Scoliosis in a Tiger Salamander (Ambystoma tigrinum) Associated with Encysted Digenetic Trematodes of the Genus Clinostomum

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1 Scoliosis in a Tiger Salamander (Ambystoma tigrinum) Associated with Encysted Digenetic Trematodes of the Genus Clinostomum Author(s): David Perpiñán, Michael M. Garner, John G. Trupkiewicz, Jennifer Malarchik, Douglas L. Armstrong, Araceli Lucio-Forster, and Dwight D. Bowman Source: Journal of Wildlife Diseases, 46(2): Published By: Wildlife Disease Association URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Journal of Wildlife Diseases, 46(2), 2010, pp # Wildlife Disease Association 2010 Scoliosis in a Tiger Salamander (Ambystoma tigrinum) Associated with Encysted Digenetic Trematodes of the Genus Clinostomum David Perpiñán, 1,5,6 Michael M. Garner, 2 John G. Trupkiewicz, 2 Jennifer Malarchik, 3 Douglas L. Armstrong, 1 Araceli Lucio-Forster, 4 and Dwight D. Bowman 4 1 Henry Doorly Zoo, 3701 South 10th Street, Omaha, Nebraska 68107, USA; 2 Northwest ZooPath, 654 W. Main, Monroe, Washington 98272, USA; 3 Russell Veterinary Hospital, 1276 Route 957, Russell, Pennsylvania 16345, USA; 4 Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Campus Road, Ithaca, New York 14853, USA; 5 Current address: Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Dr., Athens, Georgia 30602, USA; 6 Corresponding author ( david1@uga.edu) ABSTRACT: A group of 202 tiger salamanders (Ambystoma tigrinum) was brought into captivity due to habitat destruction. Half of these animals died, over two mo, showing generalized hemorrhages compatible with an infectious disease, but an etiologic agent was not determined. Encysted metacercarial stages of trematodes within the skeletal musculature, coelomic cavity, and subcutaneous space were additional necropsy findings. One salamander had scoliosis and multiple skin nodules. A radiograph showed no skeletal abnormality to explain the scoliosis; however, numerous round nodules were more radiodense than the surrounding tissue. A presumptive diagnosis of generalized trematodiasis was made, yet the salamander did not improve after a course of praziquantel and subsequently died. Necropsy revealed massive, encysted trematode infection. Histologic examination revealed marked multifocal intramuscular, subcutaneous, and coelomic trematodiasis with associated necrosis and inflammation. Based on gross morphology, the trematode was identified as a member of the genus Clinostomum. Key words: Amphibian, Clinostomatidae, deformity, disease, malformation, Strigeidida. Disease is among the factors associated with amphibian declines worldwide (Stuart et al., 2004). Both chytrid and ranavirus infections, the two most important diseases of wild amphibians, have been described in the tiger salamander (Ambystoma tigrinum; Bollinger et al., 1999; Davidson et al., 2003). Although ranavirus infection can cause lethal epizootics in tiger salamanders, chytridiomycosis rarely causes mortality in this species (Bollinger et al., 1999; Davidson et al., 2003). Parasites frequently use amphibians as both paratenic and definitive hosts, and clinical disease is common in captive animals, but relatively rare, in wild ones (Poynton and Whitaker, 2001). Digenean trematodes are commonly encountered in amphibians and may cause disease when a large number of parasites are present in relation to the volume of host tissue, or when the normal growth of developing limb buds is disrupted (Poynton and Whitaker, 2001; Stopper et al., 2002). We describe a case of massive trematode metacercarial parasitism associated with scoliosis in a tiger salamander. A group of 202 tiger salamanders was removed from the wild in an area of Polk County, Iowa, USA (41u439570N, 93u459220W) and brought to the Henry Doorly Zoo (Omaha, Nebraska, USA) to be kept over the winter. These salamanders were cm in length and were considered adults. The hibernaculum was in the foundation of an old house where construction and massive earth movement were taking place. At the Henry Doorly Zoo, salamanders were housed in RubbermaidH-brand polycarbonate tanks ( cm; Newell Rubbermaid Inc., 10B Glenlake Parkway, Atlanta, Georgia, USA) located in quarantine rooms. The tanks were inclined so they would provide both an area with water and a dry area. Full-spectrum light was provided using one, 65-watt fluorescent bulb (ESU 24 Reptile UV Aqualight Desert 7% UVB, Energy Savers Unlimited, Walnut Creek, California, USA) per cage, placed 30 cm above the floor. Temperatures in the tank ranged from C. Photoperiod was 12 hr light and 12 hr dark. Salamanders were fed six times per week with a variety 579

3 580 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 2, APRIL 2010 of food items including crickets, earthworms, waxworms, flightless fruit flies, and flour beetles. Immediately after arrival, salamanders started dying with a hemorrhagic disease characterized by edema of the gastric wall, hemorrhage in stomach, intestine and spleen, and petechial hemorrhages in tongue, palate, and skin. The cause of the disease could not be determined with viral testing, bacterial culture, and PCR for ranavirus, but an infectious etiology remains the primary differential. Mortality was 50% at 2 mo, when it stopped after the temperature of the tanks was raised. Parasitic nodules were incidentally found in most necropsies, generally in the coelomic wall, but also in the mesentery, lung, spleen, skeletal muscles, and subcutaneous space. In all cases in which parasites were found (except for the case reported herein), the infestation was considered mild, with only 1 5 parasitic nodules per animal. These parasites were identified as metacercarial stages of digenetic trematodes from the order Strigeidida; the presence of two suckers, one in front and a larger one about a third of the way down from the front, was compatible with the family Clinostomatidae. A salamander from the surviving group developed scoliosis one month after group mortality ceased (Fig. 1). This salamander weighed 33 g and had a length of 18 cm. It also had exophthalmus and skin nodules over its entire body (Fig. 1). The spine could be physically straightened, but returned to its deviated shape when force was not applied. No skeletal abnormalities associated with the vertebral column were observed on radiographic exam (Fig. 2) to explain the scoliosis. However, numerous nodules that were more radiodense than the surrounding tissues were found in the tail, limbs, and thoracic and abdominal walls (Fig. 2). In the right forelimb, a softtissue swelling was observed between the bones of the carpus, radius, and ulna, dislodging the bones involved; this softtissue swelling was believed to be a FIGURE 1. Tiger salamander with scoliosis and multiple nodules in the skin. Inset: detail of the tail showing multiple skin nodules (arrows). parasitic cyst. Based on clinical findings and previous necropsies performed on other salamanders, a presumptive diagnosis of encysted trematodiasis was made, and a course of praziquantel was started (25 mg/kg IM q7d for 3 doses). The condition of the salamander worsened throughout the treatment, and it was found dead 6 days after the last dose of praziquantel. External exam at necropsy showed marked gular edema, bilateral exophthalmus, and intramuscular and subcutaneous parasitic nodules distributed in tail, limbs, dorsal and lateral trunk, and head, including the retrobulbar region (Fig. 3). Nodules affecting toes and tail were sometimes erythematous. These cysts were about 2 mm in diameter and consisted of a clear, thin capsule containing an ovalshaped parasite identified as metacercariae of Clinostomum sp. (Fig. 4). Histologic examination revealed multiple foci of intramuscular, subcutaneous, and intracoelomic granulomatous inflammation oriented around encysted and degenerative trematodes. Where discernible, the trematodes were mm in diameter, with a thin tegument, and exhibited male and female reproductive tracts, absence of a coelomic cavity, and two ventral suckers; the caudal sucker was approximately twice as large as the rostral sucker. In the body

4 SHORT COMMUNICATIONS 581 FIGURE 2. Dorsoventral radiograph of a tiger salamander with scoliosis. Note bony changes in the the leg (arrowhead). Inset: detail of the tail showing multiple radiodense areas representing parasitic cysts (arrows). wall, head, tail, and extremities of the salamander, the parasites were associated with granulomatous inflammation and tract formation delineated by irregular zones of fibrosis that extended into adjacent muscle bundles. In these areas, muscle bundles had varying degrees of edema, hemorrhage, and atrophy, and many of the myofibers were shrunken or fragmented (Fig. 5). Additional histologic findings included moderate to marked edema, congestion, and hepatocellular vacuolar degeneration with associated disruption of hepatic cords in centrilobular regions of the liver. The lung had mild, intra-alveolar hemorrhage and the interstitium was infiltrated by low numbers of mixed lymphocytes and granulocytes. The kidney had numerous tubules that were slightly dilated, lined by necrotic epithelium, or had protein casts. Deformities in amphibians have been linked to a variety of factors including UV light, chemical contaminants, and parasites (Blaustein and Johnson, 2003). Trematode infection is an important cause of skeletal deformities in amphibians, and infection by the digenean trematode Ribeiroia ondatrae is considered an emerging disease (Johnson and Sutherland, 2003). Ribeiroia causes deformities FIGURE 3. Multiple parasitic cysts in the subcutaneous and muscular tissues of the dorsum of a tiger salamander (black arrows). A cyst is shown in the retrobulbar area of the left eye, producing exophthalmus (white arrowhead). Inset: detail of the tail showing multiple intramuscular parasitic cysts.

5 582 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 2, APRIL 2010 FIGURE 4. Clinostomum sp. recovered from one of the cysts affecting a tiger salamander. Bar50.5 mm. in larval amphibians by disrupting normal cellular growth of developing limb buds (Stopper et al., 2002). These deformities usually affect the hind limbs (Stopper et al., 2002). Microsporidia have also been associated with skeletal deformities by producing myositis and fibrosis, leading to abnormal tension of the epaxial muscles (Gamble et al., 2006). In addition, several stressors, such as immunosupression, pesticide use, and habitat modification (Kiesecker, 2002; Belden and Kiesecker, 2005; King et al., 2008), have been suggested as facilitators of parasitic infections. However, whether agriculture (and associated pesticide use) increases or reduces the populations of parasites is controversial (Gray et al., 2007; King et al., 2007; Koprivnikar et al., 2007). Therefore, the effect of habitat modification on the case reported herein is unknown. Clinostomum spp. are digenean trematodes belonging to the order Strigeidida and the family Clinostomatidae. Adults live in the oral cavity and esophagus of herons and egrets (family Ardeidae; McAllister et al., 2007). Miracidia that hatch from eggs passed in the feces of birds penetrate the tissues of a snail (first intermediate host), and the cercariae that emerge from the snails encyst as metacercariae in fish or amphibians (second FIGURE 5. Longitudinal section through the vertebral column (slightly off midline) of a tiger salamander. Note foci of granulomatous inflammation and tract formation (g) associated with an encysted trematode (t). Surrounding the tract are areas of fibrosis (f) that extend into the surrounding muscle (m) and are associated with myofiber disruption, atrophy, and hemorrhage (h). Note additional anatomic points of reference including bone of lateral facet (b), dermis (between arrows), and dermal granular glands (gl). Bar5500 mm. intermediate host). Trematodes of the genus Clinostomum (either C. attenuatum or C. complanatum) have been reported in multiple species of amphibians from North America including green frogs (Rana clamitans; Muzzall et al., 2001), bullfrogs (Rana catesbeiana; Muzzall, 1991b), cane toads (Bufo marinus; Etges, 1991), red-spotted newts (Notophthalmus viridescens; Muzzall, 1991a), cave salamanders (Eurycea lucifaga; McAllister et al., 2007), and tiger salamanders (Ambystoma tigrinum; Miller et al., 2004), among others. However, to the authors knowledge, musculoskeletal deformities have not been previously reported in association with Clinostomum spp. Deformities in amphibians occur during larval development, although the effects can sometimes be seen in adult animals as well (Stopper et al., 2002; Iwamuro et al., 2003, Witzmann, 2007). This is also true in the cases of scoliosis reported in urodeles, either caused by hemivertebra (Witzmann, 2007), toxins (Iwamuro et al., 2003), or experimental surgical induction (Emura et al., 2002). In the present case,

6 SHORT COMMUNICATIONS 583 scoliosis appeared well after the metamorphosis of this salamander, as these animals were admitted as subadults or adults and the problem was initially seen 2 mo later. Out of 100 necropsies performed from this group of salamanders during a 2-mo period, the specimen reported here was the most heavily parasitized by trematodes. A similar case was reported in San Marcos salamanders (Eurycea nana), where muscular inflammation and fibrosis were seen around microsporidial organisms, and that was believed to produce abnormal muscular tension of the epaxial muscles, leading to skeletal deformities in both young and adult animals (Gamble et al., 2006). Therefore, we believe the muscle damage produced by Clinostomum sp. cysts was responsible for the scoliosis seen in this tiger salamander, similar to the microsporidial-associated myositis causing kyphosis in San Marcos salamanders (Gamble et al., 2006) and muscular dystrophies causing scoliosis in humans (Cambridge and Drennan, 1987). Treatment of encysted trematodiasis did not produce clinical improvement. However, all of the parasites were degenerative on histologic sections, and that was likely due to treatment. This decomposition prevented identification of the parasite to species level. It is possible that decomposition worsened the granulomatous inflammation associated with the parasite and may have worsened the scoliosis. It is also possible that a large, dead trematode load may have caused enough toxemia to account for the remaining lesions in the liver and kidney, which were severe enough to have caused death. LITERATURE CITED BELDEN, L. K., AND J. M. KIESECKER Glucocorticosteroid hormone treatment of larval treefrogs increases infection by Alaria sp. trematode cercariae. Journal of Parasitology 91: BLAUSTEIN, A. R., AND P. T. J. JOHNSON The complexity of deformed amphibians. Frontiers in Ecology and the Environment 1: BOLLINGER, T. K., J. MAO, D. SCHOCK, R. M. BRIGHAM, AND V. G. CHINCHAR Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan. Journal of Wildlife Diseases 35: CAMBRIDGE, W., AND J. C. DRENNAN Scoliosis associated with Duchenne muscular dystrophy. Journal of Pediatric Orthopedics 7: DAVIDSON, E. W., M. PARRIS, J. P. COLLINS, J. E. LONGCORE, A. P. PESSIER, AND J. BRUNNER Pathogenicity and transmission of chytridiomycosis in tiger salamanders (Ambystoma tigrinum). Copeia 2003: EMURA, T., M. ASASHIMA, M. FURUE, AND K. HASHIZUME Experimental split cord malformations. Pediatric Neurosurgery 36: ETGES, F. J Clinostomum attenuatum (Digenea) from the eye of Bufo marinus. Journal of Parasitology 77: GAMBLE, K. C., M. M. GARNER, G. WEST, E. S. DIDIER, A. CALI, AND T. P. ALVARADO Kyphosis associated with microsporidial myositis in San Marcos salamanders, Eurycea nana. Journal of Herpetological Medicine and Surgery 15: GRAY, M. J., L. M. SMITH, D.L.MILLER, AND C. R. BURSEY Influences of agricultural land use on Clinostomum attenuatum metacercariae prevalence in Southern Great Plains amphibians, USA. Herpetological Conservation and Biology 2: IWAMURO, S., M. SAKAKIBARA, M. TERAO, A. OZAWA, C. KUROBE, T. SHIGEURA, M. KATO, AND S. KI- KUYAMA Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larval Xenopus laevis. General and Comparative Endocrinology 133: JOHNSON, P. T. J., AND D. R. SUTHERLAND Amphibian deformities and Ribeiroia infection: An emerging helminthiasis. Trends in Parasitology 19: KIESECKER, J. M Synergism between trematode infection and pesticide exposure: A link to amphibian limb deformities in nature. Proceedings of the National Academy of Sciences 99: KING, K. C., J. D. MCLAUGHLIN, A. D. GENDRON, B. D. PAULI, I. GIROUX, B. RONDEAU, M. BOILY, P. JUNEAU, AND D. J. MARCOGLIESE Impacts of agriculture on the parasite communities of northern leopard frogs (Rana pipiens) in southern Quebec, Canada. Parasitology 134: , A. D. GENDRON, J. D. MCLAUGHLIN, I. GIROUX, P.BROUSSEAU, D.CYR, S.M.RUBY, M. FOURNIER, AND D. J. MARCOGLIESE Shortterm seasonal changes in parasite community structure in northern leopard froglets (Rana pipiens) inhabiting agricultural wetlands. Journal of Parasitology 94:

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