paramyxovirus infection in caiman lizards (draecena guianensis)

http://vdi.sagepub.com/Investigation

Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/13/2/143The online version of this article can be found at:

DOI: 10.1177/104063870101300208

2001 13: 143J VET Diagn InvestRobert Nordhausen, Jennie W. Owens, Donald K. Nichols, Darryl Heard and Bruce Homer

Elliott R. Jacobson, Francesco Origgi, Allan P. Pessier, Elaine W. Lamirande, Ian Walker, Brent Whitaker, Ilse H. Stalis,)Draecena GuianensisParamyxovirus Infection in Caiman Lizards (

Published by:

http://www.sagepublications.com

On behalf of:

Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

can be found at:Journal of Veterinary Diagnostic InvestigationAdditional services and information for

http://vdi.sagepub.com/cgi/alertsEmail Alerts:

http://vdi.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

http://vdi.sagepub.com/content/13/2/143.refs.htmlCitations:

What is This?

- Mar 1, 2001Version of Record >>

at Universitaetsbibliothek Bern on January 16, 2013vdi.sagepub.comDownloaded from

http://vdi.sagepub.com/

http://vdi.sagepub.com/content/13/2/143

http://www.sagepublications.com

http://www.aavld.org

http://vdi.sagepub.com/cgi/alerts

http://vdi.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://vdi.sagepub.com/content/13/2/143.refs.html

http://vdi.sagepub.com/content/13/2/143.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml


143

J Vet Diagn Invest 13:143–151 (2001)

Paramyxovirus infection in caiman lizards (Draecena guianensis)

Elliott R. Jacobson, Francesco Origgi, Allan P. Pessier, Elaine W. Lamirande, Ian Walker, BrentWhitaker, Ilse H. Stalis, Robert Nordhausen, Jennie W. Owens, Donald K. Nichols, Darryl Heard,

Bruce Homer

Abstract. Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were importedinto the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizardsdemonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus thatwas consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolateof ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity with-in pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards wereplaced in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later,syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similarsyncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracyto-plasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundantfilamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed inculture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples werecollected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determinepresence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of #1:20and 10 had titers of .1:20 and #1:80. This report is only the second of a paramyxovirus identified in a lizardand is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.

Viruses in the family Paramyxoviridae are knownto infect and cause disease in a variety of snakes. Thefirst report in 1975 was of a die-off of vipers (Bothropsmoojeni) in a serpentarium in Switzerland,5,6 and sincethat time multiple virus isolates have been obtainedfrom captive snakes in Germany and the UnitedStates.2–4,10–13,17,18 This group of viruses has been col-lectively called ophidian (snake) paramyxoviruses(OPMVs).13 Transmission studies in Aruba Island rat-tlesnakes (Crotalus unicolor) demonstrated a causalrelationship between a proliferative pneumonia seen insnakes and an Aruba Island rattlesnake isolate ofOPMV.10 Recent comparative analyses of partial genesequences for the large (L) protein and hemagglutinin-neuraminidase (HN) protein of 16 reptilian paramyxo-viruses recovered from multiple species of snakes fromdifferent families indicated that there were at least 2

From the College of Veterinary Medicine, University of Florida,Gainesville, FL 32610 (Jacobson, Origgi, Heard, Homer), The De-partment of Pathology, Zoological Society of San Diego, PO Box120551, San Diego, CA 92112-0551 (Pessier, Stalis), The Depart-ment of Pathology, Smithsonian National Zoological Park, 3001Connecticut Avenue NW, Washington, DC 20008 (Lamirande, Nich-ols), The National Aquarium in Baltimore, Pier 3, 501 Pratt Street,Baltimore, MD 21202-3194 (Walker, Whitaker), The California Vet-erinary Diagnostic Laboratory System, West Health Sciences Drive,Davis, CA 95616 (Nordhausen), and the Veterinary Resources Pro-gram, Office of Research Services, National Institutes of Health,Bethesda, MD 20892 (Owens).

Received for publication February 15, 2000.

distinct subgroups of isolates and several intermediateisolates.1 The results of this work indicate that reptilianparamyxoviruses do not have a narrow reptilian hostrange.

Although OPMVs are well known, there is scantinformation on paramyxovirus infection in lizards. Aparamyxo-like virus was isolated from a false tegu(Callapistes maculatus) (Ahne W, et al.: 1991, Int Col-loq Pathol Med Reptiles Amphib 4:30–31). Basedupon partial L and HN gene sequences, this virus wasconsidered to be within an intermediate group whencomparisons were made with 16 snake isolates.1 Nogross or histologic findings were presented for the in-fected lizard. In contrast with snakes, the role of para-myxoviruses in diseases of lizards is unknown.

Caiman lizards (Dracenea spp.) are semiaquatic liz-ards of the family Tiedae. They range from Surinamto Peru and have a unique feeding preference forsnails. For the first time, groups of caiman lizards (D.guianensis) from Peru were imported into the USA inthe winter of 1998–1999. This report documents theisolation of a paramyxovirus from 3 colonies of cai-man lizards experiencing epidemic proliferative pneu-monia. This is the first report linking a paramyxovirusto an epidemic in a group of lizards.

Materials and methods

History. Three separate epidemics (I, II, and III) occurredin caiman lizards imported by reptile dealers in Florida, Cal-



144 Jacobson et al.

ifornia, and New England. In epidemic I, 50 adult caimanlizards were imported from Peru in November 1998 by areptile dealer in southern Florida. A private breeder of rep-tiles in southern Florida purchased 10 animals when theshipment arrived and placed them in an outdoor pen at hisfacility. This breeder purchased another 10 lizards 2 wk laterfrom the same shipment and placed them with his previouslypurchased group of lizards (group IA). One animal from thefirst purchase and 2 animals from the second purchase diedwithin 3 wk. No necropsies were performed. At 7 mo fol-lowing the deaths, the 17 other lizards were still alive.

Approximately 5 wk after the initial purchases, another 30caiman lizards (group IB) were purchased from the sameimporter and transferred to the property of the same reptilebreeder. These lizards were maintained separately fromgroup IA lizards. The 30 caiman lizards originated from sev-eral shipments, including lizards remaining from the originalNovember 1998 shipment. After arrival, group IB lizardsstarted dying, and over a 4–5 wk period after purchase, allbut 2 group IB lizards died. One of these 2 lizards (lizard1) exhibiting clinical signs of depression, anorexia, musclewasting, and possibly respiratory distress was sent to theCollege of Veterinary Medicine, University of Florida, forpathologic evaluation. The reptile breeder removed the re-maining group IB caiman lizard from his property and keptthe group IA lizards in quarantine.

Epidemic II involved a group of caiman lizards importedfrom Peru in late 1998 by a reptile dealer in southern Cali-fornia. Two adult female caiman lizards (lizards 2 and 3)were later transferred to a second reptile dealer prior to do-nation to the San Diego Zoo in late January 1999. On arrival,the lizards were in poor body condition and dehydrated; his-tory obtained from the dealer indicated that they had beenanorexic for approximately 3 wk. Lizard 2 was found dead5 days after arrival. Lizard 3 was persistently anorexic anddied 16 days after arrival. Twelve other caiman lizards ob-tained from either the same reptile dealership (n 5 2) ordirectly from suppliers in Peru (n 5 10) in late 1998 andearly 1999 and housed separately remained healthy.

In epidemic III, in late April 1999, 2 adult caiman lizards(lizards 4 and 5) were acquired by the National Aquarium(Baltimore, MD) from a reptile distributor in New Hamp-shire and housed in an off-exhibit holding area. In early May1999, a caiman lizard (lizard 6) that had been obtained froma private collector in Atlanta, Georgia (originating from aCalifornia reptile importer), was added to the enclosurehousing lizards 4 and 5. In mid-May, lizards 4 and 5 werefound dead without premonitory clinical signs. Lizard 6 wassubsequently examined and found to be thin but was other-wise clinically normal. In late May, lizard 6 was unexpect-edly found dead.

Pathology. Complete necropsies were performed on liz-ards 1, 2, 3, 5, and 6; lizard 4 was in an advanced state ofautolysis and was not necropsied. Portions of brain, heart,liver, lung, spleen, kidney, stomach, pancreas, small intes-tine, and large intestine were collected and fixed in neutralbuffered formalin. Tissues were subsequently embedded inparaffin, sectioned at 5–7 mm, and stained with hematoxylinand eosin (HE). For electron microscopy, portions of theformalin-fixed lung of lizard 1 were postfixed in 2% osmium

tetroxide, dehydrated through graded alcohols, and embed-ded in Spurr/Epon (1:1) resin. Ultrathin sections werestained with uranyl acetate and lead citrate and examined ona transmission electron microscope.a For lizard 3, formalin-fixed lung was minced and transferred to a modified Kar-novsky’s solution (1% paraformaldehyde, 2% glutaraldehydein 0.1 M sodium cacodylate with 0.001 M calcium chlorideat pH 7.4) overnight at room temperature. Following alde-hyde fixation, tissues were rinsed in cacodylate pH 7.4 buff-er, postfixed in 2% osmium tetroxide, dehydrated through agraded ethanol series, and embedded in Spurr resin. Ultra-thin sections were stained with uranyl acetate and lead citrateand examined on a transmission electron microscope.b

Virus isolation. From lizard 1, portions of brain, liver,kidney, and lung were aseptically collected and placed inDulbecco’s minimum essential medium/Ham’s F 12(DMEM/F12)c with streptomycin and amphotericin B andlacking fetal bovine serum (FBS). Tissue was placed in atissue grinder, and the homogenate was sonicated for 30 sec.The tubes were centrifuged at 290 3 g for 10 min, andsupernatant was harvested and passed through a 0.2-mm fil-ter. One milliliter of supernatant was diluted in 4 ml ofDMEM/F12, and the resulting 5-ml solution was placed ina 25-cm2 flask containing a confluent monolayer of terrpeneheart (TH-1) cellsd and incubated at 28 C with 5% CO2 for1 hr. For each organ, 2 flasks were infected. After 1 hr, themedium in each flask was discarded and replaced with thesame medium containing 5% FBS. The other flask waswashed 3 times with 13 Hanks’ balanced salt solution, andDMEM/F12 containing 5% FBS was added to the flask. After8 passages on TH-1 cells, the virus isolate was inoculated intoa monolayer of Vero cellsd grown in a T25 flask with DMEM;no FBS was used. Vero cells were incubated at 31 C.

At 9 days of inoculation, TH-1 cells inoculated with braintissue cultures were fixed in McDowell and Trumps fixative(phosphate buffered 4% formaldehyde–1% gluteraldehyde)overnight at 4 C. The remaining steps were completed atroom temperature. Each sample was washed several timesin phosphate-buffered saline (PBS), pH 7.2. Postfixation wasperformed with 1% buffered osmium tetroxide for 1 hr fol-lowed by a PBS wash. After several distilled water washes,the samples were dehydrated in a graded ethanol series end-ing with 100% acetone. Samples were infiltrated in a gradedepoxy resin seriese for 1 hr each and polymerized in 100%EmBed 812 for 2 days at 60 C. Ultrathin sections (70 nm)were collected on 200-mesh copper grids and poststainedwith 2% uranyl acetate and lead citrate. Specimens wereexamined on a transmission electron microscope,a and mi-crographs were taken.f

From lizards 3 and 6, samples of lung were asepticallycollected at the time of necropsy and frozen at 270 C priorto shipment on dry ice to the laboratory of 1 of the authors(DKN). Sections of lung were subsequently transferred tobasal medium Eaglec supplemented with 2% FBS and 2% L-glutamine (2% BME) and placed in a tissue grinder. Thehomogenate was centrifuged at 209 3 g for 10 min, and thesupernatant was removed and centrifuged at 2,135 3 g for15 min. The supernatant from this sample was passedthrough a 0.22-mm syringe filterg and serially diluted at 1:10 and 1:100. A 0.1-ml aliquot of undiluted sample and 0.1-



145Virus infection in caiman lizards

ml aliquots of serial dilution samples were inoculated ontomonolayers of viper heart cellsd in 25-cm2 tissue cultureflasks. The inoculated flasks and a single control uninfectedflask were incubated at 28 C for 1 hr. The cells were thenfed with 2% BME, incubated at 28 C, and observed dailyfor cytopathic effects. After observation of cytopathic ef-fects, cells were subjected to 3 freeze–thaw cycles, and al-liquots of the culture medium were negatively stained with2% phosphotungstic acid and examined with a transmissionelectron microscope.h

Immunoperoxidase staining. A polyclonal antibody raisedin rabbits against a paramyxovirus isolated from an ArubaIsland rattlesnake was used for immunoperoxidase stainingas previously described.8 For immunohistochemical evalua-tion, 5-mm sections of lung from lizard 1 were first adheredto poly-L-lysine–coated slides. Two sections of each blockwere placed on a single microscope slide. Lung tissue of anAruba Island rattlesnake with proliferative pneumonia thatwas challenged with an isolate of OPMV served as the pos-itive control.10 Infected tissue culture cells, single-well glasschamber slidesi also were prepared for immunoperoxidasestaining. The chambers were filled with 4 ml of DMEM withantibiotics and antimycotics but lacking FBS. Vero cellswere propagated in the chambers and incubated at 31 C with5% CO2. At confluency of the cell monolayer, 200 ml of viralsuspension derived from lizard 1 was added to each chamberslide and incubated at 31 C. Chamber slides were fixed inice-cold 10% buffered formalin for 30 sec and air dried assoon as the cell monolayer showed 40–60% cytopathic ef-fects. Slides were then stored in a dark, dry environmentuntil use.

A modification of the avidin–biotinylated horseradish per-oxidase macromolecular complex (ABC technique) wasused.7 Endogenous peroxidase was expended in deparaffin-ized sections and chamber slides by incubation in 3% hy-drogen peroxide for 30 min and 5 min, respectively, at roomtemperature. Nonspecific binding was blocked by incubationwith normal horse serum for 30 min at the dilution suggestedby the manufacturer.j Rabbit immune serum was diluted innormal horse serum at 1:500. The immune serum was al-lowed to incubate overnight on treated sections and on cellsgrowing in chamber slides. For histologic material, one sec-tion on each slide was incubated with immune serum whilethe other section was incubated with preimmunization se-rum. The preimmunization serum served as a negative con-trol. Chamber slides were similarly treated. Following in-cubation with the immune and negative control sera, slideswere washed 3 times in PBS for 10 min each. Detection wascompleted using a biotinylated goat anti-rabbit antibody kitj

following the directions of the manufacturer. Diaminoben-zidine (0.05% solution with 0.01% H2O2 in 0.1 M Tris, pH7.2) was used as the chromogen. Hematoxylin was used asthe counterstain.

Serology. At approximately 7 mo following submissionof the group IB caiman lizard (lizard 1), blood samples werecollected from the ventral coccygeal vessel of the 17 sur-viving group IA lizards. All lizards were manually restrainedand blood was obtained from the ventral tail vein. Plasmawas removed and frozen at 280 C.

The cells and medium in flasks of Vero cells infected with

brain homogenate and having maximal syncytia formationwere harvested and freeze–thawed twice. Using chickenerythrocytes, the hemagglutination titer of the virus was de-termined at 4 C as previously described.13,18 A hemaggluti-nation inhibition (HI) assay was used to determine the pres-ence of specific anti-virus antibodies based on the ability ofthe antisera to inhibit viral hemagglutination.11 In assayingthe samples, plasma samples were diluted 1:10 with PBS,and complement was inactivated by placing plasma in a 56C water bath for 30 min. Nonspecific agglutinins were re-moved by absorbing the diluted sera with washed and pel-leted chicken erythrocytes overnight at 5 C. Serial dilutions(1:10 to 1:1,024) of 0.05 ml of treated serum were made inPBS in a microtiter plate. Eight hemagglutination units ofantigen/0.05 ml was added to each well. After incubation atroom temperature for 60 min, 0.05 ml of a 0.5% suspensionof chicken erythrocytes was added to each well. After 3 hrof incubation at 5 C, the HI titer was read as the reciprocalof the highest dilution of serum that inhibited hemaggluti-nation.

Results

Pathology. At necropsy, lizard 1 had minimal sub-cutaneous adipose tissue. No coelomic fat bodies wereidentified and the digestive tract contained very littleingesta. The primary gross lesion was present in thelungs. The cranial half to two-thirds of both lungs werediscolored reddish purple, and the caudal portionswere pale pink. Both lungs contained yellow/gray mu-coid fluid and yellow flocculent material within thecentral airway (Fig. 1). The left lung was more se-verely affected. The liver was diffusely mottled red/brown and had smooth borders. Necropsy findings forlizards 2, 3, 5, and 6 were similar to those seen inlizard 1. The only other gross necropsy finding was anadult trematode parasite (unidentified) that was at-tached to the gastric mucosa of lizard 3.

Histologic changes were similar in all of the lizardsexamined and consisted of a proliferative heterophilicand histiocytic pneumonia. Within the central lungcavity and in multiple air spaces, there was a moder-ately cellular histiocytic and heterophilic exudate ad-mixed with low numbers of sloughed epithelial cells,erythrocytes, and rare gram-negative bacteria (Fig.2A). Faveolar septa were expanded by edema and in-filtrates of low to moderate numbers of predominantlyheterophils, with fewer macrophages and lymphocytes.Similar inflammatory cell infiltrates were present with-in the submucosal stroma surrounding terminal pul-monary smooth muscle bundles. Diffusely, there wasmarked hyperplasia and hypertrophy of respiratory ep-ithelial cells (type-2 pneumocyte hyperplasia) liningfaveoli, with increased numbers of mitotic figures pre-sent and formation of many syncytial cells (Fig. 2B).Occasional epithelial cells contained 1 or more intra-cytoplasmic 1–2 mm globular to aggregated bright eo-



146 Jacobson et al.

Figure 1. Lung; caiman lizard (Dracaena guianensis). Exudate and abundant clear fluid (with air bubbles) fills the central airway.

sinophilic hyaline inclusions. Rare epithelial cells werenecrotic, characterized by nuclear pyknosis and kary-orrhexis and increased cytoplasmic eosinophilia. Mod-erate numbers of heterophils and fewer lymphoctesand plasma cells were present in the faveolar intersti-tium. In lizard 1, there was marked proliferation andhypertrophy of pleural mesothelial cells, with scatteredbinucleate cells and scattered mitotic figures. Rarely,mesothelial cells had cytoplasmic eosinophilic hyalineinclusions.

In the pancreas of lizards 2, 3, 5, and 6, there wasa mild to moderate degree of interstitial edema. In liz-ards 2 and 5, prominent syncytial cells were observedwithin pancreatic acini. Other histologic findings in-cluded trematode ova in the lung, heart, kidney, liver,adrenal gland, stomach, small intestine, and pancreas.These ova were encapsulated by a thin rim of fibrousconnective tissue. No trematode ova were associateddirectly with inflammatory infiltrates in the lung orpancreatic syncytial cells.

Portions of lung from lizards 1 and 3 were examinedultrastructurally for evidence of virus. Viral particleswere found most commonly in type II faveolar cellsand within the faveolar spaces. Within the cytoplasm,granular amorphous inclusions (Fig. 3A) were com-

posed of loosely to densely packed nucleocapsid ma-terial (Fig. 3B) 10–14 nm in diameter. Membrane-bound cytoplasmic vacuoles up to 4 mm in diameter(Fig. 3C) and faveolar spaces (Fig. 3D) containedpleomorphic spherical and tubular forms, 150–390 nmin size. Nucleocapsid material could often be seen inthe core of spherical forms (Fig. 3D).

Virus isolation. The TH-1 cells inoculated withbrain, liver, kidney, and lung from lizard 1 showedsyncytial cell formation 6–9 days later. After 8 pas-sages in TH-1 cells, supernatant was inoculated into amonolayer of Vero cells, and syncytial cells were seen2 days later. Electron microscopic evaluation of thesecells revealed filamentous forms budding from cellmembranes (Fig. 4A, 4B).

In viper heart cells inoculated with both undilutedand serially diluted lung tissue homogenates from liz-ards 3 and 6, multiple small syncytia developed. Syn-cytia were first seen 5 days postinoculation and grewprogressively larger until day 10. Negative-stainingelectron microscopy of culture medium demonstratedabundant filamentous nucleocapsid material with aherringbone structure typical of the Paramyxoviridae(Fig. 5).

Immunoperoxidase staining. Using a polyclonal an-




Figure 2. Lung of a caiman lizard with proliferative pneumonia associated with paramyxovirus infection. A. Alveolar spaces are linedby hypertrophied and hyperplastic epithelium with scattered multinucleated epithelial cells (small arrowheads) and occasional mitotic figure(small arrow). Several alveolar spaces contain mixed leukocytic exudates (large arrow) with an occasional multinucleated histiocytic cell(large arrowhead). HE. Bar 5 67 mm. B. Higher magnification from another area of this lung reveals marked hypertrophy and hyperplasiaof alveolar epithelium. One syncytial cell has a faintly stained cytoplasmic inclusion encompassed by a narrow halo. HE. Bar 5 34 mm.

tibody raised in rabbits against an OPMV, there waspositive staining of viral antigen in epithelial cells lin-ing air-ways of lizard 1. Following inoculation of Verocells with supernatant of infected TH-1 cells, positivestaining for viral antigen was also seen in cells show-ing cytopathic effect. In both lung tissue sections andinfected Vero cell monolayers, staining was granularand was focal to diffuse within the cytoplasm.

Serology. Vero cells inoculated with supernatant ofinfected TH-1 cells (originally infected with brain tis-sue from lizard 1) developed a hemagglutination titerof 1:64. When this virus was used in an HI assay,plasma samples collected from 17 lizards (group 1B)that survived epidemic I had titers ranging from ,1:10 to 1:80 (7 had titers of #1:20; 10 had titers of .1:20 and #1:80).

Discussion

Proliferative pneumonias have been reported in rep-tiles and can result from infection with a variety ofpathogens. Proliferative pneumonia has been seen ingopher tortoises (Gopherus polyphemus) with myco-plasma infection15 and in a radiated tortoise (Geoche-lone radiata) with intranuclear coccidiosis in pulmo-nary epithelial cells (Garner MM, et al.: 1998, ProcAAZV and AAWV, pp. 71–73). In snakes, prolifera-tive pneumonias have been seen in inclusion body dis-ease, (Jacobson ER: 1997, Proc ARAV, p. 165), my-coplasmosis,16 OPMV infection,10–13,17 and reovirus in-fection.14 Proliferative pneumonia has been seen alsoby one of the authors (ERJ) in snakes infected withlungworm (Rhabdias sp.). Determining the causative



148 Jacobson et al.

Figure 3. Transmission electron micrographs of lung of a caiman lizard (Dracaena guianensis). A. Within the cytoplasm of a type IIalveolar cell, a granular amorphous inclusion can be seen. Lead citrate and uranyl acetate. B. Within the cytoplasm of a type II alveolarcell, a granular amorphous inclusion containing nucleocapsid material can be seen. Lead citrate and uranyl acetate. C. Pleomorphic sphericaland tubular virions can be seen within cytoplasmic vacuoles (A) and alveolar spaces (B). Lead citrate and uranyl acetate. D. Sphericalvirions contain nucleocapsid strands. Lead citrate and uranyl acetate.

agent requires specific light microscopic findings and,if necessary, confirmation of the diagnosis using elec-tron microscopy, microbial isolation, immunohisto-chemical staining, and serology.9

In the present report, 3 separate epidemics occurredin caiman lizards imported into the USA from Peru.All necropsied lizards had a proliferative interstitialpneumonia that was similar to that seen in snakes with

OPMV infection. Using TH-1 cells, viper heart cells,and Vero cells, an agent that hemagglutinated chickenred blood cells and produced syncytial cell formationwas isolated from multiple tissues. The highest hem-agglutinating titers were in Vero cells grown at 31 Cand inoculated with brain homogenates of 1 lizard. Us-ing a rabbit polyclonal antibody against an isolate ofOPMV, positive staining was demonstrated in the cy-




Figure 4. Transmission electron micrograph of infected TH-1 cells. Filamentous and spherical virions (arrows) can be seen buddingfrom cell membranes. Lead citrate and uranyl acetate.

toplasm of infected Vero cells. Transmission electronmicroscopy revealed intracytoplasmic inclusions thatwere composed of nucleocapsid strands. Negativestaining electron microscopy of culture medium re-vealed filamentous nucleocapsid material with a her-ringbone structure typical of the Paramyxoviridae.

Using rabbit polyclonal antibody against an isolateof OPMV, an immunoperoxidase technique demon-strated viral antigen within pulmonary epithelial cells.By electron microscopy, nucleocapsid strand-contain-ing intracytoplasmic inclusions similar to those seenin infected Vero cells were seen in pulmonary epithe-lial cells. Virus was seen budding from cell mem-branes and released into adjacent airways. Virionswere compatible in size and morphology with mem-bers of the viral family Paramyxoviridae. This repre-sents the first paramyxovirus associated with pulmo-nary disease in a lizard. Although a paramyxoviruswas isolated previously from another lizard (false tegu;Callapistes maculatus) (Ahne W, et al.: 1991, Int Col-loq Pathol Med Reptiles Amphib 4:30–31), no grossor microscopic lesions were reported.

At 7 months following the initial outbreak, bloodsamples were collected from the 17 surviving groupIA caiman lizards, and presence of antibody againstthe isolate in this report was determined using an HIassay.13 Titers ranged from ,1:10 to 1:80. Seven liz-ards had titers .1:20. When assaying snake plasmafor determining exposure to OPMV, a titer of .1:20but ,1:160 is considered in the suspect range (ERJ,personal observation). Because these lizards were sam-

pled 7 months following the initial outbreak, the peakantibody response may have been missed. In a zoo-logical collection of snakes experiencing an OPMVoutbreak, snakes sampled at 5 months following thedeath of the first snake showed elevated antibody ti-ters; 3 months later, many snakes sampled had anti-body titers of ,1:80.13 Thus, the HI antibody responsedoes not remain elevated for prolonged periods. Morework needs to be done to better define the appropriatecutoff between a positive and negative plasma sample.

This report is the first of epidemics in groups ofcaptive lizards associated with a paramyxovirus infec-tion. The susceptibility of lizards to infection and deathfrom paramyxovirus previously was unknown. The or-igin of the paramyxovirus in these caiman lizards isunclear. Although these lizards were imported into theUSA from Peru, they may have been exposed follow-ing collection from the wild in Peru, either from otherreptiles in the export facility in Peru or at the reptileimporters’ facilities in the USA. Further work is need-ed to understand the relationship of this virus withother reptile paramyxoviruses. Comparisons withsnake isolates using both western blot analysis of viralproteins and analyses of the partial L and HN genesequences is needed to better understand these affini-ties. Transmission studies are also needed to demon-strate a causal relationship between the isolate and theproliferative pneumonia in these caiman lizards.

AcknowledgementsWe thank Sylvia Tucker (Department of Small Animal

Clinical Sciences) and Karen Kelly (ICBR, Electron Mi-



150 Jacobson et al.

Figure 5. Negatively stained filamentous nucleocapsid material with a herringbone structure characteristic of the Paramyxoviridae.Phosphotungstic acid.

croscopy Core Laboratory, University of Florida) for tech-nical assistance. This article is published as University ofFlorida College of Veterinary Medicine Journal Series num-ber 567.

Sources and manufacturers

a. Hitachi H-7000, Hitachi America, Tarrytown, NY.b. Zeiss 10, Carl Zeiss, Thornwood, NY.c. Gibco BRL, Grand Island, NY.d. American Type Culture Collection, Rockville, MD.e. EmBed 812, EMS, Fort Washington, PA.f. Gatan Bio/Scan Digital Micrograph 2.5, Gatan, Pleasanton, CA.g. Millipore Corp., Bedford, MA.h. Phillips 201, FEI Co., Hillsboro, OR.i. Nalge-Nunc International, Naperville, IL.j. Vectastain, Vector Laboratories, Burlingame, CA.

References

1. Ahne W, Batts WN, Kurath G, Winton JR: 1999, Comparativesequence analyses of sixteen reptilian paramyxoviruses. VirusRes 63:65–74.

2. Ahne W, Neubert WJ: 1989, Antigenic relationship betweenthree members of Paramyxoviridae isolated from differentsnakes. Herpetopathologia 2:65–72.

3. Ahne W, Neubert WJ, Thomsen I: 1987, Reptilian viruses: iso-lation of myxovirus-like particles from the snake Elaphe oxy-cephala. J Vet Med B 34:607–612.

4. Blahak S: 1995, Isolation and characterization of paramyxovi-ruses from snakes and their relationship to avian paramyxovi-ruses. J Vet Med B 42:216–224.

5. Clark H, Leif F, Lunger P, et al.: 1979, Fer-de-lance virus(FDLV): a probable paramyxovirus isolated from a reptile. JGen Virol 44:405–418.




6. Foelsch D, Leloup P: 1982, Infection enzootique grave dans unserpentarium. In: Pathology of reptiles and amphibians, ed.Vago C, Matz G, pp. 25–31. Presse de l’Universite d’Angers,Angers, France.

7. Haines DM, Chelack BJ: 1991, Technical considerations for de-veloping enzyme immunohistochemical staining procedures onformalin-fixed paraffin-embedded tissues for diagnostic pathol-ogy. J Vet Diagn Invest 3:101–112.

8. Homer BL, Sundberg JP, Gaskin JM, et al.: 1995, Immunope-roxidase detection of ophidian paramyxovirus in snake lung us-ing a polyclonal antibody. J Vet Diagn Invest 7:72–77.

9. Jacobson ER: 1999, Diagnosis of reptilian viral infections. In:Laboratory medicine: avian and exotic pets, ed. Fudge AM, pp.229–235. WB Saunders, Philadelphia, PA.

10. Jacobson ER, Adams HP, Geisbert TW, et al.: 1997, Pulmonarylesions in experimental ophidian paramyxovirus pneumonia ofAruba Island rattlesnakes, Crotalus unicolor. Vet Pathol 34:450–459.

11. Jacobson ER, Gaskin JM, Page D: 1981, Illness associated withparamyxo-like virus infection in a zoologic collection of snakes.J Am Vet Med Assoc 179:1227–1230.

12. Jacobson ER, Gaskin JM, Simpson CF, Terrell TG: 1980, Par-

amyxo-like virus infection in a rock rattlesnake. J Am Vet MedAssoc 177:796–799.

13. Jacobson ER, Gaskin JM, Wells S, et al.: 1992, Epizootic ofophidian paramyxovirus in a zoological collection: pathological,microbiological, and serological findings. J Zoo Wildl Med 23:318–327.

14. Lamirande EW, Nichols DK, Owens JW, et al.: 1999, Isolationand experimental transmission of a reovirus pathogenic in rat-snakes (Elaphe species). Virus Res 63:135–141.

15. McLaughlin GS, Jacobson ER, Brown DR, et al.: 2000, Upperrespiratory tract disease in gopher tortoises: pathologic investi-gation of naturally ocurring disease in Florida. J Wildl Dis 36:272–283.

16. Penner JD, Jacobson ER, Brown DR, et al.: 1997, A novel My-coplasma sp. associated with proliferative tracheitis and pneu-monia in a Burmese python (Python molurus bivittatus). J CompPathol 17:283–288.

17. Potgieter LN, Sigler RE, Russell RG: 1987, Pneumonia in Ot-toman vipers (Vipera xanthina xanthina) associated with a par-ainfluenza 2–like virus. J Wildl Dis 23:355–360.

18. Richter GA, Homer BL, Moyer SA, et al.: 1996, Characteriza-tion of paramyxoviruses isolated from three snakes. Virus Res43:77–83.



paramyxovirus infection in caiman lizards (draecena guianensis)

Documents