Cytoplasmic Inclusions in Corn Snakes, Elaphe guttata, Resembling Inclusion Body Disease of Boid Snakes

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1 P a t h o l o g y P a g e s Cytoplasmic Inclusions in Corn Snakes, Elaphe guttata, Resembling Inclusion Body Disease of Boid Snakes Gregory J. Fleming1, BBA, DVM, Darryl J. Heard1, BSc, BVMS, PhD, DACZM, Elliott R. Jacobson1, DVM, PhD, DACZM, Claus Buergelt2, DVM, DACVP 1. Department of Small Animal Clinical Sciences College Veterinary Medicine Gainesville, FL 32610, USA 2. Department of Pathobiology College Veterinary Medicine Gainesville, FL 32610, USA A b str a c t - Three neonatal, captive-bred com snakes, Elaphe guttata, from a clutch of nine eggs were found dead after a short period of anorexia and opisthotonus. Histological examination of tissue samples revealed eosinophilic intracytoplasmic inclusions in hepatocytes and brain endothelial cells. These inclusions were similar in appearance to those observed in the tissues of animals affected with inclusion body disease of boids, a disease not presently recognized in colubrid snakes. Electron microscopic examination of the inclusions revealed membrane bound electron dense material inconsistent with IBD inclusions. K eyw ords: com snake, Elaphe guttata, intracytoplasmic eosinophilic inclusion, electron microscopy, inclusion body disease. In t r o d u c t io n Numerous clinical diseases of reptiles have been linked to viral pathogens based on histopathologic and electron microscopic findings (Ahne, 1977, C lark and Lunger, 1981, Axthelm, 1985, Jacobson, 1986, Collett, et al, 1988, Axthelm, 1989). Some of these viral diseases include paramyxoviruses in viperid snakes and a boelans python, Morelia boeleni, (Ahne, et al, 1987, Jacobson and Gaskn, 1987, West, et al, 2001), herpes vims in numerous turtle and tortoise species (Coward, et al, 1972, Rebell, et al, 1975, Haines and Klesse, 1977, Origgi and Jacobson 2000), poxvirus in spectacled caimans, Caiman crocodylus and Nile crocodiles, Crocodylus niloticus, (Jacobson, et al, 1979), adenovirus in bearded dragons, Pogona vitticeps, (Jacobson, et al, 1996), and inclusion body disease (IBD) of boids (Ahne, 1977, Clark and Lunger, 1981, Jacobson, 1986). Inclusion body disease affects snakes in the family Boidae and is characterized by the presence of intracytoplasm ic inclusions in visceral epithelial cells and neurons in the central nervous system (A xthelm, 1985, A xthelm, 1989, Schumacher, et al, 1994). Affected snakes may show chronic regurgitation, anorexia, incoordination, head tremors and opisthotonus resulting in either spontaneous death or euthanasia. Electron microscopic examination of affected tissues reveals viral particles with morphological characteristics similar to C-type retroviral particles (Schumacher, et al, 1994). A vims that has reverse transcriptase activity and morphologic features of viruses in the family Retroviridae has been isolated from a boa constrictor, Boa constrictor, with IBD and Koch s postulates were partially fulfilled (Jacobson, 1996). In addition a 68-kd protein has been associated with IBD in boa co n stricto rs and is hypothesized to be a v iral protein (Wozniak, et al, 2000). In addition to snakes of the family Boidae, a disease resembling IBD, with intracytoplasmic inclusion bodies has been described in captive palm vipers, Bothrops marchi (Raymond, et al, 2001). Inclusions resembling IBD have also been found in a California king snake, Lampropeltis geltus californiae, (Jacobson, et al, 1980), an eastern king snake, Lampropeltis getula getula, (Jacobson, 1996), and an Arizona mountain kingsnake, Lam propeltis pyrom elana, (Jacobson, 1989). However these three cases did not have sufficient material to confirm a C -type retro v iru s by tran sm issio n electron microscopy (TEM) (Jacobson, 1989). With the exception of IBD almost all retroviral infections in snakes have been recovered from neoplasms (Jacobson, et al, 2001). C-type retroviral particles have been identified in an embryonal rhabdomyosarcoma from a corn snake, Elaphe guttata, (Clark, et al, 1979), in cultured spleen cells from a Russell s viper, Vipera russelli, with a myxofibroma (Zeigel and Clark, 1969, Zeigel and Clark, 1971, Lunger, et al, 1974), in a boa constrictor with erythroleukosis (Konstantinov and Ippen, 1982), and several B razilian lancehead vipers, Bothrops moojeni, with renal tumors (Hoge, et al, 1995). A few non-tumor related retroviruses have also been identified in Russell s viper heart cell lines (Clark, 1973), venom gland of Jararacussu vipers, Sphenodon punctatus (Cameiro, 1992), 18 Journal of Herpetological Medicine and Surgery Volume 13, No. 1,2003

2 and IBD of boids (Schumacher, 1994). Here we describe the clinicopathological findings of IBD like inclusions in three captive bred corn snakes housed in a zoological collection in north-central Florida. C a s e S t u d y A clutch of nine fertile captive corn snake eggs were rem oved from the ad u lt s enclosure and incubated (26-28 C:79-82 F for 62 d) in a separate building within 24 hr of being laid. The eggs were incubated in an 80 x 30 x 15 cm (L x W x H) plastic container containing equal parts of vermiculite and water by weight. During the hatching process, offspring may have had contact with each other for up to 12 hr before they were removed from the incubator and individually housed. Each snake was housed in a sm all plastic container measuring 80 x 30 x 15 cm (L x W x H), with a hide box, water bowl and newspaper substrate, and fed dayold pinkie mice twice a week. The first snake died at three months of age after a onemonth period of anorexia and occasional opisthotonus. The second and third snakes appeared healthy and were eating well, but died two days apart, one month following the death of the first snake. The parents of the com snakes were housed 3 m from a pair of boa constrictors for over five years. Approximately five months prior to the death of the three com snakes, the female boa constrictor died and a diagnosis of IBD was made based on histological examination that revealed characteristic inclusions in epithelial cells of the liver, pancreas, kidney, neuron and brain. Additional findings included nephritis (lymphohistiocytic), splenitis (heterophilic), pancreatitis (lymphocytic) and esophagitis (lymphocytic). The remaining male boa constrictor appeared norm al based upon a norm al physical examination, absence of IBD inclusions from esophageal lymphoid tonsillar tissue, and absence of any neurologic or gastrointestinal signs of IBD. One author (ERJ) feels that the lymphoid tissue from esophageal tonsils is the most diagnostic sample tissue to obtain inclusions for IBD of boids. One year later the male boa constrictor is still healthy and is not showing any clinical signs of IBD. The adult com snakes did not have direct contact with the female boa constrictor and there were no reported disease or parasitic problems, including snake mites, Ophionyssus natricis, in this facility. However, indirect contact could have taken place as the same animal care staff looked after both enclosures. The adult corn snakes were given a complete physical exam and appeared to be healthy and in good body condition one year after the death of the three neonatal com snakes. Pathology Findings - Necropsy examinations on all three corn snakes and the boa constrictor were performed at the University of Florida Veterinary Medical Teaching Hospital within 24 hr of death. No gross abnormalities were noted. H istological exam ination of tissues from the first snake revealed multifocal necrotizing hepatitis with eosinophilic intracytoplasmic inclusions ranging between 1-3 pm (Figure 1). Intracytoplasmic eosinophilic inclusions were also found in the second and third snakes in blood vessel endothelium, in the brain and throughout the gastrointestinal tract (Figure 2). Figure 1. A few intracytoplasmic inclusions (arrows) are seen in otherwise healthy hepatocytes of a com snake, Elaphe guttata. H&Ex 100. Figure 2 Inclusions are present in the capillaries of the brain of a com snake, Elaphe guttata. H&E x 100. (arrows) The intracytoplasmic inclusions were interpreted to be suggestive of boid IBD. Special staining of the corn snake inclusions for DNA with Feulgen reaction, and for DNA and RNA with acridine orange were negative in both cases. Electron Microscopic Methods and Materials - Samples of the brain and liver from all three com snakes were processed for transmission electron microscopy. Tissues were fixed in M cdowell and Trumps fixative, washed with 0.1M PBS (phosphate buffer saline solution), post-fixed with 1% buffered osmium tetroxide, dehydrated in a graded ethanol series and embedded in Embed 812 epoxy resin (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were collected on coated 100 mesh copper grids, post stained with 1% aq. uranyl acetate and R eynold s lead citrate. Sections were examined with a Hitachi H-7000 (Hitachi High Technologies America, Gaithersburg, MD) transmission elec- Volume 13, No. 1,2003 Journal of Herpetological Medicine and Surgery 19

3 tron m icroscope at 75Kv accelerating voltage and images were acquired with Gatan Bioscan Digital Micrograph soft ware (G atan Inc., Pleasanton, CA). Electron microscopy revealed intracytoplasm ic inclusion bodies ranging in size from 1-4 pm (Figure 3). The first inclusion contained a homogeneous material surrounded by more disperse particu late m aterial (Figure 3, IB 1). The second inclusion was membrane bound and consisted of course electron dense fluc tu an t m ateria l (F ig u re 3, IB 2). T h ese in clu sio n s were com pared to inclusions typically found in IBD infected snakes (Figure 4). Cells containing the inclusions had an abundance of rough endoplasm ic reticulum, mitochondria and vacuoles. D is c u s s io n Figure 3. Electron micrograph of two inclusions within a blood vessel in the brain of a com snake. N=nucleus, M= mitochon dria, IB1= inclusion body 1, IB2= inclusion body 2, RER= rough endoplasmic reticulum, V= vacuolated rough endoplasmic retic ulum. Uranyl acetate and Reynold s lead citrate. Figure 4. Electron micrograph of a IBD inclusion in the brain of a boa constrictor. Small round to oval electron dense subunits are being laid down on the periphery of the inclusion (white arrows) IB=inclusion body. Uranyl acetate and Reynold s lead citrate. 20 Journal of Herpetological Medicine and Surgery A lth o u g h c lin ic a l p re s e n ta tio n and th e p re sen ce of eosinophilic intracytoplasmic inclusion bodies may have sup ported the diagnosis of IBD, additional evaluation by TEM did not demonstrate the presence of budding virions as seen in snakes with IBD. Thus it is important to note that the pres ence o f intracytoplasm ic eosinophilic inclusions by light microscopy does not guarantee a diagnosis of IBD. Intracytoplasmic inclusions found in the liver of the first com snake were found in both healthy hepatocytes and areas of necrotic hepatocytes. The necrotic hepatitis appears to be caused by the formation of the inclusions, possibly viral, as no other etiologic agent such as bacteria or chronic inflamma tory cells were seen. Necrosis of the hepatocytes was only seen in one of the three com snakes and the sample was mild ly autolyzed. Special histochemical stains may assist in determining if there is nucleic material within the inclusion which may indi cate virions may be present. M ethyl green-pryonin stain is utilized to stain RNA. Feulgen reaction stains for DNA while acridine orange stains RNA a red yellow and DNA a yellow ish green. In this case both Feulgen and acridine orange were negative for the com snake inclusions (Jacobson, 1986), sug gesting that no nucleic material was present in the com snake inclusions and that virions are not present. In m ammals there are non-viral processes that result in intracellular inclusions (Cheville, 1983). For example, some mammalian inclusion bodies, called Mallory bodies, are com posed of hyaline, a dense amorphous eosinophilic material made up of structureless proteinaceous material. Lead toxico sis can also result in intranuclear inclusions representing lead-histone complexes (Cheville, 1983, Coffin, 1996). A number of non-viral inclusions also have been found in a variety of reptile species (Jacobson, 1989). Hepatocytes from a Deckerts rat snake, Elaphe obsoleta deckerti, contained eosinophilic intranuclear inclusions. By TEM the inclusions were found to develop from vacuoles near Golgi complexes and were likely lysosom al in origin (Jacobson, 1989). A California king snake with lymphosarcoma had eosinophilic in tra n u c le a r in clu sio n s w ith in n eo p lastic lym phocytes (Jacobson, 1989). These inclusions were large and uniformly electron dense by TEM, but did not stain with Feulgen and methyl green-pyronin, suggesting a non-nucleic acid compo sition. H ow ever, ex am in atio n by TEM found particles morphologically consistent with virions. Staining techniques may be fallible and the stain may have been applied incorvolume 13, No. 2,2003

4 rectly. The use of multiple diagnostic techniques including TEM may be a more definitive tool for identifying viral inclusions. Viral disease may produce inclusions containing viral components such as capsids or nucleic m aterial how ever, inclusions without a viral component have also been noted in viral infections. T his was dem onstrated in a case of a paramyxoviral infection in a rattlesnake where eosinophilic intracytoplasmic inclusions of the pancreatic ductal epithelial cells co n sisted o f a uniform p ro tein aceo u s m aterial (Jacobson, 1989). Boa constrictors experimentally infected with IBD produced intracytoplasm ic inclusions with and without virions (Wozniak, 2000). The inclusions without virions, staining negative for nucleic material, were characterized by a membrane bound fine granular electron dense center and a unique 68-kd protein on a protein electrophoretogram. This protein band may be related to the viral replication in IBD and may be an additional method of determining etiology of the inclusions (W ozniak, 2000). This test cannot be performed on tissues preserved in formalin or paraformaldehyde and can only be preformed on fresh or frozen tissues, which were not available on the com snake samples. Retroviruses typically have a high species specificity and it is possible that a retrovirus found in a com snake may be antigenically distinct from a retrovirus found in a boa constrictor (Robbins, et al, 1984, Coffin, 1996). In the case of the three corns snakes in this report, TEM revealed membrane bound inclusions consisting of a course electron-dense fluctuant material measuring 2-4 pm (Figure 3, IB2). The IBD inclusion from a boa constrictor was a nonmembrane bound electron-dense inclusion measuring 1-4 pm, with small round to oval electron dense subunits being laid down on the periphery of the inclusion in the remaining cytoplasmic space (Figure 4, white arrow). Other IBD inclusions may also contain small electron dense structures such as stacks of filaments, electron dense subunits, or hexagonal capsids. Different cross sections seen on the TEM micrograph may result in slightly varied views. It is proposed that membrane bound inclusions are an undigested substrate in phagolysosomes or an accumulation of synthesized proteins inside cisterns of rough endoplasmic reticulum as a result of viral infection. Similar intracytoplasmic membrane bound inclusions have been demonstrated in other retroviral infections (Coward, et al, 1972, Gilka and Spenser, 1985). Thus the inclusions seen in the com snakes may be a product of viral production but contain no viral material as confirmed by the stains and TEM. In conclusion, forming a diagnosis based on clinical signs and light microscopy alone, may result in a misdiagnosis of viral disease such as IBD. If inclusions are present, samples should be submitted for evaluation by special staining for nucleic material, protein electrophoretograms to identify viral related protein, and visual comparison of TEM micrographs to compare structural morphology of the inclusions. This may further assist in distinguishing between viral and non-viral inclusions, allowing a more accurate diagnosis. However, based on limited knowledge in this emerging field, viral disease still cannot be ruled out as a cause of the corn snake inclusions. R e f e r e n c e s Ahne W Bei reptilien vorkom m ende viren. In Reichenbach-Klinke, HH (ed.) Krankheiten der reptilien. 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