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1 Dutch Wildlife Health Centre, based at Department of Virology, Erasmus Medical Centre, PO Box 1738, 3000 DR Rotterdam, The Netherlands and Faculty of Veterinary Medicine, University of Utrecht, The Netherlands;
2 Vereniging Natuurmonumenten, Noordereinde 60, 1234 JJ ’s–graveland, The Netherlands;
3 Community and Conservation Ecology, University Groningen, PO Box 14, 9750 Haren, The Netherlands
4 Corresponding author (email: t.kuiken{at}erasmusmc.nl)
ABSTRACT:
The number of free-living European rabbits (Oryctolagus cuniculus) in the Netherlands has declined dramatically in recent years. Although rabbit hemorrhagic disease virus (RHDV) infection has been implicated as a possible cause of this decline, the definitive diagnosis has not been reported. We examined three free-living rabbits found dead in the Netherlands in 2004 by use of gross pathology, histopathology, immunohistochemistry, and reverse transcriptase polymerase chain reaction. We subsequently compared the identified virus with RHDV from elsewhere in the world by phylogenetic analysis. There was widespread necrosis, hemorrhage, or both in liver, kidney, spleen, and lungs of all three rabbits, consistent with RHDV infection. The presence of RHDV in affected tissues was demonstrated by immunohistochemistry and reverse transcriptase polymerase chain reaction. The RHDV from the Netherlands showed the highest identity, 99%, with a strain from France in 2000, and fitted in genogroup G5. These results prove that RHDV infection causes mortality of free-living rabbits in the Netherlands and suggest that RHDV strains circulating in free-living rabbits in the Netherlands and France have a common source or that one has originated from the other.
Key words: Calicivirus, Oryctolagus cuniculus, phylogeny, rabbit, RHDV.
The number of free-living European rabbits (Oryctolagus cuniculus) in the Netherlands has declined dramatically in recent years (Bijlsma, 2004; Drees and Van Maanen, 2005). The reasons for this are unclear. Possible explanations include habitat loss, a series of severe winters, the emergence of an infectious disease such as rabbit hemorrhagic disease (RHD), or a combination of these factors. The rabbit used to be very common on the sandy soils of the Netherlands and especially on the coastal dunes. However, rabbit numbers on coastal dunes declined between 10% and 30% per year since 1990, resulting in an overall decrease of 90% between 1990 and 2003. Similar declines have been recorded for rabbit numbers from inland habitats (Bijlsma, 2004; Drees and Van Maanen, 2005).
Although implicated as a possible cause of the above decline, the definitive diagnosis of RHD has not been reported in free-living rabbits from the Netherlands. Rabbit hemorrhagic disease has spread rapidly throughout Europe and other continents since its emergence in 1984 in China (Liu et al., 1984), and is now endemic in most parts of the world. In Europe, the spread of RHD raised serious concerns, because the rabbit is important both as an element of the ecosystem and as a game species (Cooke, 2002). Rabbit hemorrhagic disease in free-living rabbits in the Netherlands was first suspected in 1990, when unusual mortality of free-living rabbits, some with blood-stained nasal discharge, was reported (Siebenga, 1991). At that time, RHD was diagnosed in nine of 10 rabbits by postmortem examination, but the methods were not stated (Siebenga, 1991). Subsequently, antibodies to RHDV were detected in 12% of sera from 33 free-living rabbits, collected from different regions in the Netherlands in 1991 and 1992 (Van Maanen et al., 1996). However, serology does not distinguish between RHDV and other, non-pathogenic caliciviruses (Capucci et al., 1996; White et al., 2002).
In November 2004, three carcasses of free-ranging rabbits found in the dunes of the island Goeree–Overflakkee in Zeeland Province, The Netherlands, were presented for necropsy to the Dutch Wildlife Health Centre. They were the only freshly dead rabbit carcasses found on the island. No mortality of rabbits was noted on the neighboring islands Voorne and Schouwen-Duiveland. The rabbits had probably died less then 24 hr before they were found, based on the stiffness of the carcasses. The carcasses of these adult rabbits were mildly to moderately auto-lyzed and in a moderate to good nutritional condition. Detailed postmortem examinations were performed on the carcasses and samples were taken for routine histology, as well as for immunohistochemistry and RT-PCR.
For immunohistological detection of RHDV antigen, formalin-fixed, paraffin-embedded tissue sections were deparaffinized and pre-treated with 0.1% protease (Sigma, St. Louis, Missouri, USA) for 10 min at 37 C. Guinea pig serum against RHDV (a kind gift from Per Have, Danish Institute for Food and Veterinary Research) was incubated for 1 hr at room temperature and detected with a peroxidase-labeled rabbit-anti-guinea pig immunoglobulin (DAKO, Glastrup, Denmark). Peroxidase was revealed with 3-amino-9-ethyl-carbazole (Sigma) resulting in a bright red precipitate. A negative guinea pig serum and omission of serum were included as negative controls for all tissues. Tissues were counterstained with hematoxylin and embedded in glycerol–gelatin. The following tissues were examined by this method: heart, jejunum, kidney, liver, lung, spleen, and trachea of all three rabbits, adrenal gland, cecum, colon, lymph node, skeletal muscle, skin, and stomach of two rabbits, and abdominal wall, duodenum, esophagus, ovary, urinary bladder, testis, and thyroid of one rabbit.
From all three rabbits, samples of kidney, liver, lung and spleen as well as the bladder and jejunum from one rabbit and the bladder or jejunum from the other two rabbits were preserved in lysis buffer for RNA isolation. Total nucleic acids were isolated using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany). Reverse transcriptase polymerase chain reaction was performed in a one-tube system, using primers REF: 5' CAACCTCCAGCC-CACCACCAACAC 3' and REB: 5' TGGTTGGGAGCCTGTGCCGTACTG 3' as described previously (Bascuñana et al., 1997), which amplify a 316-base pair fragment of the VP60 gene located at nucleotide positions 6482 to 6782. Automated nucleotide sequencing of PCR fragments was performed on an ABI 3130XL genetic analyser with the Big Dye terminator cycle sequencing kit (ABI, Applied Biosystems, Foster City, California, USA) using the same primers as above. The resultant nucleotide sequences were compared with published sequences of RHDV by use of the DNAstar software package (DNASTAR, Inc., Madison, Wisconsin, USA) to confirm the identity of the RT-PCR product. For phylogenetic analysis, the 316-base pair fragment of the VP60 gene of RT-PCR products obtained in this study were compared with sequences from the Genbank database representing the different genotypes using the Phylip package (Felsenstein, 2004).
The main gross changes were seen in lungs, liver, kidneys, and spleen of all three rabbits. The lungs had multiple or coalescing, dark red, firm areas, which were only partly collapsed. The liver was diffusely pale, slightly swollen, and—in one rabbit—had an enhanced lobular pattern. The kidneys were diffusely dark red to red-black. The spleen was diffusely dark red and mildly to moderately swollen. In all three rabbits, the stomach was filled with grass.
The main histological lesions in all three rabbits were found in the liver, kidney, spleen, and lung. In the liver (Fig. 1A
), there was widespread hepatocytic necrosis, often sparing the perivenular areas (zone 3). Affected hepatocytes were separated from the hepatic plates, were rounded, and had hypereosinophilic cytoplasm and fragmented or pyknotic nuclei. In the kidney, there was generalized global necrosis and thrombosis of glomeruli, characterized by pyknosis of glomerular cells and the presence of fibrin thrombi in congested glomerular capillaries. One of three rabbits also had multiple foci of cortical tubular hemorrhage. In the spleen, there was diffuse necrosis. The red pulp of the spleen was diffusely replaced by abundant fibrin mixed with a few reticular cells, erythrocytes, and cell debris. The white pulp had marked loss of lymphocytes, and contained moderate numbers of necrotic lymphocytes and cell debris. In the lung, multifocal pulmonary hemorrhage and edema were found, characterized by flooding of the alveolar lumina with serous fluid, erythrocytes, or a mixture of both. Minor histologic lesions in other tissues were few small foci of myocardial hemorrhage in the right ventricle of one of three rabbits, and mild to moderate congestion of the tracheal sub-mucosa in two of three rabbits. No significant histologic lesions were detected in the other tissues examined.
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) and kidney of all three rabbits, in the spleen of two rabbits, and in the ovaries of one rabbit. Cells expressing viral antigen included hepatocytes, glomerular cells, cortical tubular cells, large mononuclear or dendritic cells in the spleen, and ovarian stromal cells. There was specific staining in blood vessel lumina of multiple tissues, but this was not cell-bound. In liver, kidney, and spleen, RHDV antigen expression was closely associated with the presence of histologic lesions. Expression of RHDV was absent in both isotype and omission controls. The acrosomal vesicles of spermatids in the testis of one rabbit showed nonspecific light brown staining (not shown).
By RT-PCR, all samples (n = 16) but one, the jejunum of rabbit number two, showed a clear band of approximately the right size upon analysis on a 2% agarose gel. Nucleotide sequencing of PCR fragments confirmed the presence of RHDV in all three rabbits. Comparison of the 316-base pair fragment of the VP60 gene with sequences from the Genbank database showed the highest identity of 99% with a French strain from the year 2000, isolate 00–13 (Le Gall-Reculé et al., 2003). The sequences derived from the Dutch rabbits formed a cluster with the genogroup G5 sequences according to the genotypic characterization of Le Gall-Reculé and others (Le Gall-Reculé et al., 2003) (Fig. 2).
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). This suggests that the viruses from these two areas have a common source or that the one originated from the other. Possible routes of RHDV transmission between these two areas include: 1) direct transmission among contiguous populations of free-living rabbits, 2) transmission by flying insects (McColl et al., 2002), and 3) transport by humans of infected rabbits, infected insects, or contaminated fomites (Cooke, 2002). The virus is extremely resistant and remains viable in carcasses in the field for at least three months (Henning et al., 2005). We gratefully acknowledge Jolianne Rijks, Fiona Read, and Frank van der Panne for their excellent technical assistance.
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Received for publication 3 January 2006.
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