J. Comp. Path. 2015, Vol. 152, 248e259 Available online at www.sciencedirect.com

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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Pathology of a Bohle-like Virus Infection in TwoAustralian Frog Species (Litoria splendida and

Litoria caerulea)

Cor

002

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I. V. Jerrett*, R. J. Whittington† and R. P. Weir‡

*Department of Environment and Primary Industries, AgriBio Centre, Bundoora, Victoria, †Faculty of Veterinary Science,

University of Sydney, Camden, New South Wales and ‡Department of Primary Industries and Fisheries, Berrimah Veterinary

Laboratories, Northern Territory, Australia

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1-99

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Summary

Gross and histopathological examination was performed on seven captive magnificent tree frogs (Litoria splen-dida) and one green tree frog (Litoria caerulea) that had died or been humanely destroyed while naturally in-fected with Mahaffey Road virus, a Bohle iridovirus-like ranavirus. Necropsy examination revealed skinlesions consisting of multiple small pale or haemorrhagic papules and ulcers in most frogs. Other common grossfindings were perineural haemorrhage affecting the spinal nerves, hydrocoelom, hepatomegaly and splenomeg-aly with pinpoint pale foci throughout the parenchyma. On histological examination, vasculitis with promi-nent endothelial necrosis was found in a wide range of tissues. Widespread lymphoid necrosis and fibroblastnecrosis were usual findings. Multifocal epithelial cell necrosis in the epidermis, liver and pancreas was foundcommonly. Non-suppurative meningoencephalitis, myelitis and ganglioneuritis were present variably. Intra-cytoplasmic basophilic inclusion bodies were found variably in hepatocytes, renal tubular epithelium and ker-atinocytes. Immunohistochemistry demonstrated ranavirus antigen in endothelial cells, fibroblasts,macrophages, lymphocytes and epithelial cells in a wide range of tissues. The finding of widespread venousand lymphatic endothelial necrosis and demonstration of abundant endothelial antigen suggests that endothe-lial tropism of the virus plays a significant role in the pathogenesis of the infection.

Crown Copyright � 2014 Published by Elsevier Ltd. All rights reserved.

Keywords: amphibian; immunohistochemistry; pathology; ranavirus

Introduction

Amphibian populations can be affected by significantdisease events. Over the last 40 years two pathogenshave emerged as important candidates for epizooticsand, in some cases, population decline: Batrachochy-trium dendrobatidis and ranavirus. Since the original re-ports of their association with disease (Wolf et al.,1968; Berger et al., 1998) both have become topics ofintense research. This report concerns ranavirus,which has been reviewed recently (Chinchar et al.,2011). Briefly, ranavirus is a genus within the familyIridoviridae, which comprises large, icosahedral,double-stranded DNA viruses that replicate in both

ondence to: I. V. Jerrett (e-mail: ian.jerrett@depi.vic.gov.au).

75/$ - see front matter Cr

x.doi.org/10.1016/j.jcpa.2014.12.007

the nucleus and the cytoplasm. The first known andthe type species ranavirus is frog virus 3 (FV3). Itwas isolated from subclinically infected leopard frogs(Rana pipiens) in North America during research onadenocarcinoma, and was shown at the time to bepathogenic for tadpoles (Granoff and Came, 1965).FV3 and closely related viruses are now known tobe significant pathogens of fish, amphibians and rep-tiles (Whittington et al., 2010). In the case of amphib-ians, at least 72 species from 14 families are recognizedhosts for one or more ranaviruses and their distribu-tion includes Asia, Europe, North America, SouthAmerica and Australia (Miller et al., 2011). Twodifferent ranaviruses have been isolated from clini-cally diseased frogs in Australia: Bohle iridovirus(BIV) from the ornate burrowing frog (Limnodynastes

own Copyright � 2014 Published by Elsevier Ltd. All rights reserved.

Bohle-like Virus Infection in Frogs 249

ornatus) (Speare and Smith, 1992) and MahaffeyRoad virus (MHRV) from Litoria splendida and Litoria

caerulea (Weir et al., 2012). BIV has been transmittedexperimentally to six other species of frog (Cullenet al., 1995; Cullen and Owens, 2002).

MHRV was characterized from a single outbreakin a small captive frog colony in Darwin, NorthernTerritory, Australia, in 2008. Weir et al. (2012)described the circumstances in which eight of 12 L.

splendida and two of 13 L. caerulea died. Preliminaryhistopathological findings of necrosis in major organswere reported in this study. The lesions were generallyconsistent with ranavirus infection in lower verte-brates (Cunningham et al., 1996; Reddacliff andWhittington, 1996), but detailed light microscopicalfindings were not provided. As the literature onpathology associated with ranavirus infection inadult anurans is scant, we undertook a detailedinvestigation of the cases described by Weir et al.

(2012), using haematoxylin and eosin (HE)-stainedsections as well as immunohistochemistry (IHC) todefine the location of ranavirus antigens in the tissues.

Materials and Methods

Animal Background

Animals were captive frogs held in outdoor pens on aproperty in the Darwin rural area in the NorthernTerritory, Australia. Eight of a group of 12 adult L.splendida died over a 10-day period and the remainingfour frogs were humanely destroyed after appearinglethargic or developing skin lesions. Two of 13 adultL. caerulea in an adjacent pen died 1 week later.MHRV was isolated from the spleen, liver or skin offour of the L. splendida and the L. caerulea examinedin the present study, as described byWeir et al. (2012).

Necropsy Examination

Detailed systematic necropsy examination was per-formed on three L. splendida, which were found deadin a fresh state, and four L. splendida, which were hu-manely destroyed with carbon dioxide. Necropsy ex-amination was also performed on one L. caerulea,which was found dead. Two frogs that were humanelydestroyed were examined within 1 h of death and twofurther humanely destroyed frogs were frozen imme-diately after death and were examined within 8 h ofthe commencement of thawing. The interval betweendeath and necropsy examination was estimated to beless than 24 h for frogs found dead. Dissected coelomicviscera and the remainder of the carcass from eachfrog were fixed in 10% neutral buffered formalin.Bones of the axial skeleton were decalcified in 25%

formic acid solution for 72 h following formalinfixation.

Histopathology

A wide range of tissues from all frogs were processedfor histopathological examination. Formalin-fixed tis-sues were dehydrated through graded alcohols andxylene before being embedded in paraffin wax. Sec-tions (4 mm) were stained with HE.

Immunohistochemistry

IHC was performed on a wide range of tissues fromsix frogs. Sections were labelled using an immuno-peroxidase method based on Reddacliff andWhittington (1996). Briefly, sections (5 mm) weremounted on SuperFrost� Plus ground edge slides(Menzel-Glaser, Braunschweig, Germany), de-waxed in a xylene bath then rehydrated throughgraded ethanol solutions. The following steps werethen completed using an autostainer (AutostainerPlus LV-1; Dako, Carpinteria, California, USA)at room temperature. The slides were rinsed inTris buffer then H2O2 3% in methanol was addedand incubated for 5 min. After rinsing, the slideswere incubated in proteinase K solution (ready-to-use solution, Dako, catalogue number S3020) for10 min, rinsed in Tris buffer and primary antibody(affinity purified rabbit anti-EHNV Lot No. M708,OIE Reference Laboratory for Epizootic Haemato-poietic Necrosis Virus, University of Sydney)diluted 1 in 1,500 in antibody diluent (Dako, cata-logue number S0809) was added and incubated for30 min. Sections were gently air blown by the auto-mated processor to remove the reagents. Labellingof anti-EHNV antibodies was achieved using a com-mercial kit (DAKO LSAB�+ Kit, peroxidase;Dako, catalogue number K0690). Secondary anti-body (biotinylated link universal) was added andincubated for 30 min. Sections were air blownand streptavidineperoxidase was added andincubated for 30 min. Sections were air blownthen substrateechromogen solution (DAKOREAL�: Dako, catalogue number K5007) wasadded and incubated for 5 min. Slides were rinsedwith distilled water, removed from the autostainerand counterstained in Mayer’s haematoxylin for2 min. Slides were rinsed with distilled water for1 min, immersed in Scott’s bluing solution for2 min, rinsed in distilled water for 2 min, dehy-drated through graded ethanol solutions and xyleneand then mounted under coverslips using non-aqueous mounting medium. Control slides were pre-pared in the same way using non-immune rabbitserum at a dilution of 1 in 1,500.

250 I.V. Jerrett et al.

Results

Gross Findings

The results of gross examination are presented inTable 1. Skin lesions were usually multiple and con-sisted of 1e4 mm papules or ulcers, which were eitherpale pink or haemorrhagic and frequently werelocated over the entire hindlimbs (Figs. 1 and 2) oron the volar and dorsal surfaces of the extremities ofeither the hind- or forelimbs. In one frog, skin lesionswere localized to the upper lip and in another werefound only in a localized cluster on the back. Inareas where skin lesions were prevalent there wasoften associated subcutaneous oedema.

Systemic haemorrhagic lesions were most promi-nent in the perineural connective tissue of spinalnerves (Fig. 3), but a few scattered haemorrhages,1e3 mm in diameter, were also found on the parietalcoelomic serosa and in the skeletal muscle fascia.There was frequent petechiation of the oral mucousmembranes, including the tongue, hard palate andinner lip. In two frogs, haemorrhagic fluid was foundin the mouth. The presence of excess clear or lightlyblood-stained fluid in the coelom was a commonfinding. In some frogs the stomach wall was markedlyoedematous. Intestinal contents were variably hae-morrhagic.

In most frogs the spleen was enlarged, measuringup to 12 mm diameter, and contained pale foci upto 1 mm diameter. The liver was variably enlargedand mottled with a pale lobular pattern. Most frogswere in good body condition with prominent intra-coelomic fat bodies.

Table

Gross necrops

Animal number 1 2 3

Species L.s. L.s. L.s.

Found dead or humanely

destroyed

D D HD

Sex M F MSkin papules or ulcers e ++ +

Subcutaneous oedema ++ + e

Oral petechiation + +++ e

Hydrocoelom + +++ eSplenomegaly + ++ e

Splenic pale foci +++ +++ e

Hepatic mottling ++ e e

Perineural haemorrhage ++ ++ eStomach oedema +++ ++ e

Intestinal haemorrhage +++ + e

Gross lesion score: e, none; +, mild; ++ moderate; +++ severe.

L.s., Litoria splendida; L.c., Litoria caerulea; D, found dead; HD, humanely d

Histopathological Findings

Tissues from two frogs, which had been frozenfollowing humane destruction, were unsuitable forhistological examination due to freezeethaw artifact.The presence and severity of necrosis in tissues from L.

splendida (frogs 1e5) and L. caerulea (frog 8), in whichhistological preservation was suitable, is detailed inTable 2. Skin lesions were found in all frogs.Epidermal lesions consisted of irregular areas of kera-tinocyte hyperplasia with multiple scattered foci ofcell lysis and occasional formation of small vesiclesin the stratum spinosum (Figs. 4 and 5). In 2/6frogs, single or multiple intracytoplasmicamphophilic or basophilic inclusion bodies(approximately 1 mm diameter) were evident inoccasional keratinocytes. In the superficial dermisthere was multifocal oedema, fibroblast necrosis andcollagenolysis with variable infiltration ofgranulocytes and lymphocytes. Necrotizinglymphocytic vasculitis with adjacent connectivetissue oedema and necrosis was often present in thesubcutis underlying epidermal lesions (Fig. 4). Intra-vascular leucocytes were frequently karyorrhectic orlysed and clumps of cell debris, including melano-somes, were often present in the vascular lumina.Perivascular lymphocyte infiltration and accumula-tion of necrotic cell debris was variably present.Lymphatic endothelium in the subcutis wasfrequently diffusely necrotic.

Vascular endothelial necrosis, variable mononu-clear inflammatory cell infiltration and extensive hae-morrhage were found in the epineurium of spinalnerves. Multifocal axonal and endoneurial necrosis

1

y findings

4 5 6 7 8

L.s. L.s. L.s. L.s. L.c.

HD HD D HD D

F F F M F+++ + + e ++

++ e e e +

e ++ + e e

++ e +++ e ++e e ++ ++ +++

+ + + +++ +++

++ ++ e + e

e +++ +++ e +++e ++ e e e

e ++ e e ++

estroyed; M, male; F, female.

Fig. 3. Frog 8 with coelomic viscera removed.Haemorrhagic peri-neural tissue and spinal nerves are indicated (long arrow).There is petechiation of the parietal coelomic serosa. A skinulcer in the ventral neck is indicated (short arrow). Bar,1 cm.

Fig. 1. Skin papules and erosions (arrow) on the dorsum of the lefthindlimb of frog 4. Bar, 1 cm.

Bohle-like Virus Infection in Frogs 251

with lymphocyte, macrophage and granulocyte infil-tration of the endoneurium was present variablywithin the spinal nerves (Fig. 6). Free melanosomeswere often present in areas of endoneurial necrosis.Endoneurial necrosis andmononuclear inflammatorycell infiltration was present in the spinal nerve gangliaof the same frogs. Central nervous system lesions con-sisted of gliosis and multifocal neuropil necrosis in theforebrain, midbrain or brainstem (Fig. 7). Addition-ally, there was cerebrospinal meningeal vascularendothelial necrosis with patchy meningeal lympho-cyte and macrophage infiltration and the presenceof free melanosomes. Lesions in skeletal muscle werelimited to fascial oedema and multifocal necrosis inareas subadjacent to sites of subcutaneous tissue ne-crosis.

Fig. 2. Haemorrhagic skin papules (arrow) and subcutaneousoedema of the ventral surface of the left hindlimb of frog4. Bar, 1 cm.

Splenic lesions consisted of multifocal to diffuse ne-crosis of lymphoid tissue and supporting cells withconcurrent patchy, often widespread, necrosis of sinu-soidal tissue including endothelial cells, macrophagesand reticular cells (Fig. 8). Areas of necrosis weremildly infiltrated with granulocytes.

Liver lesions consisted of multifocal hepatocyte ne-crosis, widespread necrosis of sinusoidal endothelium,patchy hyperplasia of biliary epithelium and

Table 2

Occurrence and severity of histological lesions of

necrosis

Animal number 1 2 3 4 5 8

Skin + ++ + +++ + ++Spleen +++ +++ ++ +++ +++ +++

Liver +++ +++ + + ++ +++

Pancreas +++ ++ NE e ++ NEKidney +++ ++ + ++ ++ ++

Lung ++ + e + + ++

Heart + + e e + +

Gonad + (T) ++ (O) � (T) + (O) + (O) ++ (O)Tongue ++ ++ e ++ ++ e

Stomach ++ ++ e e ++ ++

Small intestine +++ ++ + ++ ++ ++

Mesentery ++ + e e ++ +++Brain + + e e + +

Spinal cord + + e e e +

Spinal nerve ++ ++ e e + e

Bone marrow ++ + ++ + + ++

Histopathological score: e, none; +, mild; ++, moderate; +++,severe; NE, not examined; T, testis; O, ovary.

Fig. 4. Hindlimb skin and subcutis of frog 4 showing epidermal hy-perplasia andmultifocal necrosis with subcutaneous vascu-litis and connective tissue necrosis.HE. Bar, 100mm. Inset isa higher magnification showing necrotizing phlebitis andperivascular inflammatory cell infiltration, predominantlylymphocytic. HE. Bar, 25 mm.

Fig. 6. Spinal nerve from frog 2 showing multifocal inflammatorycell infiltration, necrosis and haemorrhagewithin the endo-neurium. There is also epineurial haemorrhage. HE. Bar,50 mm. Inset is higher magnification showing lymphocyteand granulocyte infiltration associated with necrosis ofthe endoneurium and adjacent axons. HE. Bar, 25 mm.

252 I.V. Jerrett et al.

periportal hepatic stem cells. In one frog there wereoccasional large foci of parenchymal necrosis andloss of tissue architecture. Single or multiple intracy-toplasmic amphophilic or basophilic inclusion bodies(1e3 mm diameter) were apparent in hepatocytes in3/6 frogs (Fig. 9). Sinusoids frequently contained kar-yorrhectic leucocytes and clumps of cell debrisincluding many free melanosomes. The endotheliumof portal veins and arterioles was often necrotic. Thenumber of melanomacrophages within hepatic lob-ules varied widely between frogs and was not relatedto the severity of hepatic lesions. In the pancreas,interstitial connective tissue necrosis and vascularendothelial necrosis was prominent and was accom-

Fig. 5. Hindlimb skin from frog 4 showing epidermal hyperplasiaand multifocal necrosis with vesicle formation, dermalvasculitis and connective tissue necrosis. HE. Bar, 50 mm.

panied by multifocal interstitial lymphocyte andplasma cell infiltration. There was concurrent acinarepithelial cell pleomorphism, disorganization, dissoci-ation and multifocal necrosis (Fig. 10).

In the kidney, the most prominent findings werenecrosis of the glomerular endothelium and mesan-gium and necrosis of the interstitial connective tissue(Fig. 11). There was mild multifocal necrosis of renaltubular epitheliumwith formation of occasional gran-ular or hyaline intratubular casts. In 2/6 frogs occa-sional tubular epithelial cells contained single ormultiple intracytoplasmic basophilic inclusionbodies. Free melanosomes were variably present,but often abundant in the glomerular mesangium

Fig. 7. Brain from frog 2 showing a focus of inflammatory cell infil-tration in the neuropil of the telencephalon. HE. Bar,25 mm.

Fig. 8. Spleen from frog 4 showing necrosis of lymphocytes of thewhite pulp with only scattered reticular cells and occa-sional granulocytes remaining amongst cell debris in theseareas. There is also widespread necrosis of sinusoidal endo-thelium within the red pulp. HE. Bar, 40 mm.

Fig. 10. Pancreas from frog 1 showing interstitial oedema and ne-crosis with fibroplasia and mild lymphocyte infiltration.There is acinar epithelial pleomorphism, dissociationand necrosis. HE. Bar, 50 mm.

Bohle-like Virus Infection in Frogs 253

and in the interstitial connective tissue. In one frogthere were occasional large foci of renal parenchymalnecrosis. In the interrenal tissue of 3/6 frogs there wasnecrosis of stroma and cortical cells. Lung lesions con-sisted of multifocal necrosis of alveolar pneumocytesand interstitial stroma (Fig. 12). In these frogs theendothelium of interstitial capillaries and venuleswas often necrotic and there were free melanosomeswithin and around blood vessels. Lesions in the heartconsisted of multifocal to diffuse necrosis of endothe-lium lining the ventricles and the ventricular spacesof the spongy ventricular myocardium (Fig. 13). In

Fig. 9. Liver from frog 1 showing necrosis of the sinusoidal endo-thelium and scattered hepatocytes with hepatocyte dissoci-ation and disruption of hepatic cords. There are single ormultiple basophilic intracytoplasmic inclusions in hepato-cytes and an example is arrowed. HE. Bar, 15 mm.

the ovary of 4/4 frogs there was widespread stromalfibroblast necrosis and necrosis of venous endothe-lium. In the testis of 1/2 frogs there was multifocalinterstitial fibroblast necrosis.

Tongue lesions consisted of multifocal epithelial ne-crosis associated with vascular necrosis, fibroblast ne-crosis, lymphocyte infiltration, macrophageinfiltration and variable haemorrhage in the underly-ing lamina propria (Fig. 14). Fibroblast necrosis andvascular necrosis often extended into the interstitiumof the underlying skeletal muscle. In the stomachthere wasmucosal and submucosal oedema associatedwith widespread necrosis of venule endothelium(Fig. 15), with variable transmural vascular necrosis

Fig. 11. Kidney from frog 4 showing necrosis of the glomerularme-sangium and capillary endothelium. There is also necrosisand mixed inflammatory cell infiltration of the interstitialconnective tissue. HE. Bar, 30 mm.

Fig. 12. Lung from frog 1 showing necrosis of pneumocytes, inter-stitial vascular endotheliumand the interstitial connectivetissue. There is mild interstitial granulocyte, lymphocyteand macrophage infiltration. HE. Bar, 20 mm.

Fig. 14. Tongue from frog 1 showing necrosis of the surface epithe-lium of the tips of papillae. There is also vascular endothe-lial necrosis, haemorrhage and perivascular connectivetissue necrosis in the lamina propria. HE. Bar, 40 mm.

254 I.V. Jerrett et al.

and necrosis of perivascular stroma.Mild lymphocyteand granulocyte infiltration was present in areas ofstromal necrosis. Vascular necrosis was also evidentin the muscularis and serosa of the stomach. Encystedmetazoan parasites were found in the submucosa ofthe stomach of one frog. In the small intestine therewas necrosis of venules and stroma in the laminapropria and submucosa. Variable oedema, lympho-cyte karyorrhexis and granulocyte infiltration of thelamina propria was associated with stromal necrosis.Occasional small foci of mucosal epithelial necrosiswere present, overlying areas of proprial stromal ne-crosis. Mesenteric lesions consisted of widespreadvascular endothelial necrosis, with multifocal stromal

Fig. 13. Heart from frog 1 showing endothelial necrosis in thespongy myocardium and accumulation of necrotic celldebris in the ventricular spaces. HE. Bar, 20 mm.

necrosis and variable mild stromal infiltration withlymphocytes and macrophages.

Examination of bone marrow of the axial skeletonrevealed a moderate reduction in haemopoietic cellu-larity in most frogs. Karyorrhectic round cells resem-bling myeloid cells were evident in the marrow of allfrogs.

Immunohistochemistry

Antigen labelling was found in several cell types in awide range of organs. Labelling was most widespreadwithin vascular endothelium, but was also often foundin connective tissue, macrophages, lymphocytes and

Fig. 15. Stomach from frog 1 showing necrosis of the venular endo-thelium (V) and the perivascular connective tissue in thedeep laminapropria (LP).There is also oedemaof the sub-mucosa (S). HE. Bar, 20 mm.

Fig. 17. Subcutis of frog 4 showing ranavirus-specific labelling ofthe venous endothelium, perivascular mononuclear in-flammatory cells and fibroblasts of the subcutis. IHC.Bar, 20 mm.

Bohle-like Virus Infection in Frogs 255

epithelial cells. The presence of antigen correlatedclosely with presence of histopathological lesions. Inthe epidermis, antigen labelling was found in kerati-nocytes adjacent to and within foci of necrosis, mostlywithin the stratum spinosum (Fig. 16). In the dermisand subcutis, antigen labelling was associated withthe venous and lymphatic endothelium and waswithin perivascular mononuclear inflammatory cellsand within fibroblasts in areas of connective tissue ne-crosis (Fig. 17).

In spinal nerves, labelling occurred within fibro-blasts, vascular endothelium, mononuclear inflam-matory cells and necrotic cell debris in theendoneurium (Fig. 18) and of vascular endotheliumin the perineurium. Within spinal nerve ganglia,labelling of endoneurial fibroblasts, mononuclear in-flammatory cells and necrotic cell debris occurred.In the brain of frogs with meningoencephalitis, anti-gen labelling was found diffusely within meningealvenous endothelium and meningeal macrophages.Antigen labelling was also evident multifocally in ce-rebral venous endothelium and within mononuclearinflammatory cells in foci of gliosis (Fig. 19). No label-ling with viral antigen occurred in skeletal muscle fi-bres, although multifocal labelling of fascialconnective tissue and vascular endothelium occurredin areas subadjacent to necrotic subcutaneous tissue.

In the spleen, antigen labelling was abundant inintact and lysed endothelial cells and macrophagesin sinusoidal tissue and was also present in cell debrisin necrotic lymphoid tissue (Fig. 20). Antigen label-ling was also evident in fibroblasts within the spleniccapsule. In the liver, antigen labelling was diffuselyintense in sinusoidal and venous endothelium and

Fig. 16. Skin from the hindlimb of frog 4 showing virus antigen inscattered keratinocytes, the dermal connective tissue andthe dermal mononuclear inflammatory cells. IHC. Bar,50 mm.

was also found multifocally in clusters of hepatocytes(Fig. 21). Hepatocyte intracytoplasmic inclusionswere positive for virus antigen. Antigen labellingoccurred in hepatic portal areas in fibroblasts, freewithin collagenous tissue and multifocally withinbiliary epithelium. In the pancreas, venous endothe-lium, interstitial fibroblasts and scattered clusters ofacinar epithelium were positive for viral antigen(Fig. 22). In the kidney, glomerular and interstitialvenous endothelium labelled strongly positive forviral antigen (Fig. 23). Antigen labelling was alsoprominent within cell debris in the mesangium andinterstitial connective tissue. There was mild multi-focal antigen labelling of renal tubular epithelium.

Fig. 18. Spinal nerve from frog 2 showing ranavirus-specific label-ling of necrotic cells, cell debris and venule endotheliumwithin the endoneurium. IHC. Bar, 20 mm.

Fig. 19. Brain from frog 2 showing virus-specific labelling of mac-rophages, neuroglia and cell debris in the neuropil of thetelencephalon. IHC. Bar, 20 mm.

Fig. 21. Liver from frog 1 showing virus-specific labelling of the he-patocyte cytoplasm, nuclei and inclusion bodies. There isalso labelling of the sinusoidal endothelium and sinusoidalcell debris. IHC. Bar, 20 mm.

256 I.V. Jerrett et al.

In the lung, antigen labelling was found eitherdiffusely or multifocally within pneumocytes andmultifocally in capillary and venous endotheliumand interstitial fibroblasts (Fig. 24). In the heart, an-tigen labelling was found in the endothelium liningthe ventricles and the ventricular spaces within thespongy myocardium. In the ovary, antigen labellingoccurred within stromal fibroblasts, stromal celldebris and venous endothelium.

In the tongue there was multifocal antigen label-ling of venous endothelium, perivascular fibroblastsand perivascular cell debris within the lamina prop-ria. In the stomach and intestine antigen labelling

Fig. 20. Spleen from frog 4 showing ranavirus-specific labelling ofintact and lysed endothelial cells and macrophages in thesinusoidal tissue and labelling of cell debris in necroticlymphoid tissue. There is also labelling of capsular fibro-blasts. IHC. Bar, 50 mm. Inset shows highermagnificationof sinusoidal tissue. IHC. Bar, 25 mm.

was widespread within venous endothelium in thelamina propria, submucosa and serosa and was alsopresent within perivascular fibroblasts, mononuclearinflammatory cells and cell debris in these locations(Fig. 25). In the mesentery antigen labelling occurredmultifocally within venous endothelium and in peri-vascular cell debris within mesenteric connective tis-sue.

Discussion

Natural infection with MHRV in adult Australiananuran species was in most cases associated with

Fig. 22. Pancreas from frog 5 showing virus-specific labelling ofinterstitial fibroblasts, mononuclear inflammatory cellsand cell debris. There is also labelling of the venular endo-thelium and occasional acinar epithelial cells. IHC. Bar,50 mm.

Fig. 23. Kidney from frog 1 showing intense ranavirus-specificlabelling of the glomerular capillary endothelium, me-sangium and interstitial cell debris. There is also mildmultifocal labelling of tubular epithelial cells. IHC. Bar,50 mm.

Fig. 25. Stomach submucosa from frog 1 showing ranavirus-specific labelling of the venular endothelium and of fibro-blasts and lymphocytes within oedematous adjacent con-nective tissue. IHC. Bar, 50 mm.

Bohle-like Virus Infection in Frogs 257

grossly evident multifocal skin lesions and a range ofsystemic lesions including hydrocoelom, subcutane-ous and gastric oedema and variable widespread hae-morrhage, most prominent adjacent to spinal nerves.The gross lesions are similar to those described byCunningham et al. (2008) for the ulcerative plus sys-temic haemorrhagic form of ranavirus disease in thecommon frog (Rana temporaria), although the predilec-tion for perineural location of haemorrhage was notdescribed in that study. Previous reports of naturaland experimental ranavirus infection in Australiannative anurans (Speare and Smith, 1992; Cullenet al., 1995; Cullen and Owens, 2002) have notprovided descriptions of gross lesions. The gross

Fig. 24. Lung from frog 5 showing virus-specific labelling of pneu-mocytes, capillary endotheliumand interstitial cell debris.IHC. Bar, 20 mm.

cutaneous and systemic lesions in frogs in thepresent study have several similarities to thosedescribed for Regina ranavirus (RRV) infection insalamanders in North America (Bollinger et al., 1999).

Histologically, MHRV infection was associatedwith necrosis of multiple cell types in a wide rangeof tissues, as has been previously described for ranavi-rus infection in anurans (Wolf et al., 1968; Cullen et al.,1995; Cunningham et al., 1996; Zhang et al., 2001;Cullen and Owens, 2002; Balseiro et al., 2009;Mazzoni et al., 2009). However, widespreadendothelial necrosis, with or without transmuralnecrosis as seen in the present study, is poorlydocumented in anurans. Wolf et al. (1968) describedendothelial necrosis in glomeruli and vascular necro-sis in the submucosa and muscularis of the stomach oftadpoles infected with tadpole oedema virus. Cullenand Owens (2002) reported that for experimentalBIV infection in native Australian anurans, micro-scopical liver lesions were sometimes associated withblood vessels.Miller et al. (2011) state that endothelialnecrosis may be extensive in amphibians with ranavi-rus infection, citing unpublished work by the leadauthor. Endothelial degeneration in a range of tissueswas a feature of ranavirus infection in rainbow trout(Oncorhynchus mykiss Walbaum) and redfin perch(Perca fluviatilis L) (Reddacliff and Whittington,1996) and in sheatfish fry (Silurus glanis) (Ogawaet al., 1990) and the authors of both studies suggestedthat vascular damage may be the basis of most of thelesions seen.

In addition to endothelial necrosis there was necro-sis of epithelial cells and fibroblasts in a wide range oftissues. Epithelial cell necrosis was most prominent inthe skin and liver, with generally only mild multifocal

258 I.V. Jerrett et al.

epithelial damage in other organs such as the kidney,lung, pancreas and alimentary tract. The reportedtropism of FV3 virus for the proximal renal tubularepithelium of anurans (Robert et al., 2005) was notseen with MHRV infection in the present study. In-tracytoplasmic amphophilic or basophilic inclusionbodies were found in hepatocytes, renal tubularepithelial cells and keratinocytes of some affectedfrogs in this study, but were not a consistent featureof MHRV infection. Inflammatory cell infiltration,predominantly lymphocytic and histiocytic with alesser granulocytic component, accompanied necrosisin most of the affected tissues. Karyorrhexis of lym-phocytes and macrophages was common in sites ofcellular infiltration. Necrosis of lymphocytes was aconsistent finding in lymphoid tissue such as thespleen and gut-associated lymphoid tissue and lysisof melanomacrophages with presence of free melano-somes was a common finding in tissues such as theliver, spleen and kidney.

Meningoencephalomyelitis, as seen in some frogswith MHRV infection, has been described rarely inranavirus disease of amphibians, and ganglioneuritishas not been recorded previously. Cullen et al.

(1995) reported necrosis of the brain and spinalcord in metamorphs and tadpoles of Australian an-urans infected experimentally with BIV, but inflam-mation was not described. Meningitis and necrosisin the brain, in association with intracytoplasmic in-clusions, was reported in larval caudates with ranavi-rus infection (Docherty et al., 2003), but furtherdetails of the lesions were not provided.

Immunohistochemical labelling of cells withranavirus-specific antigen was closely correlatedwith histological evidence of necrosis and inflamma-tion. Labelling confirmed tropism of MHRV for mul-tiple cell types in a wide range of tissues, as haspreviously been described for ranavirus infections inanurans (Cullen et al., 1995; Robert et al., 2005;Cunningham et al., 2008; Balseiro et al., 2009). Incontrast to the wide range of tissues in whichlabelling of vascular endothelium occurred in thepresent study, previous studies in anurans haveeither not specifically mentioned ranavirus labellingof endothelium (Cullen et al., 1995; Robert et al.,2005) or have reported endothelial labelling only asan occasional finding in the intestine (Cunninghamet al., 2008; Balseiro et al., 2009, 2010) or as afrequent finding in the liver (Cunningham et al.,2008). In two of these studies ranavirus labelling ofglomeruli was reported (Cullen et al., 1995; Balseiroet al., 2009), but it was not stated whether labellingoccurred in the glomerular endothelium,mesangium or both. Our findings with MHRVinfection of Australian frogs concur with a study on

ranavirus infection of fish in which strong ranaviruslabelling of endothelial cells was found in manytissues (Reddacliff and Whittington, 1996). Ranavi-rus labelling of inflammatory cells in the brain andof inflammatory and supportive cells in the gangliaand spinal nerves, as seen in the present study, hasnot previously been reported in anurans; however,labelling of vascular endothelial and connective tissuecells in the meninges has been reported in fish infectedwith ranavirus (Reddacliff and Whittington, 1996).

In MHRV infection of anurans, endothelialtropism evidenced by widespread microscopicalvascular damagewith corresponding strong ranaviruslabelling of vascular endothelium, is likely a majorcontributor to lesions such as widespread oedema, sys-temic haemorrhage, hydrocoelom and skin ulcera-tion. Concurrent tropism for epithelium, fibroblasts,lymphocytes and macrophages further contributedto systemic lesions and the rapid demise of affectedfrogs. It was not possible to define the route of infec-tion based on the distribution of lesions in this studyand no temporal course can be inferred based on com-parison of dead and humanely destroyed frogs.Despite this, it seems clear that this was a systemicinfection with potential for widespread disseminationof the virus within the vascular system of the frogs.

Acknowledgments

We are grateful to R. Wilson of the Berrimah Veteri-nary Laboratories for technical assistance. Thanks arealso due to E. Chew and K. Barnes at the Faculty ofVeterinary Science, University of Sydney, for assis-tance with immunohistochemistry.

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