J. Comp. Path. 2002, Vol. 126, 231–234doi:10.1053/jcpa.2001.0534, available online at http://www.idealibrary.com on

SHORT PAPER

Subacute Liver Necrosis after ExperimentalInfection with Rabbit Haemorrhagic Disease

Virus (RHDV)

J. P. Teifke, I. Reimann and H. Schirrmeier∗

Institut fur Infektionsmedizin and ∗Institut fur Virusdiagnostik, Bundesforschungsanstalt fur Viruskrankheiten der Tiere

(BFAV), Friedrich-Loeffler-Institute, Insel Riems, Germany

Summary

Rabbit haemorrhagic disease (RHD) is usually peracute to acute, while subacute to chronic disease israre. This paper describes gross and histopathological findings in four out of 20 rabbits aged 14 weeks,experimentally infected with one of two German field isolates of RHD virus. Eight rabbits survived theinfection for 10 days and were killed after four of them, infected with 100 to 10 000 haemagglutinationunits, had started to develop progressive jaundice. Histopathologically, icteric livers showed severe subacutecentrilobular bridging necrosis with calcification, and proliferation of periportal hepatocytes and bile ducts.Positive-strand RHDV RNA was detected by in-situ hybridization, mainly in periportal macrophages.Loss of the normal hepatic architecture, reparation (fibrosis) and hepatocellular regeneration, togetherwith moderate inflammatory reaction, are signs of liver cirrhosis. These signs, observed in young rabbitsgiven small doses of RHD virus, are interpreted as an unusual outcome of experimental inoculation.

2002 Elsevier Science Ltd

results in a haemorrhagic syndrome (Park et al.,Rabbit haemorrhagic disease (RHD), a highly con-1997; Alonso et al., 1998). We report here lesionstagious and usually fatal disease, affects domestic,that developed in four rabbits with an unusualfarmed and wild rabbits of the species Oryctolagus(subacute) form of the infection. They formed partcuniculus (reviewed by Kroneman and Horzinek,of a group of 20 ZIKA-hybrid rabbits (E. Zim-1994; Chasey, 1996). Rabbit haemorrhagic diseasemermann, Untergroningen, Germany) aged 14virus (RHDV) is a positive-stranded RNA virusweeks which were inoculated in sub-groups of fourwhich has recently been classified as the type specieswith 1, 10, 100, 1000, or 10 000 haemagglutinationof the genus Lagovirus, family Caliciviridae (Greenunits (HAU) of RHDV, each 1-ml dose beinget al., 2000). RHD is characterized by a peracutedivided equally between the intramuscular andto acute course with high morbidity (100%) andoral routes of inoculation. In each sub-group, twomortality (40–90%) in adult rabbits. Ex-rabbits received the “Eisenhuttenstadt” isolate andperimentally infected rabbits exhibit the first clin-two the “Triptis” isolate. The molecular and an-ical signs 30 h post-inoculation (p.i.) and usuallytigenic properties of both these isolates were de-die within 3 days. Animals younger than 45 daysscribed by Schirrmeier et al. (1999).are susceptible to RHDV infection but do not show

During the first 3 days p.i., 12 rabbits died (threeclinical signs (Xu and Chen, 1989; Marcato et al.,from each of the 10 000, 1000 and 10 HAU sub-1991; Mikami et al., 1999). Necrosis of liver, whichgroups; two from the 100 HAU sub-group; andis central to the pathogenesis of RHD, is followedone from the 1 HAU sub-group), but the survivingby primary or secondary depletion of coagulation

factors; this, together with endothelial lesions, eight animals showed no clinical signs during this

0021-9975/02/020231+04 $35.00 2002 Elsevier Science Ltd

J. P. Teifke et al.232

period. On day 4 p.i., however, four animals (two severely disrupted the sinusoidal and lobular ar-chitecture of the liver (Fig. 4). Portal tracts showedfrom the 100 HAU and one each from the 1000 andmarked proliferation of bile ducts accompanied by10 000 sub-groups) became anorectic and lethargicmoderate to severe lymphocytic and histiocyticand developed jaundice, which progressed untilinfiltration and mild fibrosis (Fig. 5). In the liver,they and the four symptomless animals were killedRHDV positive-strand RNA was located mainly inon day 10 p.i. Of the four rabbits with subacuteRAM11-labelled macrophages of periportal areasdisease, two had received the Eisenhuttenstadt isol-and, albeit rarely, in periportal hepatocytes (Figsate (100 and 10 000 HAU) and two the Triptis5 and 6). Macrophages and mononuclear cellsisolate (100 and 1000 HAU).located in the sinuses and red pulp of the spleenAt necropsy, each of the four diseased rabbitswere also positive for RHDV-RNA. In only a fewwas seen to be severely icteric. All lobes of thepulmonary intravascular and alveolar macrophagesdiffusely brownish–yellow liver showed multifocalwas RHDV-RNA detectable. Interestingly, no hy-to confluent, sharply demarcated, irregularlybridization signals were present in endothelial cellsshaped, reticulated, greyish-white to red, grittyof the kidney, lung, liver or spleen. No RHDV-areas (Fig. 1). The kidneys were enlarged and brightRNA was detected in any of the four symptomlessyellow. The thymus was atrophic, the spleen wasanimals.moderately enlarged, and the lungs showed diffuse,

Subacute and chronic forms of RHD are verysevere, alveolar emphysema. Paraffin-wax sectionsuncommon (Marcato et al., 1991; Fuchs and Weis-(3 �m) were stained with haematoxylin and eosinsenbock, 1992). After introduction of RHD into an(HE), and with Gomori and von Kossa stain. Inarea previously free of the disease, rabbits areaddition, RHDV-RNA was detected by in-situ hy-highly susceptible and die from peracute infectionbridization (ISH) with RHDV antisense and sensewithout clinical signs. Experimentally, peracuteriboprobes. For this method, a 698 bp fragmentRHD can be consistently induced in adult rabbitslocated in the conserved ORF2 and ORF3 (VP60/by intramuscular inoculation with clarified liverVP10 gene) of RHDV isolate “Bosdorf” was amp-homogenate containing >10 000 HAU (Alonso etlified by reverse transcriptase-polymerase chain re-al., 1998; Schirrmeier et al., 1999). Because little isaction (RT-PCR) with primer RHDV-6422 (5′-known about the pathogenesis and pathology ofTGC AGG CCT ATG AGT TAG-3′ [nucleotidesthe subacute disease, the aim of this study was to6422–6439]) and primer RHDV-7120R (5′-TTG investigate liver lesions induced by RHDV doses ofTAG TTG CAG CTC TTG CC-3′ [com- <10 000 HAU. A combination of the intramuscularplementary to nucleotides 7120–7101; nucleotide and the oral routes of infection was chosen because

position according to Meyers et al., 1991]). The natural infection occurs via the nasopharynx, andDNA was cloned into the pGEM-T-Easy vector alveolar macrophages are thought to be importantand in-vitro transcription was performed with the for initial viral replication (Kimura et al., 2001). InRiboMAX system (Promega Corp., Madison, the four rabbits with subacute disease, killed onWI, USA). Macrophages were immunolabelled day 10 p.i., general and hepatic icterus togetherwith the mouse monoclonal anti-rabbit macro- with cholemic nephrosis were the predominantphage/monocyte specific antibody RAM11 (Tsu- gross findings. Macroscopically visible hepatickada et al., 1986; Dako Corp., Carpinteria, CA, necrosis, unusual in RHD, was associatedUSA). histopathologically with centrilobular fatty

Microscopically, periportal and midzonal hepa- degeneration, bridging necrosis and mineralizationtocytes were variably increased in size, and had of hepatocytes. Lesions in the liver and kidneys arebasophilic cytoplasm with large, chromatin-poor interpreted as the final stage of temporary severenuclei with at least two prominent nucleoli. They hypoxia to the liver and renal ischaemia, coincidingwere therefore considered to be proliferating or with disseminated intravascular coagulation (DIC)activated. Most midzonal hepatocytes contained during the acute phase of disease (Fuchs and Weis-large cytoplasmic vacuoles (lipid) and sometimes senbock, 1992). The possible cause of the pro-bilirubin droplets. Centrilobular hepatocytes were tracted nature of the infection in the four subacutelyhypereosinophilic, with pyknotic or karyorrhectic infected rabbits may have been related to suchnuclei, and showed dystrophic, granular cal- factors as immaturity, innate immunity or breed,cification as demonstrated by von Kossa staining singly or in combination. In acutely infected rab-(Figs 2 and 3). Multifocally, areas of necrosis and bits, Gelmetti et al. (1998) found that, at 44 hmineralization were surrounded by large numbers p.i., hepatocytes of the periportal areas were the

primary target cells for RHDV replication, andof histiocytes and lymphocytes. Bridging of necrosis

Liver Necrosis in Rabbit Haemorrhagic Disease 233

Fig. 1. Liver, rabbit inoculated with 100 haemagglutination units of RHDV isolate “Eisenhuttenstadt”; 10 days p.i. Multifocalareas of necrosis, haemorrhage and mineralization are present in all lobes of the moderately enlarged liver. The sameliver is illustrated in Figs 2–6.

Fig. 2. Subacute centrilobular bridging necrosis of hepatocytes, with moderate periportal lymphocytic and histiocytic infiltration,numerous binucleated hepatocytes and biliary hyperplasia. HE. Bar, 100 �m.

Fig. 3. Bridging necrosis of hepatocytes with pronounced mineralization. Von Kossa. Bar, 100 �m.Fig. 4. Disrupted lobular architecture with collapse and condensation of the reticulin network, mild fibrosis and regeneration

of hepatocytes. Gomori. Bar, 50 �m.Fig. 5. Numerous positive-strand RHDV-RNA positive cells (presumed to be macrophages) in the periportal area, with biliary

hyperplasia. Nonradioactive in-situ hybridization. Bar, 50 �m.Fig. 6. Numerous RAM11-positive cells (monocytes, macrophages) in the periportal area. Immunohistochemistry, ABC

technique. Bar, 50 �m.

Kimura et al. (2001) detected both positive- and liver of the four subacutely infected rabbits, andalmost exclusively in macrophages, suggestingnegative-strand (in smaller amounts) RHDV-RNA.

We found positive-strand RHDV-RNA only in the phagocytosis rather than viral replication (Kimura

J. P. Teifke et al.234

Clarke, I. N., Estes, M. K., Matson, D. O., Nakata,et al., 2001). Hepatic diseases leading to severeS., Neill, J. D., Studdert, M. J. and Thiel, H.-J. (2000).tissue necrosis stimulate fibrinogen synthesis andTaxonomy of Caliciviruses. Journal of Infectious Diseases,release massive amounts of tissue thromboplastins181 (Suppl. 2), 322–330.(Park et al., 1997). The defective clearance of ac- Kimura, T., Mitsui, I., Okada, Y., Furuya, T., Ochiai,

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� Received, May 21st, 2001

Accepted, October 14th, 2001�Methods, 72, 219–226.Green, K. Y., Ando, T., Balayan, M. S., Berke, T.,