J. Matsumoto). 0304-4017/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2014.01.021
some species of birds could be second intermediate hosts. However, first intermediate host of this parasite is still unknown. Adult Mesocestoides spp. worms reside in the intestinal tracts of their final hosts, which include carnivores, birds and occasionally humans (Soulsby, 1982). Infection with adult worms is usually asymptomatic. In contrast, the third stage larvae, which are called tetrathyridium, live in the serosal cavities of their intermediate hosts, which include amphibians, reptiles, birds, and rodents. Dogs and cats can also harbor tetrathyridia in their peritoneal cavities (Soulsby, 1982). Interestingly, the tetrathyridia of several (but not all) species belonging to the genus Mesocestoides,
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
155
Fig. 1. Lateral abdominal radiography of the patient illustrating groundglass appearance.
including Mesocestoides vogae (syn. M. corti), can proliferate asexually (Specht and Voge, 1965; Conn, 1990; Padgett and Boyce, 2004). Active proliferation of tetrathyridia in the peritoneal cavity of dogs causes canine peritoneal larval cestodiasis (CPLC). Dogs affected by CPLC usually show no symptoms at all or nonspecific symptoms such as ascites, anorexia and decreased physical activity (Crosbie et al., 1998; Caruso et al., 2003; Boyce et al., 2011). Indeed, although it has been reported that CPLC can be fatal, subclinical infections have been detected incidentally during surgery for unrelated issues (Crosbie et al., 1998; Caruso et al., 2003; Papini et al., 2010; Boyce et al., 2011). 2. Case report In December 2010, a 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Kanagawa, Japan. The dog had been owned in Kanagawa prefecture, Japan. The dog had a history of chronic cough and diagnosed with chronic bronchitis. Leukocytosis (20,800 cells/l) and eosinophilia (22%) were detected on blood examination. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging (Fig. 1). Upon removal of 20 ml of ascitic fluid using a syringe with a 21G winged
Fig. 2. Tetrathyridia obtained from ascitic fluid from the abdominal cavity of the patient.
needle, a number of parasites were seen in the fluid. The parasites appeared whitish, with sizes of less than 1 mm each (Fig. 2). Soon after the removal of ascitic fluid, the parasites were observed under an optical microscope and morphologically identified as tetrathyridia of a Mesocestoides sp. Such parasites were characterized by the presence of four suckers, calcareous corpuscles and the absence of hooks or rostellum (Fig. 3). Based on the above findings, the patient was diagnosed as a typical CPLC case caused by tetrathyridia of a Mesocestoides sp., a diagnosis that has never been reported in Japan previously. Some of the parasite material was washed three times in saline, and then stored at −80 ◦ C until use for molecular identification. Molecular identification of the tetrathyridia was conducted by targeting both the mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 (CO1) DNA as it is difficult to identify the species of Mesocestoides based on the morphological characteristics of the tetrathyridia alone. Genomic DNA was extracted from the
Fig. 3. Close-up appearance of tetrathyridium parasites. A: invaginated scolex and calcareous corpuscles in the tissue are visible. B: a flattened tetrathyridium. Four suckers are present (arrows).
156
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
parasites using a DNeasy® Blood & Tissue Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. First, a PCR was performed using mt 12S rDNA cestode-specific primers, which amplify a 314 bp fragment, as reported previously (von Nickisch-Rosenegk et al., 1999). The PCR primers had the following sequences: 60.for (5 -TTAAGATATATGTGGTACAGGATTAGATACCC-3 ) and 375.rev (5 -AACCGAGGGTGACGGGCGGTGTGTACC-3 ). The reaction condition was comprised of denaturation for 10 min at 95 ◦ C, 49 cycles at 93 ◦ C for 1 min, 55 ◦ C for 1.5 min, 73 ◦ C for 2 min and a final extension at 72 ◦ C for 5 min. Then we performed another PCR targeting the mt CO1 DNA that is commonly used for cestode identification. The reaction condition was similar to Okamoto et al. (1995). The used primers were pr-a (5 -TGGTTTTTTGTGCATCCTGAGGTTTA-3 ) and pr-b (5 AGAAAGAACGTAATGAAAATGAGCAAC-3 ); these primers are expected to yield a 391 bp fragment. The reaction condition was comprised of denaturation for 1.5 min at 94 ◦ C, 35 cycles at 94 ◦ C for 50 sec, 42 ◦ C for 1.5 min, 72 ◦ C for 1.5 min and a final extension at 72 ◦ C for 7 min. The amplicons obtained for each PCR were cloned into a pCR® 2.1-TOPO® Vector using a TOPO TA cloning® kit (Invitrogen, Carlsbad, CA). Each construct was sequenced using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions before analysis using an ABI 3130XL Genetic Analyzer (Applied Biosystems). The sequences obtained were deposited in DDBJ under accession numbers AB848990 for mt 12S rDNA and AB848991 for mt CO1. The sequences were compared with sequences from related species using publically available databases. The sequence obtained for mt 12S rDNA shared 99.6% identity with AB031363 and L49448, both sequences of which were deposited for M. vogae. The mt CO1 DNA sequence shared 99.7% identity with AB033413 from M. vogae. Based on these results, the tetrathyridia collected from the patient were confirmed as M. vogae. To the best of our knowledge, this is the first time that M. vogae has been isolated from a naturally infected host in Japan. Clinically, the patient was treated following the method of Papini et al. (2010). This involved subcutaneous administration of a standard dose (5 mg/kg) of praziquantel twice at an interval of 14 days. Although anorexia and decreased physical activity were observed temporarily, the CPLC symptoms were not detected thereafter and recrudescence was not clinically observed after the second dose of the drug was administered. After that, the patient received regular treatment for chronic respiratory disease at the animal medical center and survived more than two years until dying from a cause unrelated to CPLC.
3. Discussion Mesocestoides require at least two intermediate hosts to complete their life cycles (Loos-Frank, 1991; Padgett and Boyce, 2004) although the complete lifecycle is not known for any species within this genus. The putative first intermediate host is ground-dwelling coprophagous arthropods
that ingest oncospheres and accommodate the development of cysticercoides. Tetrathyridia, the metacestode form of Mesocestoides spp., are found in the abdominal cavity of a variety of intermediate hosts such as amphibians, reptiles, birds and rodents. After ingestion by a definitive host (i.e., dogs, cats and wild carnivores), tetrathyridia usually grow into adult worms in the host’s intestine. However, some of the ingested parasites can penetrate the intestinal wall and invade the celomic cavities during the tetrathyridium stage (Specht and Voge, 1965; Siles-Lucas and Hemphill, 2002). Thus, Mesocestoides tetrathyridia can potentially invade the peritoneal cavity, the pleural cavity and the testicular cavity of their definitive host and cause CPLC (Crosbie et al., 1998; Caruso et al., 2003). It is still unclear whether the final hosts could harbor tetrathyridia and suffer from CPLC after ingestion of a first intermediate host. Although the typical symptoms of CPLC are non-specific, they can be severe and fatal. Ascites, which was obvious in the present case, is one of most common symptoms observed in CPLC. In CPLC cases, it is usually difficult to remove tetrathyridia parasites completely from the tetrathyridia-infected patients. Thus, a long-term followup is strongly recommended because asexual proliferation of the remaining tetrathyridia may cause recrudescence of the disease. To date, fenbendazole (Crosbie et al., 1998; Caruso et al., 2003; Boyce et al., 2011) and praziquantel (Papini et al., 2010) have been reported to be effective against CPLC. Boyce et al. (2011) reported that the risk of death due to CPLC was reduced to less than 20% by using high doses of fenbendazole alone or by surgery/lavage combined with any recommended dose of fenbendazole. However, CPLC can reoccur even after treatment with fenbendazole (Crosbie et al., 1998; Papini et al., 2010; Boyce et al., 2011). Similarly, the efficacy of praziquantel is unreliable. In the work reported in 1998, Crosbie et al. did not recognize the effectiveness of praziquantel treatment for CPLC, whereas Papini et al. (2010) observed that the disease was completely cured by praziquantel treatment. In the present case study, the symptoms associated with CPLC were clearly improved and recrudescence of the disease was never observed following praziquantel treatment. Further information, based on robust clinical findings, is required before we can establish an effective long-term treatment for this poorly understood disease. According to previous reports in Japan, other cases of natural infection with Mesocestoides spp. have occurred. For example, the adult worms of M. lineatus, M. paucitesticulus, M. litteratus were found in various host species including dogs, cats, raccoon dogs, common raccoons, foxes, martens, and humans (Ito et al., 1959; Sawada and Kugi, 1973; Saito et al., 1993nd Kugi, 1973; Saito et al., 1993; Sato et al., 1999; Sato and Suzuki, 2006). In these previous studies performed in Japan, the identification of parasites was based mainly on morphological characteristics of adult specimens. It is not necessarily easy to identify Mesocestides spp. to species level as they have similar morphological characteristics. In this study, we applied PCR-based molecular techniques for the identification of the parasite. This approach enabled us to confirm the first case of natural infection with M. vogae in Japan.
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
Recently, we had another suspected case of immature adult M. vogae infection in a dog (manuscript in preparation by Dr. Tamura and his colleagues at Hokkaido Univ.). This case was found in a veterinary teaching hospital in Sapporo city, located about 800 km north from Kanagawa prefecture, Japan. This finding, together with the present report, suggests that the life cycle of M. vogae is maintained widely in Japan, possibly among wild animals. The dog described in this study is supposed to have gotten infected by accidental ingestion of a natural intermediate host of the parasite. Further investigations using molecular identification techniques are expected to reveal distribution of Mesocestoides spp. in Japan. Acknowledgement This work was supported in part by Grant-in-Aid for Young Scientists (B) (to JM) from the Japanese Society for the Promotion of Science. References Boyce, W., Shender, L., Schultz, L., Vickers, W., Johnson, C., Ziccardi, M., Backett, L., Padgett, K., Crosbie, P., Sykes, J., 2011. Survival analysis of dogs diagnosed with canine peritoneal larval cestodiasis (Mesocestoides spp.). Vet. Parasitol. 180, 256–261. Caruso, K.J., James, M.P., Fisher, D., Paulson, R.L., Christopher, M.M., 2003. Cytologic diagnosis of peritoneal cestodiasis in dogs caused by Mesocestoides sp. Vet. Clin. Pathol. 32, 50–60. Conn, D.B., 1990. The rarity of asexual reproduction among Mesocestoides tetrathyridia (Cestoda). J. Parasitol. 76, 453–455. Crosbie, P.R., Boyce, W.M., Platzer, E.G., Nadler, S.A., Kerner, C., 1998. Diagnostic procedures and treatment of eleven dogs with peritoneal infections caused by Mesocestoides spp. J. Am. Vet. Med. Assoc. 213, 1578–1583. Ito, J., Watanabe, K., Noguchi, M., Mochizuki, H., Kono, T., 1959. An investigation of the helminth parasites of dogs in Shizuoka prefecture.
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Cestodes. Jpn. J. Parasitol. 8, 649–654 (written in Japanese with English summary). Kugi, G., 1983. Development and life cycle of the cestode, Mesocestoides paucitesticulus. Jpn. J. Vet. Med. Assoc. 36, 533–537 (written in Japanese with English summary). Loos-Frank, B., 1991. One or two intermediate hosts in the life cycle of Mesocestoides (Cyclophyllidea, Mesocestoididae)? Parasitol. Res. 77, 726–728. Okamoto, M., Bessho, Y., Kamiya, M., Kurosawa, T., Horii, T., 1995. Phylogenetic relationships within Taenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochrome c oxidase subunit I gene. Parasitol. Res. 81, 451–458. Padgett, K.A., Boyce, W.M., 2004. Life-history studies on two molecular strains of Mesocestoides (Cestoda: Mesocestoididae): identification of sylvatic hosts and infectivity of immature life stages. J. Parasitol. 90, 108–113. Papini, R., Matteini, A., Bandinelli, P., Pampurini, F., Mancianti, F., 2010. Effectiveness of praziquantel for treatment of peritoneal larval cestodiasis in dogs: a case report. Vet. Parasitol. 170, 158–161. Saito, T., Morishige, K., Tongu, Y., 1993. A case of Mesocestoides sp. infection in a domestic cat. Bull. Sch. Health Sci. Okayama Univ. 4, 19–21 (written in Japanese with English summary). Sato, H., Ihama, Y., Inaba, T., Yagisawa, M., Kamiya, H., 1999. Helminth fauna of carnivores distributed in north-western Tohoku, Japan, with special reference to Mesocestoides paucitesticulus and Brachylaima tokudai. J. Vet. Med. Sci. 61, 1339–1342. Sato, H., Suzuki, K., 2006. Gastrointestinal helminths of feral raccoons (Procyon lotor) in Wakayama prefecture, Japan. J. Vet. Med. Sci. 68, 311–318. Sawada, I., Kugi, G., 1973. A new cestode, Mesocestoides paucitesticulus, from a badger, Nyctereutex Procyonoides in Japan. Jpn. J. Parasitol. 22, 45–47. Siles-Lucas, M., Hemphill, A., 2002. Cestode parasites: application of in vivo and in vitro models for studies on the host-parasite relationship. Adv. Parasitol. 51, 133–230. Soulsby, E.J.L., 1982. Helminths, Arthropods and Protozoa of Domesticated Animals, 7th ed. Bailliere Tindall, London, pp. 127–128. Specht, D., Voge, M., 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. J. Parasitol. 51, 268–272. von Nickisch-Rosenegk, M., Lucius, R., Loos-Frank, B., 1999. Contributions to the phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S rDNA. J. Mol. Evol. 48, 586–596.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Case report: First confirmed case of canine peritoneal larval cestodiasis caused by Mesocestoides vogae (syn. M. corti) in Japan Takuya Kashiide a , Jun Matsumoto a,∗ , Yoshiki Yamaya b , Aya Uwasawa a , Ai Miyoshi b , Kazuo Yamada c , Toshihiro Watari b , Sadao Nogami a a Laboratory of Medical Zoology, Department of Veterinary Medicine, Faculty of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan b Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, Faculty of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan c Yamada Animal Hospital, Asahi-ku, Yokohama, Kanagawa 241-0822, Japan
a r t i c l e
i n f o
Article history: Received 7 September 2013 Received in revised form 22 January 2014 Accepted 24 January 2014
Keywords: Mesocestoides vogae (syn. M. corti) Tetrathyridium Canine peritoneal larval cestodiasis (CPLC)
a b s t r a c t Canine peritoneal larval cestodiasis (CPLC) is an unusual parasitic disease in dogs that is caused by asexual proliferation of larval Mesocestoides. A 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Japan. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging. In addition, a number of parasites were observed in the ascitic fluid collected by abdominal paracentesis. Each of the whitish colored parasites was less than 1 mm in size. The parasites were morphologically identified as Mesocestoides sp. tetrathyridia. The parasites had four suckers and calcareous corpuscles, but no hooks or rostellum. Mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 DNA amplified from the tetrathyridia were used for molecular identification to species level. DNA sequence analysis showed that the tetrathyridia shared more than 99% identity with M. vogae (syn. M. corti) for each gene. The patient was treated with a standard dose (5 mg/kg) of praziquantel, which was administered subcutaneously twice at an interval of 14 days. This resulted in successful deworming. This is the first case that CPLC was diagnosed in a dog that had never been taken outside of Japan, indicating that M. vogae is distributed in this country. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cestodes of the genus Mesocestoides belong to the order Cyclophyllidea. Unlike other Cyclophyllidea, Mesocestoides require at least two intermediate hosts to complete their life cycles (Loos-Frank, 1991; Padgett and Boyce, 2004). However, the complete lifecycle is not known for any species within this genus. In Japan, Kugi (1983) tried to elucidate complete life cycle of M. paucitesticulus and found
∗ Corresponding author. Tel.: +81 466 84 3964; fax: +81 466 84 3964. E-mail address: [email protected] (J. Matsumoto). 0304-4017/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2014.01.021
some species of birds could be second intermediate hosts. However, first intermediate host of this parasite is still unknown. Adult Mesocestoides spp. worms reside in the intestinal tracts of their final hosts, which include carnivores, birds and occasionally humans (Soulsby, 1982). Infection with adult worms is usually asymptomatic. In contrast, the third stage larvae, which are called tetrathyridium, live in the serosal cavities of their intermediate hosts, which include amphibians, reptiles, birds, and rodents. Dogs and cats can also harbor tetrathyridia in their peritoneal cavities (Soulsby, 1982). Interestingly, the tetrathyridia of several (but not all) species belonging to the genus Mesocestoides,
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
155
Fig. 1. Lateral abdominal radiography of the patient illustrating groundglass appearance.
including Mesocestoides vogae (syn. M. corti), can proliferate asexually (Specht and Voge, 1965; Conn, 1990; Padgett and Boyce, 2004). Active proliferation of tetrathyridia in the peritoneal cavity of dogs causes canine peritoneal larval cestodiasis (CPLC). Dogs affected by CPLC usually show no symptoms at all or nonspecific symptoms such as ascites, anorexia and decreased physical activity (Crosbie et al., 1998; Caruso et al., 2003; Boyce et al., 2011). Indeed, although it has been reported that CPLC can be fatal, subclinical infections have been detected incidentally during surgery for unrelated issues (Crosbie et al., 1998; Caruso et al., 2003; Papini et al., 2010; Boyce et al., 2011). 2. Case report In December 2010, a 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Kanagawa, Japan. The dog had been owned in Kanagawa prefecture, Japan. The dog had a history of chronic cough and diagnosed with chronic bronchitis. Leukocytosis (20,800 cells/l) and eosinophilia (22%) were detected on blood examination. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging (Fig. 1). Upon removal of 20 ml of ascitic fluid using a syringe with a 21G winged
Fig. 2. Tetrathyridia obtained from ascitic fluid from the abdominal cavity of the patient.
needle, a number of parasites were seen in the fluid. The parasites appeared whitish, with sizes of less than 1 mm each (Fig. 2). Soon after the removal of ascitic fluid, the parasites were observed under an optical microscope and morphologically identified as tetrathyridia of a Mesocestoides sp. Such parasites were characterized by the presence of four suckers, calcareous corpuscles and the absence of hooks or rostellum (Fig. 3). Based on the above findings, the patient was diagnosed as a typical CPLC case caused by tetrathyridia of a Mesocestoides sp., a diagnosis that has never been reported in Japan previously. Some of the parasite material was washed three times in saline, and then stored at −80 ◦ C until use for molecular identification. Molecular identification of the tetrathyridia was conducted by targeting both the mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 (CO1) DNA as it is difficult to identify the species of Mesocestoides based on the morphological characteristics of the tetrathyridia alone. Genomic DNA was extracted from the
Fig. 3. Close-up appearance of tetrathyridium parasites. A: invaginated scolex and calcareous corpuscles in the tissue are visible. B: a flattened tetrathyridium. Four suckers are present (arrows).
156
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
parasites using a DNeasy® Blood & Tissue Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. First, a PCR was performed using mt 12S rDNA cestode-specific primers, which amplify a 314 bp fragment, as reported previously (von Nickisch-Rosenegk et al., 1999). The PCR primers had the following sequences: 60.for (5 -TTAAGATATATGTGGTACAGGATTAGATACCC-3 ) and 375.rev (5 -AACCGAGGGTGACGGGCGGTGTGTACC-3 ). The reaction condition was comprised of denaturation for 10 min at 95 ◦ C, 49 cycles at 93 ◦ C for 1 min, 55 ◦ C for 1.5 min, 73 ◦ C for 2 min and a final extension at 72 ◦ C for 5 min. Then we performed another PCR targeting the mt CO1 DNA that is commonly used for cestode identification. The reaction condition was similar to Okamoto et al. (1995). The used primers were pr-a (5 -TGGTTTTTTGTGCATCCTGAGGTTTA-3 ) and pr-b (5 AGAAAGAACGTAATGAAAATGAGCAAC-3 ); these primers are expected to yield a 391 bp fragment. The reaction condition was comprised of denaturation for 1.5 min at 94 ◦ C, 35 cycles at 94 ◦ C for 50 sec, 42 ◦ C for 1.5 min, 72 ◦ C for 1.5 min and a final extension at 72 ◦ C for 7 min. The amplicons obtained for each PCR were cloned into a pCR® 2.1-TOPO® Vector using a TOPO TA cloning® kit (Invitrogen, Carlsbad, CA). Each construct was sequenced using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions before analysis using an ABI 3130XL Genetic Analyzer (Applied Biosystems). The sequences obtained were deposited in DDBJ under accession numbers AB848990 for mt 12S rDNA and AB848991 for mt CO1. The sequences were compared with sequences from related species using publically available databases. The sequence obtained for mt 12S rDNA shared 99.6% identity with AB031363 and L49448, both sequences of which were deposited for M. vogae. The mt CO1 DNA sequence shared 99.7% identity with AB033413 from M. vogae. Based on these results, the tetrathyridia collected from the patient were confirmed as M. vogae. To the best of our knowledge, this is the first time that M. vogae has been isolated from a naturally infected host in Japan. Clinically, the patient was treated following the method of Papini et al. (2010). This involved subcutaneous administration of a standard dose (5 mg/kg) of praziquantel twice at an interval of 14 days. Although anorexia and decreased physical activity were observed temporarily, the CPLC symptoms were not detected thereafter and recrudescence was not clinically observed after the second dose of the drug was administered. After that, the patient received regular treatment for chronic respiratory disease at the animal medical center and survived more than two years until dying from a cause unrelated to CPLC.
3. Discussion Mesocestoides require at least two intermediate hosts to complete their life cycles (Loos-Frank, 1991; Padgett and Boyce, 2004) although the complete lifecycle is not known for any species within this genus. The putative first intermediate host is ground-dwelling coprophagous arthropods
that ingest oncospheres and accommodate the development of cysticercoides. Tetrathyridia, the metacestode form of Mesocestoides spp., are found in the abdominal cavity of a variety of intermediate hosts such as amphibians, reptiles, birds and rodents. After ingestion by a definitive host (i.e., dogs, cats and wild carnivores), tetrathyridia usually grow into adult worms in the host’s intestine. However, some of the ingested parasites can penetrate the intestinal wall and invade the celomic cavities during the tetrathyridium stage (Specht and Voge, 1965; Siles-Lucas and Hemphill, 2002). Thus, Mesocestoides tetrathyridia can potentially invade the peritoneal cavity, the pleural cavity and the testicular cavity of their definitive host and cause CPLC (Crosbie et al., 1998; Caruso et al., 2003). It is still unclear whether the final hosts could harbor tetrathyridia and suffer from CPLC after ingestion of a first intermediate host. Although the typical symptoms of CPLC are non-specific, they can be severe and fatal. Ascites, which was obvious in the present case, is one of most common symptoms observed in CPLC. In CPLC cases, it is usually difficult to remove tetrathyridia parasites completely from the tetrathyridia-infected patients. Thus, a long-term followup is strongly recommended because asexual proliferation of the remaining tetrathyridia may cause recrudescence of the disease. To date, fenbendazole (Crosbie et al., 1998; Caruso et al., 2003; Boyce et al., 2011) and praziquantel (Papini et al., 2010) have been reported to be effective against CPLC. Boyce et al. (2011) reported that the risk of death due to CPLC was reduced to less than 20% by using high doses of fenbendazole alone or by surgery/lavage combined with any recommended dose of fenbendazole. However, CPLC can reoccur even after treatment with fenbendazole (Crosbie et al., 1998; Papini et al., 2010; Boyce et al., 2011). Similarly, the efficacy of praziquantel is unreliable. In the work reported in 1998, Crosbie et al. did not recognize the effectiveness of praziquantel treatment for CPLC, whereas Papini et al. (2010) observed that the disease was completely cured by praziquantel treatment. In the present case study, the symptoms associated with CPLC were clearly improved and recrudescence of the disease was never observed following praziquantel treatment. Further information, based on robust clinical findings, is required before we can establish an effective long-term treatment for this poorly understood disease. According to previous reports in Japan, other cases of natural infection with Mesocestoides spp. have occurred. For example, the adult worms of M. lineatus, M. paucitesticulus, M. litteratus were found in various host species including dogs, cats, raccoon dogs, common raccoons, foxes, martens, and humans (Ito et al., 1959; Sawada and Kugi, 1973; Saito et al., 1993nd Kugi, 1973; Saito et al., 1993; Sato et al., 1999; Sato and Suzuki, 2006). In these previous studies performed in Japan, the identification of parasites was based mainly on morphological characteristics of adult specimens. It is not necessarily easy to identify Mesocestides spp. to species level as they have similar morphological characteristics. In this study, we applied PCR-based molecular techniques for the identification of the parasite. This approach enabled us to confirm the first case of natural infection with M. vogae in Japan.
T. Kashiide et al. / Veterinary Parasitology 201 (2014) 154–157
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