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.

220 Chang and Jacobson

sions in histologically processed and H&E-stained bi-opsy specimens. Boid snakes have well-developedesophageal tonsils11 (Fig 10), and in snakes with IBDthe tonsils may contain lymphoid cells or mucous epi-thelial cells with intracytoplasmic inclusions (Fig 5).Using a flexible endoscope with a biopsy device, esoph-ageal tonsils are easily biopsied, fixed, and routinelyprocessed for light microscopy. Liver and kidney bi-opsy specimens can also be obtained for histologicalevaluation. For a more rapid diagnosis, cytologicalimpression smears of the liver and renal biopsy sam-ples can be stained with H&E (Table 1; Fig 11)and/or Wright-Giemsa (Fig 12).12,13 In the authors’experience, inclusions are easier to identify in H&E-

Figure 10. Reticulated python. Pythonidae. Esophageal tonsils (ar-rows) are raised ovoid structures with a central cleft and covered bya mucous epithelium. Courtesy of CRC Press.

Table 1. Hematoxylin and eosin–stainingrecommendations for impression smears

and blood films1. Fix 1 minute in 10% neutral buffered formalin2. Stain 3 minutes in Harris hematoxylin3. One dip into acid alcohol4. Briefly wash in running water5. Dip into 0.5% ammonia to blue the nuclei6. Wash in running water7. Counterstain in eosin for 40 seconds8. Dehydrate in a series of alcohols: 95%-100%-

100%9. Place in xylene and mount as with a paraffin-

embedded section

stained preparations. Inclusions may be seen inerythrocytes (Fig 13), lymphocytes (Figs 13 and 14),and heterophils (Fig 15) in peripheral blood films ofsnakes with IBD.

Cost can be a limiting factor in using the diagnos-tic test above. Tissue biopsies from living snakes areoften processed and evaluated for less than US $150,and full necropsy examinations on dead snakes maycost US $300-$500 per case. Blood smears from sus-pect cases can be examined for presence of inclu-sions in lymphocytes and heterophils, but it is notknown how many snakes that are diagnosed with IBDalso have inclusions in circulating white blood cells.Nonetheless, submission of a blood smear to deter-

Figure 11. Boa constrictor. IBD. Photomicrograph of a cytologicalimpression of liver. Numerous eosinophilic intracytoplasmic inclu-sions can be seen. H&E stain. Courtesy of CRC Press.

Figure 12. Boa constrictor. IBD. Photomicrograph of cytologicalimpression of liver. Numerous basophilic intracytoplasmic inclusions

(arrows) can be seen. Wright-Giemsa stain.

Inclusion Body Disease 221

mine if inclusions are present is relatively inexpen-sive (approximately US $50).

Cause and Transmission

Retroviruses have been isolated from boas with IBD,and several of these viruses have been partially char-acterized.4 Using transmission electron microscopy,viral morphogenesis in cell culture was described.4

Evaluation of viper heart 2 (VH2) cells infected with

Figure 13. Boa constrictor. IBD. Peripheral blood film with anerythrocyte (arrow) and lymphocyte (arrowhead) containing eosino-philic-staining inclusions. H&E stain.

Figure 14. Boa constrictor. IBD. Peripheral blood film with a lym-phocyte containing an eosinophilic-staining inclusion (arrow). H&E

stain.

tissues from IBD-positive snakes revealed intracyto-plasmic and extracellular virions (Fig 16). Based onsize (80-110 nm) and morphology, the virus resem-bled C-type retroviruses. Reverse-transcriptase activ-ity was measured in infected cell cultures, and highactivity levels were further evidence that the isolatedvirus was a retrovirus. Retroviruses also have beenobserved in transmission electron microscopy of tis-sue sections from IBD-infected snakes.4,5,7 However,the VH-2 cells that were infected with the virus didnot form inclusions. Huder and coworkers isolatedand sequenced endogenous retroviruses from Bur-

Figure 15. Boa constrictor. IBD. Peripheral blood film with a het-erophil containing an eosinophilic-staining inclusion (arrow). H&Estain.

Figure 16. Boa constrictor. Transmission electron photomicrographof primary kidney cells from a snake with IBD. Extracellular retroviralparticles are seen on the surface of kidney cells. Uranyl acetate and

lead citrate. Courtesy of CRC Press.

222 Chang and Jacobson

mese and blood pythons (P. curtus).14 The Burmesepythons from which the endogenous retroviruseswere sequenced belonged to a collection of snakes inwhich clinical signs of IBD had been identified,whereas no background information was providedfor the blood pythons. Other species of boid snakestested, whether they had IBD or not, were virusnegative. Therefore, it was concluded that there wasno causal link between this virus and IBD.14

Although retroviruses have been observed in in-clusion-bearing tissues, many hours of investigation,using the electron microscope, are needed to locatemature retroviral particles. For a number of IBDcases, neither viral particles nor reverse-transcriptaseactivity have been demonstrated in tissues of affectedsnakes. Transmission studies have been performedin Burmese pythons and boa constrictors by inocu-lating young Burmese pythons with the supernatantof primary cultured kidney cells taken from an in-fected boa constrictor, resulting in the developmentof clinical signs and microscopic lesions associatedwith IBD.2 In a second study, boa constrictors thatwere administered filtered liver homogenate ob-tained from an IBD-infected boa eventually devel-oped intracytoplasmic inclusions in hepatocytes.5 Be-cause purified virus was not used in these studies, itis impossible to implicate a retrovirus as the under-lying etiology of inclusion formation in the inocu-lated snakes. IBD may represent a protein-storagedisease induced by viral infection, or the proteinitself may be behaving in a manner similar to that ofa prion-like disease. The protein and the isolatedviruses must be sequenced to gain a better under-standing of a possible causal relationship.

The route of transmission of IBD between snakeshas not been determined, although it is believed thatdirect contact is involved. Because the snake mite(Ophionyssus natricis) (Fig 17) is present in manysnake colonies experiencing an IBD outbreak, mitesmay be associated in the transmission of the infec-tious agent. Thus, preventing mites from entering acollection and eliminating established infestationsare essential components of a preventive medicineprogram. It is also possible the causative agent ispassed through vertical transmission from mother toyoung in both egg-laying and live-bearing snakes.

IBD Risk Reduction

There is no simple protocol to follow when trying toprevent IBD from entering a snake collection ormanaging a breeding operation where the diseasehas been diagnosed. However, several approaches

can reduce the risk of introducing IBD into a snake

collection. First and foremost, a solid preventativemedicine program should be established. Althoughno such program is 100% effective, the main objec-tive is reducing the risk of IBD entering a collection.Having a veterinarian knowledgeable about reptilesor specializing in reptile medicine is also a veryimportant component to a preventative medicineprogram.

It is very helpful to have knowledge about thehistory of the previous owner or breeder’s collectionfrom which the purchased snake is derived. Procur-ing information regarding the history of a breedingoperation, including annual mortality and causes ofmortality in a collection, is often difficult or impos-sible to uncover. Because of the ease at which snakesare sold over the internet and purchased at reptileexpositions, new snake “breeders” establish them-selves, seemingly, on a daily basis. Based on theauthors’ experience, the vast majority of snakes thatdie in a collection never have a thorough necropsyexamination. Every collection inevitably has animalsthat become ill and die, with no collection beingpathogen-free. Therefore, buyers of new animalsshould reduce the risk of introducing an infectiousagent by purchasing animals from well-established,recognized breeders that have preventive medicineprograms in place rather than purchasing animalsbecause they are being sold for a low price from an

Figure 17. Snake mite, Ophionyssus natricis. Photomicrograph of amite removed from a snake. A single egg can be seen within themite. Courtesy of CRC Press.

unknown individual.

Inclusion Body Disease 223

Quarantining new animals is essential for risk re-duction in relation to infectious disease. A quaran-tine period of at least 90 days is recommended fornew snake acquisitions and animals that have beento reptile swaps, although some new animals mayhave to be quarantined for longer periods of time.Ideally, quarantined animals should be housed in abuilding that is separate from the main collection. Atrue quarantine, which is rarely done, takes placewhen all new animals enter the facility on the sameday and leave on the same day, 90 days later. For IBDevaluation, blood is the easiest diagnostic sample tocollect in snakes, and blood smears can be examinedfor the presence of IBD inclusions in blood cells. Ifsnake-keepers have the financial resources, biopsysamples can be obtained from a snake’s tonsils, liver,and kidneys. Collection of biopsy samples is notpractical in all situations, but antemortem diagnostictesting improves the owner’s ability to identify aninfected animal. In the end, the cost of recom-mended quarantine programs and the use of diag-nostic testing depend on either the owner’s financialsituation or the importance they attach to such pre-ventative medicine programs.

Finally, snakes showing clinical signs of illness oranorexia and weight loss should never be added toan established collection. Only mite-free animalswith a good appetite and excellent body conditionshould be added after the quarantine period is over.Once mites infest a large snake collection, they maybe impossible to totally eliminate.

Managing a Collection

Seeing a single lone IBD-positive snake in a collec-tion is uncommon. Typically, where there is one caseof IBD there are others. As mentioned before, sub-clinical cases of IBD are common. So what should bedone in a situation where IBD has been identified ina snake collection? In extreme cases, some peoplehave depopulated entire collections of susceptiblesnakes where IBD had been diagnosed and where alarge number of the snakes shared the same room orhad been in contact with one another. Other ownerswill be in denial despite evidence that IBD exists intheir collection. Snake owners in denial often decideto conduct business as usual. This wide range ofowner responses is typical for any type of animal-breeding operation when a potential infectious dis-ease has been identified.

What has made a difference in controlling andmanaging infectious disease outbreaks in domesticanimals is 2-fold. First, there is the ability to develop

very sensitive and specific diagnostic tests for deter-

mining exposure to a specific pathogen. Currently,demonstration of inclusions using H&E staining isthe only diagnostic method being used to identifycases of IBD. The sensitivity and specificity of thisstaining technique for IBD have not been estab-lished. Second, there is the development of vaccinesthat can protect animals from infection and disease.Unfortunately, there is no vaccine available for pro-tection against IBD or any effective treatment. Vac-cine research and development are extremely costly,and the odds that the money will become availablefor an IBD project are extremely low.

There are still options available to both veterinar-ians and owners to determine the IBD status of theanimals they are presented with or own. Biopsy sam-ples and blood smears can be obtained from ex-posed animals to screen for inclusions. If it is finan-cially impossible to obtain samples from all animalswithin a collection, then select a subset for evalua-tion. Snakes showing signs of IBD should be imme-diately removed from the main collection andplaced in a separate room, or submitted for a micro-scopic and/or necropsy evaluation. Cages of ill ordead animals should be disinfected with bleach andallowed to dry in the sunlight for a few days. House-hold bleach is the best overall disinfectant and mustcome into contact with cleaned surfaces. Although itwill not kill every pathogen known to snakes andother reptiles, it is highly recommended.

A question that has been asked many times iswhether the agent causing IBD can be transmitted toneonates of either live-bearing or egg-laying snakes. Wehave very little information to determine whether ver-tical transmission of this disease is possible. There areanecdotal reports that recently hatched ball pythonswere diagnosed with IBD, but microscopic slides werenot forwarded to the authors for substantiation. Oneof the authors (ERJ) received a series of neonate boaconstrictors that had an IBD-positive mother, butinclusions of the disease were not found in the off-spring of that animal. The problem with trying todetermine if vertical transmission of IBD is possibleis related to a lack of appropriate diagnostic tests;molecular-screening tools that typically have a highdegree of sensitivity and specificity are currently notavailable. The conservative viewpoint is to assumethat the agent can be transmitted to offspring; there-fore, neonates from known IBD-positive femalesshould not be kept, sold, or given away.

Present and Future Research Needs

A study by Wozniak and coworkers demonstrated

that inclusion bodies in IBD are composed of an

224 Chang and Jacobson

antigenically unique 68-kd protein.5 Monoclonal anti-bodies (MAB) against IBDP have been produced thatrecognize an IBDP band in a western blot and IBDPantigen in frozen tissue sections using immunohisto-chemical (IHC) staining. Unfortunately, the originalanti-IBD MAB was lost. Recently, we have isolated in-clusions (Fig 18) from a case of IBD and, using hybrid-oma technology, have produced a new anti-IBDP MABthat stains inclusions in paraffin-embedded tissues (Fig19, A and B). This MAB is going through rigid testingand validation. Staining using IHC could be a veryhelpful first step in developing an immunologic-basedor molecular-based diagnostic method that will allowus to identify infected snakes that are in an earlystage of infection. This MAB will also be helpful indistinguishing inclusions consisting of IBDP fromthose inclusions resembling but distinct from IBDinclusions.15

It is unknown whether all IBD cases in differentsnake species consist of exactly the same protein or ifvariability occurs that is species dependent. Westernblot is another practical test that can be developedfor making an antemortem diagnosis. Using Westernblot technology, inclusion protein in small amountsobtained from biopsied tissue or isolated peripheralwhite blood cells from a blood sample can be tested.Studies to evaluate the sensitivity and specificity ofthe diagnostic testing methods described above areplanned.

Some of the authors’ current work centers on abetter understanding of the IBD protein composi-tion and the development of a test to identify itspresence. Identifying the amino acid sequence ofIBDP will be a critical step in eventually understand-ing the pathogenesis of IBD. Once the sequencing

Figure 18. Boa constrictor. A semipurified preparation of inclusionsobtained from the liver of a boa constrictor with IBD. H&E staining.

data for IBDP are obtained, bioinformatic analysis

can be used to predict its origin, structure, andbiochemical properties. Through peptide synthesis,the antigenic region of the IBDP can be producedand used as a better source of antigen for developingan enzyme-linked immunosorbent assay or other im-muno-based molecular diagnostic tests. Concur-rently, retroviruses isolated from tissue of IBD-in-fected snakes4 have been recovered and are growingin culture. Once the virus is purified and sequenced,specific primers against the viruses will be made andused in a polymerase chain reaction test. The use ofa polymerase chain reaction–based test will be in-valuable in determining if there is consistent associ-ation of this retrovirus and IBD.

Figure 19. (A) Boa constrictor. Liver. Negative-control staining. TheMAB prepared against IBD inclusion protein was excluded from theIHC staining protocol. Several heterophils with light nonspecificstaining can be seen (arrows). Immunoperoxidase stain. (B) Boidae.Liver. Positive-control staining. The MAB prepared against IBD in-clusion protein was included in the immunohistochemical stainingprotocol. Positive staining of inclusions can be seen (arrows). Im-

munoperoxidase stain.

Inclusion Body Disease 225

If there is any hope or desire for IBD to be man-aged in breeding operations of snakes sold for thepet trade, the need for such molecular-based tests isclear. The main problem scientists have regardingIBD research is that research dollars needed to per-form the investigations have been extremely limited.Although several reptile expositions and a numberof private individuals have made contributions tothis work, the total number of dollars received hasbeen inappropriate for the studies needed to have asignificant impact on determining the cause of IBDand developing timely, cost-effective, scientificallyvalidated diagnostic tests.

Concluding Remarks

IBD is one of the few worldwide diseases of captivesnakes. The ease at which snakes can be shippedaround the world is probably responsible for itsspread. Because many IBD-positive snakes may besubclinically infected, infected snakes consideredhealthy have been sold. It is possible that subclinicalIBD infections can persist for long periods. It isunknown where this disease originated becausethere are no reports of IBD in wild populations. Asensitive and specific blood-based immunodiagnos-tic and/or molecular test is needed to screen bloodsamples of both captive and wild populations of boidsnakes. Maintaining healthy and disease-free animalsshould be the primary responsibility for all whokeep, breed, and intend to release captive bred andrehabilitated animals back to the wild.

Acknowledgments

Portions of this article and figures are from: Jacob-son E (ed): Infectious Diseases and Pathology of Reptiles:A Color Atlas and Text. Boca Raton, FL, CRC Press,2007; and Jacobson E: Catching an insidious killer.Reptiles Magazine December:58-61, 2008. The authorsthank Marjorie Chow, Linda Green, Diane Duke,Edward Wozniak, and Ann Dongtao Fu for technicalhelp.

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