A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit

Abstract

Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac^® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.

Keywords: European rabbit; Oryctolagus cuniculus; atypical clinical course; pet rabbit; rabbit haemorrhagic disease; subacute.

Figure 1

Simplified schematic representation of the genomic RNA of RHDV2. Nucleotide positions were calculated from sequence KF442964.2. The genome includes two ORFs. ORF1 encodes a polyprotein that is cleaved to form the non-structural proteins p16, p23, the helicase, p29, VPg, a protease and the viral polymerase, and the major structural protein VP60. ORF 2 encodes a minor structural protein VP10.

Figure 2

Ultrasound is compatible with hepatitis. The image on the left shows peri-lobular mesenteric reactivity (arrow). On the right, free fluid in the abdomen is visible (arrow).

Figure 3

Liver. Severe haemorrhagic necrosis consistently affects perilobular areas or acinar zone 1, conspicuous due to the bright red colour close to the portal areas (H&E, 40×).

Figure 4

Liver. Apart from perilobular haemorrhagic necrosis, single-cell necrosis is present in dispersed hepatocytes in acinar zones 2 and 3, which surround the periacinar vein in the upper right. Inset-magnification of the acinar zones 2 and 3, showing various cells with the fragmentation of the nucleus-karyorrhexis (arrows) (H&E, 100×, inset 400×).

Figure 5

Spleen. Severe diffuse fibrinoid necrosis of the red pulp. The eosinophilic material fibrinoid material is very abundant between the lymphoid tissue that surrounds blood vessels (H&E, 40×).

Figure 6

Duodenum. Necrotic enteritis affecting the upper mucosa. Note the absence of villi and the eosinophilic deposits of fibrin in the upper mucosa. The intestinal glands are disorganized and the inflammatory infiltrate of the mucosal lamina propria by mononuclear cells (lymphocytes mostly) is scant (H&E, 100×).

Figure 7

Phylogenetic analysis. (A) Maximum likelihood analysis using 14 RdRp gene partial (436 nt) nucleotide sequences, namely GI.4 (NP1–non-pathogenic caliciviruses similar to RCV-A1 are the most likely donors of non-structural proteins) representatives (a-MG763946, MG763944, MG763954 and a’-MG763952), GI.1b (G1) representatives (c-MG763939, MG763938, MG763947 and MG763953), GI.3 (NP2-non-pathogenic caliciviruses similar to CBAnd1 are the most likely donors of non-structural proteins) representatives (e-MG763942, MG763949, MG763943 and MG763945; GI.1d (G3-G5) representatives (d-MH190418) and non-pathogenic rabbit calicivirus Australia 1 (RCV-A1) representatives (b-EU871528). (B) Maximum likelihood analysis using 14 complete VP60 gene nucleotide sequences including, GI.1b representatives (a-MG763939 and MG763938 and a’-MG763953 and MG763947), GI.4 (NP1) representatives (b-MG763952 and MG763954 and b’-MG763946 and MG763944), GI.3 (NP2) representatives (c-MG763949, MG763942, MG763945 and MG763943; GI.1d representatives (d-MH190418) and RCVA-A1 representatives (e-EU871528). (C) Maximum likelihood analysis using 14 sequences comprising the terminus of the RdRp gene and the complete VP60 gene, namely GI.1b representatives (a-MG763938 and MG763939, a’-MG763947, and a’’-MG763953), GI.3 (NP2) representatives (c-MG763949, MG763942, MG763945 and MG763943), GI.4 (NP1) representatives (d-MG763954, MG763946, MG763944 and d’-MG763952) GI.1d representatives (b-MH190418) and RCVA1 representatives (e-EU871528). The nomenclature used is in accordance with Silvério et al., 2018 [8], Abrantes et al., 2020 [31]. Designations only used by Silvério et al., 2018 are underlined in the phylogenetic trees.