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Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/14/4/332The online version of this article can be found at:

 DOI: 10.1177/104063870201400411

2002 14: 332J VET Diagn InvestDae Young Kim, Mark A. Mitchell, Rudy W. Bauer, Rob Poston and Doo-Youn Cho

Sp.)IsosporaDependovirus and Coccidial Protozoa () Coinfected withPogona VitticepsAn Outbreak of Adenoviral Infection in Inland Bearded Dragons (

  

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332 Brief Communications

J Vet Diagn Invest 14:332–334 (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 (48hours) of weakness and lethargy. The most common clinical signs were head tilt and circling. Six beardeddragons with neurological signs were euthanized, and postmortem examination revealed no gross abnormalities.Microscopically, severe, randomly distributed hepatocellular necrosis with large basophilic intranuclear inclu-sion bodies in numerous hepatocytes was noted. Small-intestinal enterocytes contained intracytoplasmic coc-cidial protozoa (Isospora sp.) and occasional enterocytes had basophilic intranuclear inclusion bodies. Trans-mission electron microscopy revealed both 80- and 20-nm-diameter viral particles, which were consistent withadenoviruses and dependoviruses, respectively. Adenoviral outbreaks in groups of animals are uncommon. Anadverse synergistic effect of the coccidiosis with the adenoviral infection may have played a critical role in thehigh morbidity and mortality in this case.

Adenoviruses (Family Adenoviridae) are well-knownpathogens in several mammalian and avian species. Ade-noviruses are double-stranded DNA viruses, 70–90 nm indiameter, and have a characteristic nonenveloped, icosahe-dral structure. Generally, adenoviruses are host specific andare transmitted by the fecal-oral route or direct contact viaoronasal secretions. Often, mammalian infections are sub-clinical, except for infectious canine hepatitis. Adenoviraldisease generally occurs in immunocompromised or younganimals. Outbreaks in groups of animals are uncommon. Re-cently, adenoviral infections also have been reported in sev-eral reptilian species, including crocodiles, snakes, and liz-ards.3,4,6–12,15,16

The Australian inland bearded dragon,Pogona vitticeps(Pogon: bearded in Greek), is one of the most popular rep-tiles in the pet trade. Two isolated cases of adenoviral infec-tion in 4 neonatal inland bearded dragons have been report-ed, but the infections were limited mainly to the individualsaffected.10 No outbreak of adenoviral infection in a group ofreptiles has been reported. This report describes an outbreakof adenoviral infection in a group of captive-bred inlandbearded dragon hatchlings coinfected with dependovirus andIsospora sp. coccidia.

Two hundred hatchling captive-bred inland bearded drag-ons, which had been purchased at different times by a reptileimporter from different captive breeding populations, wereplaced in a holding facility. The ages of the dragons wereuncertain; however, based on their weight (�5 g), the esti-mate age was less than 1 month. The bearded dragons weremaintained at an environmental temperature of 29–32 C andwere fed commercially obtained crickets and lettuce. Thirtyof the 200 (15%) bearded dragons were found dead after ashort period (48 hours) of weakness and lethargy. The most

From the Departments of Pathobiological Sciences (Kim, Cho)and Veterinary Clinical Sciences (Mitchell), School of VeterinaryMedicine, Louisiana State University, and The Louisiana VeterinaryMedical Diagnostic Laboratory (Bauer, Poston), Baton Rouge, LA70803.

Received for publication August 27, 2001.

common clinical signs were head tilt and circling. Physicalexamination of tympanic bullae revealed no significant find-ings. Six of the bearded dragons with neurological signswere euthanized and submitted for necropsy to the LouisianaVeterinary Medical Diagnostic Laboratory, Baton Rouge,Louisiana.

At necropsy, no significant gross abnormalities were notedin any of the 6 bearded dragons. Sections from major organswere fixed in 10% neutral buffered formalin, routinely pro-cessed, sectioned at 4�m thick, and stained with hematox-ylin and eosin. For electron microscopy, 2 methods wereused. At first, the liver specimens from 3 bearded dragonswere pooled together, homogenized, and centrifuged at 800� g for 30 minutes. The supernatant was collected and cen-trifuged at 98,000� g for 1 hour. The formed pellet washarvested after discarding the supernatant. The pellet wassuspended in 100�l of phosphate-buffered saline (PBS) so-lution, stained with 100�l of 4% phosphotungstic acid,placed on Formvar- and carbon-coated grids, air-dried, andexamined with a transmission electron microscope (TEM).Second, formalin-fixed liver tissue was trimmed, dehydrated,postfixed in 1% phosphotunstic acid (w/v), embedded in LRWhite,a sectioned, and stained with uranyl acetate and leadcitrate for TEM. The contents of the large intestines weresubmitted for parasitologic examination.

Significant microscopic changes were similar in all 6dragons and were limited to the liver and small intestine.Severe, randomly distributed hepatocellular necrosis involv-ing �50% of the parenchyma was accompanied by mild tomoderate infiltration of lymphocytes and histiocytes. Nu-merous hepatocyte nuclei were expanded (2–4-fold) bylarge, basophilic, glassy, intranuclear inclusion bodies andmarginated chromatin (Fig. 1). Small intestinal villi wereatrophied, and many enterocytes contained variable stages ofcoccidia in the cytoplasm. Enterocytes occasionally had ba-sophilic intranuclear inclusion bodies similar to those seenin the liver (Fig. 2). Electron microscopy revealed viral par-ticles of 2 distinct sizes. Larger viral particles were approx-imately 80 nm in diameter, nonenveloped, and had hexago-nal outlines and electron-dense or electron-lucent cores.

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333Brief Communications

Figure 1. Liver of inland bearded dragon with numerous inclu-sion bodies within enlarged nuclei of hepatocytes. Bar� 40 �m.

Figure 2. Small intestine of inland bearded dragon with an in-tranuclear inclusion body (asterisk) and various stages of coccidiain the cytoplasm of enterocytes including microgamonts (short ar-rows), a macrogamont (long arrow), zygotes (open arrow), and un-sporulated oocysts (arrow heads). Bar� 25 �m.

Figure 4. Transmission electron micrograph of a hepatocytewith a nucleus filled with viral particles. The cytoplasm is markedlyvacuolated. Bar� 2.5 �m. Inset, The viral particles (approximately80 nm in diameter) are arranged in paracrystalline arrays. Bar� 0.3�m.

Figure 3. Transmission electron micrograph of viral particlesfrom the pooled liver homogenate. Note viral particles of two dis-tinctly different sizes. Bar� 150 nm.

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

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334 Brief Communications

(Fig. 3). Direct smear and flotation of the intestinal contentsrevealed large numbers ofIsospora sp.

Histopathologic findings and the shape and size of thenonenveloped larger viral particles in paracrystalline arrayswere consistent with adenovirus.2,5 All 6 lizards had acutesevere coccidiosis throughout the small intestines. In the ab-sence of significant morphologic changes in the brain, theneurologic signs (head tilting and circling) in some of thelizards may be attributed to an acute hepatoencephalopathydue to the severe hepatocellular necrosis.

Adenoviral infection has been identified in several rep-tiles, including Nile crocodiles (Crocodylus niloticus),7 rosyboas (Lichanura rosefusca),16 boa constrictors (Boa constric-tor),4,8,15 a 4-lined rat snake (Elaphe quatuorlineata),4 a gab-oon viper (Bitis gabonica),4 an aesculapian snake (Elaphelongissima),4 a Savannah monitor (Varanus exanthemati-cus),9 a Jackson’s chameleon (Chamaeleo jacksoni),6 amountain chameleon (Chameleo montium),12 bearded drag-ons (Pogona [� Amphibolurus] barbatus and Pogona vitti-ceps),10,11 and Rankin’s dragons (Pogona henrylawsoni).3

Generally, typical large intranuclear adenovirus inclusionswere found in the liver or upper gastrointestinal system, in-cluding esophagus, or both. With the long replication cycleof 32–36 hours, adenoviruses form large, dense intranuclearinclusion bodies consisting of massive numbers of virions inthe infected cells.5 Common clinical problems were inap-petence, vomiting, neurological signs, such as disorientation,head tremor, and opisthotonos, and sudden death withoutclinical signs.

The smaller viral particles found in the liver homogenatewere compatible with dependoviruses based on the morpho-logic characteristics, especially the virion size (20 nm), andcopresence with adenoviruses. No dependoviruses, however,were identified by TEM in the formalin-fixed liver speci-mens due to possible inadequate prefixation in formalin. De-pendoviruses (Family Parvoviridae) are small (18–28 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 (15–22 nm).14 The Picornaviridaeviruses are larger than the smaller viruses found in the drag-ons, and circoviruses have not been reported in reptiles. De-pendoviruses are commonly referred to as adeno-satellite vi-ruses or adeno-associated viruses (AAV) because of theirdependence on helper viruses (adenoviruses or herpes virus-es) for replication.1,2 Some of the early-stage genes fromadenovirus, as a helper virus, are required for the synthesis,transport, and translation of dependovirus mRNA. Both vi-ruses, however, do not share the same set of viral proteins,such as DNA binding proteins, DNA polymerase, etc., fortheir replication.1 Interestingly, recent studies have shownthat pretreatment of several cell lines with toxic agents re-sults in dependovirus replication in the absence of a helpervirus.1

According to others,10 coccidiosis is a common problemin captive-bred inland bearded dragons; however, in that re-port, only 1 of 4 neonatal dragons coinfected with adeno-virus-like and dependovirus-like viruses had coccidiosis.Isospora amphiboluri has been identified in the inland beard-ed dragon,13 but the pathologic significance of these coccidia

has not been fully elucidated. In this current outbreak, all 6hatchling bearded dragons examined had adenoviral inclu-sions as well as variable stages of coccidial organismsthroughout the small intestines. The coccidia were identifiedas Isospora sp. Common clinical manifestations of coccidi-osis in animals are sudden onset of bloody diarrhea withfever, followed by dehydration, emaciation, and occasionaldeath, especially in severely infected young animals. Al-though no obvious clinical evidence of coccidial infectionwas observed in these dragons, it is possible that an adversesynergistic effect of the coccidiosis and adenoviral infectionresulted in the high morbidity and mortality in this case.

Sources and manufacturers

a. LR White, London Resin Company Limited, Berkshire, UK.

References

1. Berns KI: 1991, Parvoviridae and their replication.In: Funda-mental virology, eds. Fields BN, Knipe DM, Chanock RM,Hirsch MS, Milnick JL, Monath TP, 2nd ed., pp. 817–837. Ra-ven Press, New York, NY.

2. Doane FW, Anderson N: 1987, Electron microscopy in diag-nostic virology. Cambridge University Press, Cambridge, UK.

3. Frye FL, Munn RJ, Gardner M, et al.: 1994, Adenovirus-likehepatitis in a group of related Rankin’s dragon lizards (Pogonahenrylawsoni). J Zoo Wildl Med 25:167–171.

4. Heldstab A, Bestetti G: 1984, Virus associated gastrointestinaldiseases in snakes. J Zoo An Med 15:118–128.

5. Horwitz MS: 1991, Adenoviridae and their replication.In: Fun-damental virology, eds. Fields BN, Knipe DM, Chanock RM,et al., 2nd ed., pp. 771–813. Raven Press, New York, NY.

6. Jacobson ER, Gardiner CH: 1990, Adeno-like virus in esopha-geal and tracheal mucosa of a Jackson’s chameleon (Chamaeleojacksoni). Vet Pathol 27:210–212.

7. Jacobson ER, Gardiner CH, Foggin CM: 1984, Adenovirus-likeinfection in two Nile crocodiles. J Am Vet Med Assoc 185:1421–1422.

8. Jacobson ER, Gaskin JM, Gardiner CH: 1985, Adenovirus-likeinfection in a boa constrictor. J Am Vet Med Assoc 187:1226–1227.

9. Jacobson ER, Kollias GV: 1986, Adenovirus-like infection in aSavannah monitor. J Zoo An Med 17:149–151.

10. Jacobson ER, Kopit W, Kennedy FA, et al.: 1996, Coinfectionof a bearded dragon,Pogona vitticeps, with adenovirus- anddependovirus-like viruses. Vet Pathol 33:343–346.

11. Julian AF, Durham PJK: 1982, Adenoviral hepatitis in a femalebearded dragon (Amphibolurus barbatus). NZ Vet J 30:59–60.

12. Kinsel MJ, Barbiers RB, Manharth A, Murnane RD: 1997,Small intestinal adeno-like virus in a mountain chameleon (Cha-meleo montium). J Zoo Wildl Med 28:498–500.

13. McAllister CT, Upton SJ, Jacobson ER, et al.: 1995, A descrip-tion of Isospora amphiboluri (Apicomplexa: Eimeriidae) fromthe inland bearded dragon,Pogona vitticeps (Sauria: Agami-dae). J Parasitol 81:281–284.

14. Murphy FA: 1996, Virus taxonomy.In: Fields virology, eds.Fields BN, Knipe DM, Howley PM, et al., pp. 15–57. Lippi-nocott-Raven Publishers, Philadelphia, PA.

15. Ramis A, Fernandez-Bellon H, Majo N, et al.: 2000, Adenovirushepatitis in a boa constrictor (Boa constrictor). J Vet DiagnInvest 12:573–576.

16. Schumacher J, Jacobson ER, Burns R, et al.: 1994, Adenovirus-like infection in two rosy boas (Lichanura trivirgata). J ZooWildl Med 25:461–465.

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Erratum

450578 JVDXXX10.1177/1040638712450578

Corrigendum

Stegelmeier, BL, et al.: 2010, Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats. J Vet Diagn Invest. 22: 570–577

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 physi-ologic 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†) Dose Day 0 Day 3 Day 6 Day 7

Creatinine kinase (< 350 U/l) 0 226 ± 93 107 ± 6 73 ± 16a 66 ± 30a

10 226 ± 160 118 ± 8 206 ± 184a 495 ± 623ab

20 967 ± 1233 306 ± 276 240 ± 113a 497 ± 277ab

40 125 ± 18 117 ± 24 6,699 ± 5,329b 16,270 ± 11,054b

60 202 ± 93 202 ± 124 2,987 ± 3,701a 10,433 ± 4,326ab

Cardiac troponin-I (<0.40 U/l‡) 0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 10 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 <0.02 ± 0.0 20 <0.02 ± 0.0 0.17 ± 0.26 0.05 ± 0.03 <0.02 ± 0.0 40 <0.02 ± 0.0 <0.02 ± 0.0 1.98 ± 3.39 1.79 ± 2.97 60 <0.02 ± 0.0 <0.02 ± 0.0 1.38 ± 2.31 0.13 ± 0.18Aspartate aminotransferase (<125 U/l) 0 96 ± 7 91 ± 6 83 ± 2a 72 ± 3a

10 147 ± 69 104 ± 11 89 ± 8a 97 ± 13a

20 164 ± 82 284 ± 248 293 ± 252ab 376 ± 256a

40 112 ± 17 102 ± 12 991 ± 184c 3,277 ± 1,556b

60 96 ± 13 115 ± 31 819 ± 571bc 2,095 ± 1,333b

Alanine aminotransferase (<55 U/l) 0 39 ± 3 37 ± 3 38 ± 0a 43 ± 18a

10 44 ± 1 42 ± 3 39 ± 2a 37 ± 1a

20 41 ± 9 57 ± 34 63 ± 38ab 61 ± 25a

40 46 ± 2 44 ± 4 134 ± 24a 333 ± 127b

60 40 ± 7 44 ± 5 118 ± 84ab 267 ± 176b

Lactate dehydrogenase (<1,560 U/l) 0 1,061 ± 145 1,075 ± 62 875 ± 213a 573 ± 115a

10 1,334 ± 668 1,050 ± 223 942 ± 265a 709 ± 182a

20 1,650 ± 1,546 2,617 ± 2,685 1,185 ± 449a 753 ± 447a

40 1,054 ± 201 1,162 ± 130 5,996 ± 2,491b 9,891 ± 3,210b

60 1,026 ± 287 1,277 ± 348 3,623 ± 2,924ab 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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