Topics in Medicine and SurgeryTopics in Medicine and Surgery

Inclusion Body Disease, A Worldwide InfectiousDisease of Boid Snakes: A ReviewLi-Wen Chang, BVM,

and Elliott R. Jacobson, DVM, PhD, Dip. ACZM

ease (IBD)

216

Abstract

A disease called inclusion body disease (IBD) is seen worldwide in snakes that aremembers of the families Boidae and Pythonidae. Snakes affected by this diseaseoften have neurological signs. A diagnosis is based on light microscopic examina-tion of tissues for the presence of intracytoplasmic inclusions that consist of aunique protein that has been termed inclusion body disease protein (IBDP). In somecases, inclusions are found exclusively in the central nervous system, whereas inothers they may be diffusely disseminated in multiple tissues. In a few cases, theIBD inclusions have overlapped in appearance with other types of nonviral intra-cytoplasmic inclusions. The specific etiologic agent of IBD remains unknown.Because the underlying cause of IBD is unknown, recent research has focused onunderstanding the formation and nature of IBDP. A monoclonal antibody has beendeveloped against IBDP and is currently being used in immunodiagnostic assays todevelop a reliable diagnostic test for IBD. This monoclonal antibody is goingthrough rigorous validation testing and will provide more specificity and sensitivitythan currently available routine histological diagnostics. Because large numbers ofboas and pythons are being bred and sold for the pet trade, better diagnostic testsare needed to help develop IBD-free breeding colonies of these snakes. Further,because IBD is one of the few worldwide diseases of captive snakes, there isconcern in many countries (e.g., Australia, where it has been identified in captivepythons) that it will become established in native wild populations. Thus, there isconservation value in developing better diagnostic tools for screening snakesintended for release as part of reintroduction programs. Copyright 2010 ElsevierInc. All rights reserved.

Key words: Boa; IBD; IBDP; monoclonal antibody; protein sequencing; python

From the Department of Small Animal Clinical Sciences, College ofVeterinary Medicine, University of Florida, Gainesville, FL USA.

Address correspondence to: Elliott Jacobson, DVM, PhD, Dip.ACZM, Department of Small Animal Clinical Sciences, Box100126, College of Veterinary Medicine, University of Florida,Gainesville, FL 32610. E-mail: jacobsone@vetmed.ufl.edu.

© 2010 Elsevier Inc. All rights reserved.1557-5063/10/1903-$30.00

Snakes make up approximately 19% of all rep-tiles maintained as pets in the U.S.1 Of these,various boid snakes (members of the families

Boidae and Pythonidae) are bred in large numbersfor the pet trade. Although accurate numbers arenot available, it is believed that several million ofthese snakes are classified as pets or maintained inbreeding operations within the United States. Ofillnesses affecting boid snakes, inclusion body dis-

has surfaced as the most important world- doi:10.1053/j.jepm.2010.07.014

Journal of Exotic Pet Medicine, Vol 19, No 3 ( July), 2010: pp 216–225

Inclusion Body Disease 217

wide disease, a condition characterized by the for-mation of intracytoplasmic inclusions.2 In Australia,where IBD has been identified in captive pythons,3

and in other countries where boid snakes are beingbred for release to the wild, there is concern that thisdisease will become established in native wild popu-lations. Boid snakes with IBD may have a subclinicalinfection. It is not known what percentage of in-fected snakes will develop clinical signs of disease inrelation to those that will appear unaffected. It ispossible that latent infections can persist for longperiods of time. Currently, a presumptive diagnosisof IBD is based on the light microscopic identifica-tion of intracytoplasmic inclusions in one or moretissues. However, some snakes have very few inclu-sions in tissue sections because they are easy to over-look, especially if limited to the central nervous sys-tem (CNS). Although several viruses, including ret-roviruses,4 have been identified and isolated fromsnakes with IBD, the causative agent remains un-known. Because the inclusions consist of a uniqueprotein (inclusion body disease protein, IBDP),5 un-derstanding the cause of IBD will be based on deter-mining the composition and factors affecting theformation of this protein. To better understand thenature of IBDP, this protein needs to be entirely orpartially sequenced. Eventually the sequencing ofthis protein will allow the creation of peptides thatcan be used in the development of better immuno-diagnostic tests to screen individuals and colonies ofsnakes for IBDP.

History, Hosts, and Geographic Range

In the 1970s, IBD was first identified in the UnitedStates, where it affected multiple species of boidsnakes in private and zoological collections.2 Whenfirst recognized, Burmese pythons (Python bivittatus)were the most common boid snake diagnosed withIBD. In 1998, IBD was reported in captive nativecarpet (Morelia spilota variegata) and diamond py-thons (M. spilota spilota) in Australia,3 in captive boaconstrictors in the Canary Islands, Spain,6 and sub-sequently in Belgium.7 Beginning in the early 1990s,more cases of IBD were diagnosed in boa constric-tors than pythons, but the cause of this epidemio-logic shift is unknown. Additional species diagnosedwith IBD include the green anaconda (Eunectes mu-rinus), yellow anaconda (Eunectes notaeus), rainbowboa (Epicrates cenchris), Haitian boa (Epicrates stria-tus), Madagascan boa (Acranthophis madagascarien-sis), Indian python (P. molurus molurus), reticulatedpython (P. reticulatus), and ball python (P. regius). In

addition, a disease resembling IBD was diagnosed in

an eastern king snake (Lampropeltis getula) that washoused with boa constrictors,4 and in a zoologicalcollection of palm vipers (Bothriechis marchi).8 How-ever, the correlation of the inclusions in the kingsnake and viper cases to IBD has not been confirmedwith molecular techniques or immunological re-agents.

Clinical Signs

From the late 1970s and extending into the mid-1980s, Burmese pythons were the most commonboid snake seen with IBD. Clinical signs of the dis-ease in Burmese pythons primarily involved CNSabnormalities (e.g., torticollis, disequilibrium, opis-thotonos, inability to right itself when placed in dor-sal recumbency, flaccid paralysis).2 Beginning in theearly 1990s, more cases were diagnosed in boa con-strictors in relation to Burmese and other pythons.Boa constrictors affected by IBD also regurgitatedfood items within several days of feeding, in additionto the CNS disease signs described for pythons (Figs1 and 2). Although some snakes die within severalweeks of first manifesting illness, others may survivefor months. Other clinical signs observed in affectedsnakes were stomatitis, regurgitation, pneumonia,lymphoproliferative disorders, and round cell tu-mors. Regurgitation was not a disease sign identifiedin Burmese pythons.

Hematological and selected biochemical analytevalues of acutely affected boa constrictors diagnosedwith IBD included leukocytosis, relative lymphocyto-

Figure 1. Boa constrictor. Boidae. IBD. This abnormal posture is a

sign of CNS disease.

218 Chang and Jacobson

sis, lower total protein and globulin values, and sig-nificantly higher aspartate transaminase values com-pared with those of chronically affected snakes.2

Postmortem Diagnosis

A postmortem diagnosis of IBD is based on the lightmicroscopic identification of variably sized eosino-philic to amphophilic intracytoplasmic inclusions inhematoxylin and eosin (H&E)–stained tissue sec-tions. The tinctorial characteristics of the inclusionsmay vary with the type of hematoxylin used anddifferences in staining methods.9 In pythons, inclu-sions are mostly found within neurons in the CNS(Figs 3 and 4). In boa constrictors, inclusions arealso commonly observed in neurons and glial cells in

Figure 2. Boa constrictor. IBD. When placed in dorsal recumbency,this snake was unable to right itself. Courtesy of CRC Press.

Figure 3. Boa constrictor. IBD. Photomicrograph of eosinophilicintracytoplasmic inclusions (arrows) in neurons and glial cells in the

brain. H&E stain.

the CNS, with or without an associated inflamma-tion. If identified, encephalitis is generally more se-vere in pythons compared with that in boa constric-tors. In boa constrictors, inclusions also are com-monly seen in: 1) mucosal epithelial cells adjacent toand overlying esophageal tonsils, 2) lymphoid cellsin esophageal tonsils (Fig 5), 3) epithelial cells liningthe gastrointestinal tract, 4) epithelial cells lining therespiratory tract, 5) hepatocytes (Fig 6), 6) pancre-atic acinar cells (Fig 7), and 7) renal tubular epithe-lial cells.

Electron Microscopic FindingsUsing transmission electron microscopy, intracyto-plasmic inclusions identified within CNS visceral ep-ithelial cells and nerve cells begin as polyribosome-

Figure 4. Boa constrictor. IBD. Photomicrograph of amphophilicintracytoplasmic inclusions in neurons of the brain. H&E stain.Courtesy of Nikos Gurfield and CRC Press.

Figure 5. Boa constrictor. IBD. Photomicrograph of an esophagealtonsil from a necropsied snake showing numerous eosinophilicintracytoplasmic inclusions (arrows) within submucosal lymphoid

cells. H&E stain. Courtesy of CRC Press.

Inclusion Body Disease 219

derived clusters of small round subunits (Fig 8).10

Inclusions that enlarge as additional subunits aredeposited on the periphery of individual inclusions(Fig 9). In some sections the inclusions have concen-tric profiles, with subunits observed on the surface. Aunique protein (IBDP) was identified as a 68-kdband on a protein electrophoretogram of IBD-in-fected tissues.5 Although in some cases the subunitshave an ultrastructural appearance resembling viralparticles, the current findings indicate that the in-clusions are nonviral and mainly consist of IBDP.Beyond this protein, the chemical composition ofthe inclusions remains unknown.

Figure 6. Boa constrictor. IBD. Photomicrograph of the liver show-ing hepatocytes containing eosinophilic intracytoplasmic inclusions(arrows). H&E stain. Courtesy of CRC Press.

Figure 7. Boa constrictor. IBD. Photomicrograph of the pancreasshowing acinar cells containing eosinophilic intracytoplasmic inclu-

sions. H&E stain. Courtesy of CRC Press.

Antemortem Diagnosis

An antemortem diagnosis is made by demonstratingeosinophilic to amphophilic intracytoplasmic inclu-

Figure 8. Boa constrictor. Transmission electron photomicrographof an enterocyte in the small intestine of a snake with inclusion bodydisease. During the initial stage of inclusion formation, proteinsubunits from polyribosomes start accumulating in the adjacentcytoplasm. Uranyl acetate and lead citrate stain. Courtesy of CRCPress.

Figure 9. Boa constrictor. Transmission electron photomicrographof an inclusion in an enterocyte. Deposited protein subunits have avirus-like appearance. Uranyl acetate and lead citrate stain. Courtesy

of CRC Press.