The first reported case of rabbit hemorrhagic disease in Canada

Abstract

In March 2011, rabbit hemorrhagic disease (RHD) was suspected in a 1-year-old male neutered lop-eared rabbit that had acute onset liver failure. Gross pathology, histopathology, immunohistochemistry, partial nucleic acid sequencing and phylogenetic analysis of the major capsid protein (VP60) and animal inoculation studies all supported this diagnosis making it the first confirmed case of RHD in Canada.

Introduction

Rabbit hemorrhagic disease (RHD) is a highly contagious and acutely fatal disease of the European rabbit (Oryctolagus cuniculus) caused by rabbit hemorrhagic disease virus (RHDV), a member of the family Caliciviridae, genus Lagovirus. The disease is characterized by liver necrosis, splenomegaly, and hemorrhagic lesions affecting the liver and lung. Clinical signs appear after an incubation period of 1 to 3 d and typically include pyrexia (> 40°C), anorexia, dullness, prostration, nervous signs, vocalization, dyspnea, and cyanosis of the mucous membranes (1). Death typically occurs within 12 to 36 h after the onset of fever. The European rabbit is the only species susceptible to RHD, but animals less than 2 mo of age are unaffected. The first reports of RHD were from the People’s Republic of China in 1984 (2) but outbreaks have subsequently been reported in over 40 countries including the USA.

Based on reports obtained from the World Animal Health Information Database (WAHID) that interfaces with the OIE (Office international des épizooities — World Organization for Animal Health) Web site, the most recent outbreaks of RHD in the USA occurred in 2008 and 2010. These both involved single premises; a private residence involving 4 mature pet rabbits in Maryland in 2008, and a private residence that received donated pet rabbits for use as a food source at a wildlife center in Minnesota in 2010. Prior to March 2011, RHD was never reported in Canada.

On March 31, 2011 a 1-year-old grey and white neutered male 2.6-kg lop-eared rabbit was submitted for necropsy to the Manitoba Veterinary Diagnostic Services Laboratory. This was a rescue animal of unknown origin that was a pet in a home with 2 other unaffected rabbits.

Sudden onset of liver failure was suspected by the referring veterinarian. The rabbit was recumbent, jaundiced, with nasal discharge, respiratory difficulty, and yellow serum observed after blood collection. The rabbit seizured and went into cardiac arrest with unsuccessful cardiopulmonary resuscitation (CPR).

On gross examination the rabbit was in excellent body condition with ingesta in the stomach and focal petechial hemorrhages on the mucosal surface. The ocular conjunctiva was mildly icteric. There was bilateral suppurative otitis media. At the jugular venipuncture site there was focal hemorrhage and a small blood clot. Bilaterally there were multiple red pitted foci within the renal cortical surfaces. The lungs were diffusely moderately congested. The liver was diffusely pale tan yellow. Various tissue specimens were fixed in 10% formalin solution, paraffin-embedded, stained with hematoxylin and eosin (H&E) and examined by light microscopy.

The most significant histological lesion was observed in the liver, and consisted of acute diffuse periportal to midzonal necrosis and moderate multifocal periportal nonsuppurative hepatitis. Other lesions, including lymphoplasmacytic meningoencephalitis of the brain and interstitial lymphoplasmacytic nephritis in the kidneys, were consistent with Encephalitozoon cuniculi infection. Bacteriology and special stains on the liver were negative for known infectious bacteriological agents. With no history of exposure to hepatic toxins, frozen liver was submitted to the Canadian Food Inspection Agency National Center Foreign Animal Disease to rule out rabbit hemorrhagic disease.

Materials and methods

Nucleic acid extraction and RT-PCR

Total RNA was extracted from fresh liver and whole blood. For fresh liver, ~1 g was homogenized in 9 mL of sterile phosphate buffered saline (PBS) using a closed tissue grinder system (Biomedical Polymers, Gardner, Massachusetts, USA). The homogenate was centrifuged for 15 min at 2500 × g, 100 μL of the clarified homogenate was added to 900 μL TriPure Reagent (Roche Canada, Laval, Quebec) and then mixed by vortexing. A 200 μL volume of chloroform was added, the mixture was vortexed for 15 s, and then incubated for 10 min at room temperature. Aqueous and organic phases were then separated by centrifuging for 10 min at 10 000 × g. The upper aqueous phase was transferred to an RNeasy spin column (Qiagen, Mississauga, Ontario) and the membrane-bound RNA was washed with buffers RW1 and RPE, following the manufacturer’s guidelines (RNeasy Mini Handbook, Qiagen). The RNA was eluted from the membrane with 50 μL of sterile nuclease-free water. In the case of whole blood, 100 μL was mixed with 900 μL of TriPure Reagent with subsequent steps the same as for fresh liver.

A highly conserved 316 nucleotide region of the gene encoding the VP60 capsid protein of RHDV (amino acids 386–491) was amplified in a 1-step RT-PCR assay using a modification of the method described by Basuňana et al (3). Briefly, RT-PCR was performed using a One-Step RT-PCR Kit (Qiagen) with 2 μM each of primers REF (5′-CAACCTCCAGCCCACCACCAACAC-3′) and REB (5′-TGGTTGGGAGCCTGTGCCGTACTG-3′) using the following cycling conditions: 50°C for 30 min; 95°C for 15 min; 35 cycles of 94°C for 45 s, 60°C for 45 s, 72°C for 1 min; and a final extension at 72°C for 7 min. RT-PCR products were resolved on a 2% agarose gel.

Nucleotide sequencing and phylogenetic analysis

RT-PCR reaction products were purified, using a QIAquick PCR Purification Kit (Qiagen) and ~10 ng of product were used in cycle sequencing reactions employing BigDye Terminator chemistry (Applied Biosystems, Foster City, California, USA). Cycle sequencing products were run on an Applied Biosystems 3130xl Genetic Analyzer. Similar sequences in GenBank were detected by BLAST analysis (4). Sequences were aligned with Clustal W (MEGA 4.0.2) and the phylogeny inferred by the maximum parsimony method utilizing the close-neighbor interchange method and 500 bootstrap replicates (5).

Animal inoculations

Five 9- to 10-week-old, female, New Zealand White (NZW) rabbits (Charles River, Saint Constant, Quebec) were acclimated for 1 wk prior to inoculation. Three rabbits housed in a BSL3 animal containment cubicle were inoculated intramuscularly with 1 mL of 10% liver homogenate obtained from the index case. One rabbit housed in a separate BSL3 animal containment cubicle was inoculated intramuscularly with 1 mL of whole blood from a rabbit that was experimentally infected with RHDV 2719 Korea. This rabbit served as a positive control. One rabbit housed in a separate BSL2 animal area was inoculated intramuscularly with 1 mL of tissue culture medium and served as a negative control. All animal manipulations were in compliance with Canadian Council for Animal Care guidelines and the Institutional Animal Care Committee.

Rabbits were monitored twice daily for clinical signs and when possible, humanely euthanized by intravenous pentobarbital injection when moribund. Complete necropsies were performed and tissues harvested for RT-PCR, electron microscopy, histopathology, and immunohistochemistry.

Electron microscopy

Frozen 10% homogenate of rabbit liver was thawed and clarified by centrifuging at 2000 × g for 10 min. A 20-μL volume of the supernatant was adsorbed to a carbon-coated formvar film on a 400-mesh copper grid for 1 min. The grid was negatively contrasted with 2% phosphotungstic acid (SPi Supplies, West Chester, Pennsylvania, USA). The specimen was imaged in a Philips CM-120 transmission electron microscope (TEM) operated at 80 kV, and at nominal instrument magnification of 60 000. Digital images of the specimen were acquired by an AMT XR-611-M CCD camera (AMT, Woburn, Massachusetts, USA).

Histopathology and immunohistochemistry

Tissues were fixed in 10% neutral phosphate buffered formalin, routinely processed and stained with H&E for histopathologic examination. For immunohistochemistry, paraffin tissue sections were quenched for 10 min in aqueous 3% hydrogen peroxide, followed by pretreatment with proteinase K for 10 min. Primary antibody was a mouse monoclonal antibody specific for RHDV (6F9, Istituto Zooprofilattico Sperimentale Della Lombardia E Dell’Emilia, Italy) and was used at a 1:250 dilution for 1 h. The sections were treated with a horseradish peroxidase labeled polymer, Envision + system (anti-mouse) (Dako, Carpinteria, California, USA) and the chromogen diaminobenzidine (DAB) then counterstained with Gill’s hematoxylin.

Serology

Inhibition enzyme-linked immunoborbent assay (ELISA) was performed between specific antibodies bound to the solid phase and antibodies in the liquid phase from sera, as described by Capucci et al (6). Briefly, Maxisorb plates (Nunc) were coated overnight at 4°C with 50 μL of anti-RHDV polyclonal serum at 1/500 dilution in 0.05 M carbonate buffer (pH 9.6). The wells were washed 3 times for 3 to 5 min each time using PBS with 0.05% Tween 20 (PBST).

Samples and controls were diluted in a non-absorptive plate in duplicate, starting at a 1/10 dilution with subsequent doubling dilutions across the plate in PBST containing 1% Bovine Serum Albumin (BSA). Twenty-five microliters of each dilution were then transferred to the washed ELISA plate and 25 μL of RHDV antigen at 1/300 added to each well. The ELISA plates were incubated at 37°C for 1 h with gentle mixing. Following incubation the plates were washed as described, 50 μL of horseradish-peroxidase conjugated mAb were added at a dilution of 1/500 in PBST and again the plate was incubated at 37°C for 1 h with gentle mixing. After washing as described and 1 additional wash (total 4 washes), 50 μL/well of O-phenylenediamine dihydrochloride (OPD) substrate in 0.15 M citrate phosphate buffer and 0.02% H₂O₂ was used for color development and the reaction was halted after 5 min by the addition of 50 μL/well of 1 M H₂SO₄. A serum sample was considered negative when the A₄₉₂ value of the first dilution (1/10) decreased by less than 15% of the reference value (negative serum at 1/10), while it was positive when it decreased by more then 25%. Intermediate values were considered suspicious. The titer of specific antibodies in the serum was calculated from the dilution that reduced the A₄₉₂ by 50% compared with the negative control.

Epidemiological investigation

An epidemiological investigation of the index premises, referring veterinary clinic, Manitoba Veterinary Diagnostic Services Laboratory, and frequented supply and feed sources was carried out between April 15 and April 18, 2011.

Results

The RT-PCR with total RNA extracted from the 10% liver homogenate from the suspect case resulted in a 316 bp amplicon, as did the total RNA from control whole blood from a rabbit experimentally infected with RHDV Korea 2719. The positive PCR results were supported by electron microscopic examination of the 10% liver homogenate; aggregates of ~30 nm particles with morphologic characteristics of calicivi-ruses were observed (Figure 1). In areas in which hepatic necrosis occurred, RHDV antigen was detected by immunohistochemistry within the nucleus and cytoplasm of approximately 40% of hepatocytes (Figure 2). Sanger sequencing was performed on the RT-PCR amplicons obtained from the liver sample and the positive control. Closest match of the sequence obtained from the liver samples was with the capsid protein VP60 gene of RHDV isolate YL from China (GenBank accession number DQ530363.1; 100% coverage; 0 gaps; 98% identity). Closest match of the RHDV Korea 2719 control sequence was with the capsid protein VP60 genes of RHDV isolates Korea 90 and Korea 93 (GenBank accession numbers U65341.1 and U65342.1; 99% coverage; 0 gaps; 99% identity). By comparison, only 93% identity was observed between the suspect liver and positive control sequences. Based on the 316 nucleotides of the VP60 gene, the evolutionary relationships between this Canadian RHDV isolate, the positive Korea 2719 control along with other isolates was inferred by using the maximum parsimony method (Figure 3).

Figure 1.

Transmission electron micrograph of 10% homogenate of liver sample showing ~30 nm particles with morphologic characteristics of calicivirus virions. These characteristics include nonenveloped particles with icosahedral symmetry that are 27 to 40 nm in diameter by negative stain electron microscopy.

Figure 2.

A — Nonsuppurative periportal hepatic necrosis. H&E stain, bar = 50 μm. B — Positive immunostaining of the liver for RHDV antigen was extensive and followed the pattern of necrosis. Bar = 200 μm.

Figure 3.

Phylogenetic analysis of RHDV based on a 316 nucleotide segment of the capsid protein gene. The tree was generated with MEGA software using the maximum parsimony method and 500 bootstrap replicates. The scale indicates the number of nucleotide changes.

Nonpathogenic rabbit caliciviruses have been reported in Europe (6) and the USA (7); the latter, designated as Michigan rabbit calicivirus (MRCV), was associated with subclinical infections in Oryctolagus cuniculi. This MRCV could be detected in tissues of experimentally infected rabbits by RT-PCR and immunohistochemistry, but was not associated with clinical disease. Consequently, the virulence of the virus that we isolated from rabbit liver for Oryctolagus cuniculi was evaluated. Three NZW rabbits were each inoculated intramuscularly with 1 mL of clarified 10% liver homogenate that was derived from the suspect case. All 3 inoculated rabbits showed clinical signs of disease and were moribund within 24 to 48 hours.

Grossly, the livers of all rabbits were pale with a slightly granular surface (Figure 4). In 2 of the rabbits, the livers had a slightly accentuated acinar pattern. Viral RNA was detected by RT-PCR in 10% homogenates prepared from all 3 liver samples. Multifocal pulmonary edema and hemorrhage were observed in all inoculated animals (Figure 4). Individual rabbits variably showed multifocal renal cortical hemorrhage, gastric hemorrhage with ulceration, and petechial hemorrhages of the epicardium and thymus. The major histologic finding was multifocal to coalescing mid-zonal to periportal hepatocellular necrosis. In all 3 animals there was diffuse microthrombosis of renal glomerular capillaries, splenic congestion and multifocal pulmonary edema and hemorrhage with evidence of microthrombosis.

Figure 4.

Macroscopic lesions observed in a New Zealand White rabbit inoculated with the Canadian RHDV isolate at 3 d post-infection. Multifocal pulmonary hemorrhages were a consistent finding. The liver is pale with a slightly granular surface and there is a fine reticular pattern of necrosis.

Electron microscopic examination of the 10% liver homogenate from the inoculated animals showed aggregates of ~30 nm particles with morphologic characteristics of caliciviruses.

Epidemiological investigation of the index premises revealed that the affected rabbit was purchased from a local pet store in the spring of 2010. Two other rabbits living at the premises, a female white and tan long-haired acquired from the local humane society 2 years previously and a female lop-eared acquired through a relative 3 years before, were both in good health. All 3 rabbits shared a cage and litter box and were allowed to run free. None of the rabbits had left the premises during the previous 2 mo except when the affected animal became ill and was taken to the veterinary clinic. All supplies including feed and litter were purchased from a local pet store, although commercial rabbit pellets were purchased from a local veterinary clinic. The owners had not been outside of the country and the only visitors had been the owner’s mother who also had had recent travel history. The rabbit cage had been disinfected with bleach after the affected rabbit had died. On May 3, 2011, whole blood and serum were collected from the 2 remaining animals for testing. Investigation of the referring veterinary clinic revealed that all areas that had had contact with the affected rabbit were disinfected; Virkon was used in the examination room while Proxiguard was used in the treatment area. The clinic sees a moderate number of rabbits annually and a follow-up of all clients that had visited the practice since March 30, 2011 was carried out to ensure that all rabbits were still healthy. Follow-up investigation of the Manitoba Veterinary Diagnostic Services Laboratory confirmed that the laboratory was appropriately disinfected and that none of the laboratory employees who had had contact with the affected rabbit had rabbits at their residences. Feed purchased by the owners originated either from Canada or the United States with no evidence of origins from other countries. The pet store from which the affected rabbit was purchased was found to usually source all their young rabbits from a supplier in British Columbia. However, in the case of the affected rabbit, it was determined that it had been surrendered to the store by a previous owner; therefore, its origin could not be determined.

Blood that was collected from the surviving 2 rabbits tested negative for RHDV nucleic acid and antibody (Figure 5).

Figure 5.

Graphic presentation of inhibition ELISA results showing titration of positive control serum, negative control serum, and the sera collected from the 2 cohort rabbits.

Discussion

This is the first reported case of RHD in Canada. Clinical presentation and gross pathological lesions, supported by histopathology, immunohistochemistry, nucleic acid sequencing coupled with phylogenetic analysis, and animal inoculation studies clearly demonstrate that this is a pathogenic rabbit calicivirus and not one of the non-pathogenic rabbit caliciviruses that have been reported in Europe and most recently the USA. Furthermore, phylogenetic analysis of the capsid protein gene indicates that this RHDV isolate clusters with RHDVs that have been previously isolated from the USA and are related to a pandemic antigenic variant strain known as RHDVa (8).

Although the epidemiological investigation was not able to identify any potential conventional sources of infection, it is interesting to note that the most recent documented outbreak of RHD in the USA occurred in Minnesota in 2010. The geographic proximity of these 2 cases may be more than circumstantial. We can also speculate on some of the less conventional ways the virus may have been introduced in this unusual case. Reports of virulent RHDV establishing non-lethal infections in rabbits have been reported (9,10) in which viral RNA persists. This has led to the hypothesis that virulent RHDV is capable of establishing persistent or possibly latent infections in some rabbits. As noted in the introduction, rabbits < 2-months old are clinically unaffected. The role that this age related susceptibility may play in establishing persistent infections is not known. It is also not known whether or not reactivation of this persistent form of RNA is possible. Of particular interest in this case is that despite the 2 other rabbits in the household being in continuous close contact with the affected rabbit, there was no evidence of prior or concurrent illness in these animals, nor was there any PCR or serologic evidence indicating that they had been infected. One possible explanation for this is that the 2 surviving rabbits are not of the Oryctolagus cuniculus susceptible species. Alternatively, the affected rabbit may not have shed virus into the environment.

Although this case presents with many unanswered questions it is still important for Canadian veterinary practitioners to be aware of the clinical and pathological presentation of RHD and consider it as a rule out in cases involving sudden death.