Detection and Confirmation of Reptilian Adenovirus Infection by In Situ Hybridization

Laura E. Leigh Perkins^1,2, Raymond P. Campagnoli², Barry G. Harmon², Christopher R. Gregory³, W.L. Steffens², Susan Clubb⁴, Maria Crane⁵

¹Southeast Poultry Research Laboratory, USDA/ARS, Athens, GA 30605; University of Georgia College of Veterinary Medicine, ²Department of Veterinary Pathology, ³Department of Small Animal Medicine, Athens, GA USA 30602; ⁴ Hurricane Aviaries, 3319 East Road, Loxahatchee, FA USA 33470; ⁵ Zoo Atlanta, 800 Cherokee Ave, S.E. , Atlanta, GA 30315

Abstract: Adenovirus infections are documented in at least 12 different species of the class Reptilia. The diagnosis of reptilian adenovirus infections is largely dependent upon light and electron microscopy for the identification of intranuclear viral inclusions associated with histopathologic lesions. In contrast to their mammalian and avian counterparts, reptilian adenoviruses are not well characterized as to their pathogenic potential and their ability to cause primary disease. In this communication, we present two different species of snake in which adenovirus infection was confirmed by the application of DNA in situ hybridization. In addition, both snakes featured concurrent diseases, suggesting that the adenovirus may not have been the primary pathogen in these cases.

Keywords: Adenovirus, In situ hybridization, Snake, Reptile, Boa, Rattlesnake, Boa constrictor, Crotalus scutulatus

Introduction

Adenoviruses are reported in at least 12 different species of reptiles of the orders Crocodylia (crocodiles, alligators, caimans) and Squamata, including both the Serpentes (snakes) and Suaria (lizards) suborders. The reptiles in which adenoviruses have been identified include those belonging to zoological collections and commercial breeders. In these species, adenoviruses are incriminated as the cause of gastroenteritis, hepatitis, nephritis, pneumonia, and encephalitis. ^{1,5-9,11,12,15,16} Adenovirus infection is reported in reptiles with^5,6,12 and without^5,7-9,15,16 concurrent disease, and in the majority of communications, only individual animals are afflicted. ^{5,7,9,11,12,15,16} Therefore, the pathogenic potential of the adenoviruses and their ability to cause primary disease within the class Reptilia remains uncertain.

Confirmation of adenovirus infection in the majority of the cases is based upon morphological features of the virus or viral inclusions as determined by electron microscopy. In these accounts, tissues suitable for virus isolation were not available. In three reports, successful isolation and propagation of a reptilian adenovirus in cell culture, with a virus-induced cytopathogenic effect, are described. ^7,11,15 However, in only one investigation were Koch’s postulates fulfilled, specifically identifying the isolated adenovirus as the primary etiologic agent of hepatitis and subsequent death in a common boa. ⁷

In contrast to reptilian adenoviruses, the pathogenesis and disease-causing potential of human and mammalian-origin (Mastadenovirus) and avian-origin adenoviruses (Aviadenovirus) are well characterized. In the diagnostic setting, DNA in situ hybridization (ISH) is a useful adjunct to microscopic and ultrastructural identification of mastadenoviruses and aviadenoviruses. ^2,3,14,19 Furthermore, in contrast to light and electron microscopy, ISH is specific, highly sensitive, rapid, and requires minimal specialized equipment or technical assistance. ¹³

Here we report the diagnosis of adenovirus infections in a common boa and a Mojave rattlesnake using ISH with an aviadenovirus-specific oligoprobe. ¹³

Case Histories

The first case was an adult male common boa (Boa constrictor imperator) from a zoological collection that presented with general malaise, listlessness, dehydration, anorexia, and dermatitis of an unknown duration. Treatment with Naxcel^® was ineffectual, and the snake was found dead shortly after treatment.

The second submission was an adult male Mojave Rattlesnake (Crotalus scutulatus scutulatus) from a zoological collection. The snake was in good health; however, it was found dead the day after an abdominal mass was identified.

Except fort the abdominal mass in the rattlesnake, post-mortem lesions were not reported. Formalin-fixed tissues from both snakes were independently submitted to the Department of Pathology of the University of Georgia College of Veterinary Medicine.

Materials and Methods

Fixed tissues from both snakes were routinely processed and paraffin-embedded. Three micrometer sections were stained with hematoxylin and eosin, and examined by light microscopy. Replicate unstained sections of the liver and gastrointestinal tracts were placed on ProbeOn Plus slides^a for DNA in situ hybridization tests. The digoxigenin-labeled adenoviral-specific oligoprobes, designated as FN-23 and FN-96, had the following nucleic acid sequences:¹³

FN-23: 5'-TCGGACATCGGGGTCAAGTTCGACACGCGCAACTTC-3'

FN-96: 5'-CGCCTTCAACCGCTTTCCCGAAAACGAGATTCTGAAGCAA-3'

These probes are based upon nucleotide sequences that code for a structural capsid protein of a chicken adenovirus (type 10) (GenBank accession M87008). ¹⁸ The technique for hybridization, using either of the two oligoprobes, was described previously. ¹³ Nitroblue tetrazolium (NBT) dye and fast green were utilized as the substrate chromagen and counterstain, respectively. Two avian polyomavirus (APV) oligoprobes were used as negative probe controls. Sections of chicken liver containing confirmed adenoviral inclusions were used as positive controls. Deposition of a blue-black chromagen indicated detection of adenoviral nucleic acid.

Formalin-fixed liver from the common boa was submitted for examination by transmission electron microscopy. The tissue was re-fixed in 2% paraformaldehyde, 2% glutaraldehyde, and 0.2% picric acid in 0.1M cacodylate-HCl buffer (pH 7.2), rinsed, and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate-HCl. The tissue was then infiltrated and embedded in an epon-araldite mixture and allowed to polymerize in a 75° C oven overnight. Blocks were trimmed and 1 µm sections were stained with 1% toluidine blue in 1% sodium borate and evaluated for the presence of inclusions. Tissues containing viral inclusions were sectioned at approximately 65 nm and placed on 200-mesh copper hex grids. The grids were then post-stained with 5% methanolic uranyl acetate and Reynold’s lead citrate and viewed using a JEM-1210 transmission electron microscope. ^c

Results

Multifocal to confluent hepatic necrosis and hemosiderin deposition in hepatocytes and Kupffer cells were observed in the liver sections from the boa. Anisokaryosis of hepatocytes and marked increases in sinusoidal inflammatory cells, which were predominantly macrophages, also were observed. Large basophilic intranuclear inclusion bodies were present in hepatocytes, Kupffer cells, and occasionally in endothelial cells (Figure 1). In addition the snake had concurrent enterohepatic amebiasis with corresponding ulcerative enteritis and lymphoid hyperplasia. Eosinophilic intracytoplasmic inclusion bodies were observed in the epithelial cells of renal tubules, tracheal mucosa, lung, and gastric mucosa (Figure 2). These inclusions were consistent in morphology to those identified in previously described cases of retroviral-induced wasting disease of boa constrictors. ¹⁷ In the liver sections, ISH revealed adenoviral DNA in hepatocytes, Kupffer cells, and sinusoidal endothelial cells (Figure 3). The chromagen deposition was granular in nature and predominantly nuclear. Intranuclear round to hexagonal virus particles, with both electron dense and electron lucent cores, were observed with transmission electron microscopy. These viral particles ranged in size from 65 to 70 µm and were typical of adenoviral particles (Figure 4).

Figure 1. Liver, common boa, H and E stain. Intranuclear inclusion bodies in endothelial cell (arrow), Kupffer cell (arrowhead), and hepatocyte (asterisk). Bar = 10 µm.

Figure 2. Tracheal epithelium, common boa, H and E stain. Eosinophilic intracytoplasmic retroviral inclusions (arrows) in epithelial cells. Bar = 10 µm.

Figure 3. Liver, common boa, ISH with FN-96 aviadenovirus oligoprobe and fast green counterstain.   Adenovirus DNA in nuclei of Kupffer cells and hepatocytes. Bar = 10 µm.

Figure 4. Liver, common boa, transmission electron microscope. Adenovirus particles in the nucleus of a hepatocyte. Bar = 200 nm.

In the Mojave rattlesnake, the most striking lesions were in the gastrointestinal tract. Severe multifocal hemorrhage and ulceration with intralesional amoeba and basophilic intranuclear inclusion bodies in enterocytes were observed (Figure 5). Additional lesions included bacterial nephritis and cellulitis, splenic fibrosis, myocardial necrosis with pericardial edema, and a renal carcinoma in the abdomen. In the intestinal tract, adenoviral DNA was demonstrated within scattered enterocytes cells by ISH (Figure 6).

Figure 5. Intestinal tract, Mojave rattlesnake, H and E stain. Intranuclear inclusion bodies in an enterocyte. Bar = 10 µm.

Figure 6. Intestinal tract, Mojave rattlesnake, ISH with FN-23 aviadenovirus oligoprobe and fast green counterstain. Adenovirus DNA in the nucleus of an enterocyte. Bar = 10 µm.

Discussion

Replicating mastadenoviruses and aviadenoviruses characteristically form large basophilic intranuclear inclusions in infected cells that can be detected microscopically. Similar intranuclear inclusions have been consistently identified in reptilian tissues infected with adenovirus. Tissues in which adenovirus inclusions are identified include liver (the present report)^{1,6,7,9,12,16}, biliary ductular epithelium¹², pancreatic acinar epithelium⁵, renal tubular epithelium^1,9,16, gastrointestinal mucosa (the present report)^5,8, respiratory epithelium^1,8, Kupffer cells of the liver (the present report), splenic macrophages⁵, and endothelium (the present report). ^9,12 However, other nuclear-replicating reptilian viruses, such as herpesviruses, also are capable of forming similar intranuclear inclusions. ^4,20 In addition, nonviral intranuclear inclusions are reported in reptilian tissues. ¹⁰ Presently, ultrastructural evaluation and/or virus isolation are the main diagnostic tools for the positive identification of the causative agents of the inclusions. This report describes an additional method to detect adenoviral DNA using an ISH in formalin-fixed paraffin-embedded reptilian tissues. In comparison with light microscopic examination of hematoxylin and eosin stained tissue sections, ISH is highly sensitive and specific for the identification of adenovirus-infected cells in tissue sections. ISH also is quicker, less tedious, and less expensive than transmission electron microscopy and can be incorporated with minimal expense into existing histopathology laboratories. Therefore, ISH is a useful means of obtaining a definitive diagnosis in cases where inclusion bodies are observed in the tissues of captive reptiles.

In this and previous reports, adenovirus inclusions have been associated with epithelial necrosis regardless of the specific anatomic location. In addition, an adenovirus isolate, originally obtained from a moribund boa, was shown to cause similar disease and death when experimentally inoculated into a neonatal boa. ⁷ Thus, adenoviruses appear to be capable of causing severe disease in reptiles. Several reptiles in previous reports also were undergoing stress, such as reproduction and recent importation, or were experiencing coexisting infections, such as enteric protozoa. Likewise, in the present report, concurrent amebiasis and retroviral infection (common boa) and amebiasis and neoplasia (Mojave rattlesnake) were present. This suggests that, as with the pathogenesis of some mammalian and avian adenoviruses, immunosuppression may play a role in adenoviral-induced disease in reptiles. However, there are incidences of adenoviral infections in reptiles without detectable stress or disease. ^6,8-10,15,16 Therefore, the disease-causing potential of adenoviruses in reptiles cannot be determined based on the rather limited number of cases in which this virus has been confirmed. Further research is required to define the epidemiology and virulence of adenoviruses in captive reptiles. Because of the high sensitivity of ISH³, this technique may be useful in studies for determining the prevalence and pathogenic potential of adenovirus infections.

Acknowledgments

We are grateful to Mary Ard for processing tissues for electron microscopy and printing the electron micrographs. Dr. Ken Latimer provided the DNA probes and laboratory facilities for DNA in situ hybridization, allowing specific diagnosis of adenovirus in these snakes.

Sources and Manufacturers

a. Microprobe Work Station, Fischer Scientific, Pittsburgh, PA

b. Reichert Ultracut S Ultramicrotome, Leice, Inc. Deerfield, IL

c. JEM-1210 Transmission Electron Microscope purchased through JOEL USA, Inc. , Peabody, MA

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