Journal of Veterinary Diagnostic Investigation

http://vdi.sagepub.com/ An Outbreak of Adenoviral Infection in Inland Bearded Dragons (Pogona Vitticeps) Coinfected with Dependovirus and Coccidial Protozoa ( Isospora Sp.)
Dae Young Kim, Mark A. Mitchell, Rudy W. Bauer, Rob Poston and Doo-Youn Cho J VET Diagn Invest 2002 14: 332 DOI: 10.1177/104063870201400411 The online version of this article can be found at: http://vdi.sagepub.com/content/14/4/332

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J Vet Diagn Invest 14:332334 (2002)

An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.)
Dae Young Kim, Mark A. Mitchell, Rudy W. Bauer, Rob Poston, Doo-Youn Cho
Abstract. Thirty of 200 (15%) hatchling inland bearded dragons were found dead after a short period (48 hours) of weakness and lethargy. The most common clinical signs were head tilt and circling. Six bearded dragons with neurological signs were euthanized, and postmortem examination revealed no gross abnormalities. Microscopically, severe, randomly distributed hepatocellular necrosis with large basophilic intranuclear inclusion bodies in numerous hepatocytes was noted. Small-intestinal enterocytes contained intracytoplasmic coccidial protozoa (Isospora sp.) and occasional enterocytes had basophilic intranuclear inclusion bodies. Transmission electron microscopy revealed both 80- and 20-nm-diameter viral particles, which were consistent with adenoviruses and dependoviruses, respectively. Adenoviral outbreaks in groups of animals are uncommon. An adverse synergistic effect of the coccidiosis with the adenoviral infection may have played a critical role in the high morbidity and mortality in this case. Adenoviruses (Family Adenoviridae) are well-known pathogens in several mammalian and avian species. Adenoviruses are double-stranded DNA viruses, 7090 nm in diameter, and have a characteristic nonenveloped, icosahedral structure. Generally, adenoviruses are host specic and are transmitted by the fecal-oral route or direct contact via oronasal secretions. Often, mammalian infections are subclinical, except for infectious canine hepatitis. Adenoviral disease generally occurs in immunocompromised or young animals. Outbreaks in groups of animals are uncommon. Recently, adenoviral infections also have been reported in several reptilian species, including crocodiles, snakes, and lizards.3,4,612,15,16 The Australian inland bearded dragon, Pogona vitticeps (Pogon: bearded in Greek), is one of the most popular reptiles in the pet trade. Two isolated cases of adenoviral infection in 4 neonatal inland bearded dragons have been reported, but the infections were limited mainly to the individuals affected.10 No outbreak of adenoviral infection in a group of reptiles has been reported. This report describes an outbreak of adenoviral infection in a group of captive-bred inland bearded dragon hatchlings coinfected with dependovirus and Isospora sp. coccidia. Two hundred hatchling captive-bred inland bearded dragons, which had been purchased at different times by a reptile importer from different captive breeding populations, were placed in a holding facility. The ages of the dragons were uncertain; however, based on their weight (5 g), the estimate age was less than 1 month. The bearded dragons were maintained at an environmental temperature of 2932 C and were fed commercially obtained crickets and lettuce. Thirty of the 200 (15%) bearded dragons were found dead after a short period (48 hours) of weakness and lethargy. The most
From the Departments of Pathobiological Sciences (Kim, Cho) and Veterinary Clinical Sciences (Mitchell), School of Veterinary Medicine, Louisiana State University, and The Louisiana Veterinary Medical Diagnostic Laboratory (Bauer, Poston), Baton Rouge, LA 70803. Received for publication August 27, 2001.

common clinical signs were head tilt and circling. Physical examination of tympanic bullae revealed no signicant ndings. Six of the bearded dragons with neurological signs were euthanized and submitted for necropsy to the Louisiana Veterinary Medical Diagnostic Laboratory, Baton Rouge, Louisiana. At necropsy, no signicant gross abnormalities were noted in any of the 6 bearded dragons. Sections from major organs were xed in 10% neutral buffered formalin, routinely processed, sectioned at 4 m thick, and stained with hematoxylin and eosin. For electron microscopy, 2 methods were used. At rst, the liver specimens from 3 bearded dragons were pooled together, homogenized, and centrifuged at 800 g for 30 minutes. The supernatant was collected and centrifuged at 98,000 g for 1 hour. The formed pellet was harvested after discarding the supernatant. The pellet was suspended in 100 l of phosphate-buffered saline (PBS) solution, stained with 100 l of 4% phosphotungstic acid, placed on Formvar- and carbon-coated grids, air-dried, and examined with a transmission electron microscope (TEM). Second, formalin-xed liver tissue was trimmed, dehydrated, postxed in 1% phosphotunstic acid (w/v), embedded in LR White,a sectioned, and stained with uranyl acetate and lead citrate for TEM. The contents of the large intestines were submitted for parasitologic examination. Signicant microscopic changes were similar in all 6 dragons and were limited to the liver and small intestine. Severe, randomly distributed hepatocellular necrosis involving 50% of the parenchyma was accompanied by mild to moderate inltration of lymphocytes and histiocytes. Numerous hepatocyte nuclei were expanded (24-fold) by large, basophilic, glassy, intranuclear inclusion bodies and marginated chromatin (Fig. 1). Small intestinal villi were atrophied, and many enterocytes contained variable stages of coccidia in the cytoplasm. Enterocytes occasionally had basophilic intranuclear inclusion bodies similar to those seen in the liver (Fig. 2). Electron microscopy revealed viral particles of 2 distinct sizes. Larger viral particles were approximately 80 nm in diameter, nonenveloped, and had hexagonal outlines and electron-dense or electron-lucent cores.

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These viral particles were arranged in paracrystalline arrays and occupied most of the nucleus (Figs. 3, 4). Smaller viral particles were approximately 20 nm in diameter, nonenveloped, and had hexagonal outlines and electron-dense cores

Figure 1. Liver of inland bearded dragon with numerous inclusion bodies within enlarged nuclei of hepatocytes. Bar 40 m.

Figure 3. Transmission electron micrograph of viral particles from the pooled liver homogenate. Note viral particles of two distinctly different sizes. Bar 150 nm.

Figure 2. Small intestine of inland bearded dragon with an intranuclear inclusion body (asterisk) and various stages of coccidia in the cytoplasm of enterocytes including microgamonts (short arrows), a macrogamont (long arrow), zygotes (open arrow), and unsporulated oocysts (arrow heads). Bar 25 m.

Figure 4. Transmission electron micrograph of a hepatocyte with a nucleus lled with viral particles. The cytoplasm is markedly vacuolated. Bar 2.5 m. Inset, The viral particles (approximately 80 nm in diameter) are arranged in paracrystalline arrays. Bar 0.3 m.

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(Fig. 3). Direct smear and otation of the intestinal contents revealed large numbers of Isospora sp. Histopathologic ndings and the shape and size of the nonenveloped larger viral particles in paracrystalline arrays were consistent with adenovirus.2,5 All 6 lizards had acute severe coccidiosis throughout the small intestines. In the absence of signicant morphologic changes in the brain, the neurologic signs (head tilting and circling) in some of the lizards may be attributed to an acute hepatoencephalopathy due to the severe hepatocellular necrosis. Adenoviral infection has been identied in several reptiles, including Nile crocodiles (Crocodylus niloticus),7 rosy boas (Lichanura rosefusca),16 boa constrictors (Boa constrictor),4,8,15 a 4-lined rat snake (Elaphe quatuorlineata),4 a gaboon viper (Bitis gabonica),4 an aesculapian snake (Elaphe longissima),4 a Savannah monitor (Varanus exanthematicus),9 a Jacksons chameleon (Chamaeleo jacksoni),6 a mountain chameleon (Chameleo montium),12 bearded dragons (Pogona [ Amphibolurus] barbatus and Pogona vitticeps),10,11 and Rankins dragons (Pogona henrylawsoni).3 Generally, typical large intranuclear adenovirus inclusions were found in the liver or upper gastrointestinal system, including esophagus, or both. With the long replication cycle of 3236 hours, adenoviruses form large, dense intranuclear inclusion bodies consisting of massive numbers of virions in the infected cells.5 Common clinical problems were inappetence, vomiting, neurological signs, such as disorientation, head tremor, and opisthotonos, and sudden death without clinical signs. The smaller viral particles found in the liver homogenate were compatible with dependoviruses based on the morphologic characteristics, especially the virion size (20 nm), and copresence with adenoviruses. No dependoviruses, however, were identied by TEM in the formalin-xed liver specimens due to possible inadequate prexation in formalin. Dependoviruses (Family Parvoviridae) are small (1828 nm), nonenveloped viruses that have icosahedral symmetry.1,2 Other viruses that are similar in size are Picornaviridae (24 30 nm)2,14 and Circoviridae (1522 nm).14 The Picornaviridae viruses are larger than the smaller viruses found in the dragons, and circoviruses have not been reported in reptiles. Dependoviruses are commonly referred to as adeno-satellite viruses or adeno-associated viruses (AAV) because of their dependence on helper viruses (adenoviruses or herpes viruses) for replication.1,2 Some of the early-stage genes from adenovirus, as a helper virus, are required for the synthesis, transport, and translation of dependovirus mRNA. Both viruses, however, do not share the same set of viral proteins, such as DNA binding proteins, DNA polymerase, etc., for their replication.1 Interestingly, recent studies have shown that pretreatment of several cell lines with toxic agents results in dependovirus replication in the absence of a helper virus.1 According to others,10 coccidiosis is a common problem in captive-bred inland bearded dragons; however, in that report, only 1 of 4 neonatal dragons coinfected with adenovirus-like and dependovirus-like viruses had coccidiosis. Isospora amphiboluri has been identied in the inland bearded dragon,13 but the pathologic signicance of these coccidia

has not been fully elucidated. In this current outbreak, all 6 hatchling bearded dragons examined had adenoviral inclusions as well as variable stages of coccidial organisms throughout the small intestines. The coccidia were identied as Isospora sp. Common clinical manifestations of coccidiosis in animals are sudden onset of bloody diarrhea with fever, followed by dehydration, emaciation, and occasional death, especially in severely infected young animals. Although no obvious clinical evidence of coccidial infection was observed in these dragons, it is possible that an adverse synergistic effect of the coccidiosis and adenoviral infection resulted in the high morbidity and mortality in this case.

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References
1. Berns KI: 1991, Parvoviridae and their replication. In: Fundamental virology, eds. Fields BN, Knipe DM, Chanock RM, Hirsch MS, Milnick JL, Monath TP, 2nd ed., pp. 817837. Raven Press, New York, NY. 2. Doane FW, Anderson N: 1987, Electron microscopy in diagnostic virology. Cambridge University Press, Cambridge, UK. 3. Frye FL, Munn RJ, Gardner M, et al.: 1994, Adenovirus-like hepatitis in a group of related Rankins dragon lizards (Pogona henrylawsoni). J Zoo Wildl Med 25:167171. 4. Heldstab A, Bestetti G: 1984, Virus associated gastrointestinal diseases in snakes. J Zoo An Med 15:118128. 5. Horwitz MS: 1991, Adenoviridae and their replication. In: Fundamental virology, eds. Fields BN, Knipe DM, Chanock RM, et al., 2nd ed., pp. 771813. Raven Press, New York, NY. 6. Jacobson ER, Gardiner CH: 1990, Adeno-like virus in esophageal and tracheal mucosa of a Jacksons chameleon (Chamaeleo jacksoni). Vet Pathol 27:210212. 7. Jacobson ER, Gardiner CH, Foggin CM: 1984, Adenovirus-like infection in two Nile crocodiles. J Am Vet Med Assoc 185: 14211422. 8. Jacobson ER, Gaskin JM, Gardiner CH: 1985, Adenovirus-like infection in a boa constrictor. J Am Vet Med Assoc 187:1226 1227. 9. Jacobson ER, Kollias GV: 1986, Adenovirus-like infection in a Savannah monitor. J Zoo An Med 17:149151. 10. Jacobson ER, Kopit W, Kennedy FA, et al.: 1996, Coinfection of a bearded dragon, Pogona vitticeps, with adenovirus- and dependovirus-like viruses. Vet Pathol 33:343346. 11. Julian AF, Durham PJK: 1982, Adenoviral hepatitis in a female bearded dragon (Amphibolurus barbatus). NZ Vet J 30:5960. 12. Kinsel MJ, Barbiers RB, Manharth A, Murnane RD: 1997, Small intestinal adeno-like virus in a mountain chameleon (Chameleo montium). J Zoo Wildl Med 28:498500. 13. McAllister CT, Upton SJ, Jacobson ER, et al.: 1995, A description of Isospora amphiboluri (Apicomplexa: Eimeriidae) from the inland bearded dragon, Pogona vitticeps (Sauria: Agamidae). J Parasitol 81:281284. 14. Murphy FA: 1996, Virus taxonomy. In: Fields virology, eds. Fields BN, Knipe DM, Howley PM, et al., pp. 1557. Lippinocott-Raven Publishers, Philadelphia, PA. 15. Ramis A, Fernandez-Bellon H, Majo N, et al.: 2000, Adenovirus hepatitis in a boa constrictor (Boa constrictor). J Vet Diagn Invest 12:573576. 16. Schumacher J, Jacobson ER, Burns R, et al.: 1994, Adenoviruslike infection in two rosy boas (Lichanura trivirgata). J Zoo Wildl Med 25:461465.

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JVDXXX10.1177/1040638712450578

Erratum

Corrigendum

Journal of Veterinary Diagnostic Investigation 24(4) 813 2012 The Author(s) Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638712450578 http://jvdi.sagepub.com

Stegelmeier, BL, et al.: 2010, Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats. J Vet Diagn Invest. 22: 570577

In the article Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats by Bryan L. Stegelmeier et al., the published mean body weight and the means and statistics of serum biochemistries were carried out on groups of 4 animals, not 3, as described in the Material and Methods section. The additional animal in each group was part of an auxiliary physiologic study and though the animals were dosed and treated the same, they were not necropsied and were not included in the histologic study. To correct this oversight, the corrected weight and chemistry table (shaded cells indicate corrected numbers) are listed below. The differences are minimal and do not alter the conclusions. In addition, reference 7 has been deleted. Material and Methods: Fifteen, yearling, female Spanish goats weighing 29.4 3.4 kg (mean standard deviation) were randomly divided into 5 groups with 3 animals per group.

References: Reference 7 should be deleted

Corrected Table 1. Selected mean serum biochemical data from groups of 3 goats dosed with rayless goldenrod (Isocoma pluriflora) to obtain benzofuran ketone doses of 0, 10, 20, 40, and 60 mg/kg body weight for 7 days.* Serum result (mean standard deviation) Serum test (reference range) Creatinine kinase (< 350 U/l) Cardiac troponin-I (<0.40 U/l) Aspartate aminotransferase (<125 U/l) Alanine aminotransferase (<55 U/l) Lactate dehydrogenase (<1,560 U/l) Dose 0 10 20 40 60 0 10 20 40 60 0 10 20 40 60 0 10 20 40 60 0 10 20 40 60 Day 0 226 93 226 160 967 1233 125 18 202 93 <0.02 0.0 <0.02 0.0 <0.02 0.0 <0.02 0.0 <0.02 0.0 96 7 147 69 164 82 112 17 96 13 39 3 44 1 41 9 46 2 40 7 1,061 145 1,334 668 1,650 1,546 1,054 201 1,026 287 Day 3 107 6 118 8 306 276 117 24 202 124 <0.02 0.0 <0.02 0.0 0.17 0.26 <0.02 0.0 <0.02 0.0 91 6 104 11 284 248 102 12 115 31 37 3 42 3 57 34 44 4 44 5 1,075 62 1,050 223 2,617 2,685 1,162 130 1,277 348 Day 6 73 16 206 184a 240 113a 6,699 5,329b 2,987 3,701a <0.02 0.0 <0.02 0.0 0.05 0.03 1.98 3.39 1.38 2.31 83 2a 89 8a 293 252ab 991 184c 819 571bc 38 0a 39 2a 63 38ab 134 24a 118 84ab 875 213a 942 265a 1,185 449a 5,996 2,491b 3,623 2,924ab
a

Day 7 66 30a 495 623ab 497 277ab 16,270 11,054b 10,433 4,326ab <0.02 0.0 <0.02 0.0 <0.02 0.0 1.79 2.97 0.13 0.18 72 3a 97 13a 376 256a 3,277 1,556b 2,095 1,333b 43 18a 37 1a 61 25a 333 127b 267 176b 573 115a 709 182a 753 447a 9,891 3,210b 7,011 5,205a

*Different means (<0.05) between groups are indicated with superscript letters. Estimates of normal range were determined as 2 standard deviations from mean values of control goats and pretreatment samples. These ranges are probably laboratory and assay specific. Cardiac troponin-I concentrations below detection limits are reported as <0.02 ng/ml.

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