animals Brief Report A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit Fábio A. Abade dos Santos 1,2,3,* , Carolina Magro 4, Carina L. Carvalho 2, Pedro Ruivo 5, Margarida D. Duarte 1,2 and Maria C. Peleteiro 1 1 Centre for Interdisciplinary Research in Animal Health (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; [email protected] (M.D.D.); [email protected] (M.C.P.) 2 Instituto Nacional de Investigação Agrária e Veterinária (INIAV, I.P.), Av. da República, Quinta do Marquês, 2780-157 Oeiras, Portugal; [email protected] 3 Instituto Universitario de Biotecnología de Asturias (IUBA), Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33006 Oviedo, Spain 4 VetOeiras, Hospital Médico-Veterinário, Estrada de Oeiras n18-20, 2780-114 Oeiras, Portugal; [email protected] 5 Instituto de Medicina Molecular João Lobo Antunes (IMM), Faculdade de Medicina, Universidade de Lisboa, 1070-312 Lisbon, Portugal; [email protected] * Correspondence: [email protected] Simple Summary: We report an unusual clinical case in a pet rabbit vaccinated against rabbit haemorrhagic disease virus (RHDV, GI.1), that developed a prolonged hepatic disease, and was diagnosed RHDV2 (GI.2) positive post-mortem. This finding is a warning to all veterinarians that rabbit haemorrhagic disease should also be considered for differential diagnosis despite the history of RHDV vaccination and the need to update vaccination programs against the current RHDV2 circulating strains. Abstract: Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European Citation: Abade dos Santos, F.A.; wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Magro, C.; Carvalho, C.L.; Ruivo, P.; Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised Duarte, M.D.; Peleteiro, M.C. A over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the Potential Atypical Case of Rabbit previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. Haemorrhagic Disease in a Dwarf RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of Rabbit. Animals 2021, 11, 40. RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. https://dx.doi.org/10.3390/ Although this poses a threat to the already endangered European rabbit species, the available vaccines ani11010040 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, Received: 26 October 2020 although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via Accepted: 21 December 2020 insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) Published: 28 December 2020 or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV Publisher’s Note: MDPI stays neu- through a commercially available bivalent vaccine with RHDV antigen has been extensively used ® tral with regard to jurisdictional claims (Nobivac Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper in published maps and institutional protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we affiliations. 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 Copyright: © 2020 by the authors. Li- non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the censee MDPI, Basel, Switzerland. This article is an open access article distributed etiological agent was only made after death, the clinical signs and analytic data were very suggestive under the terms and conditions of the of RHD. Creative Commons Attribution (CC BY) license (https://creativecommons.org/ Keywords: European rabbit; Oryctolagus cuniculus; pet rabbit; rabbit haemorrhagic disease; licenses/by/4.0/). atypical clinical course; subacute Animals 2021, 11, 40. https://dx.doi.org/10.3390/ani11010040 https://www.mdpi.com/journal/animals Animals 2020, 10, x 2 of 12 Animals 2021, 11, 40 2 of 12 Keywords: European rabbit; Oryctolagus cuniculus; pet rabbit; rabbit haemorrhagic disease; atypical clinical course; subacute 1. Introduction Introduction Originating in the Iberian Peninsula [1], [1], the European rabbit was widely introduced firstfirst in Europe, and subsequently in all other continents except Antarctica. The European domestic rabbit hashas beenbeen usedused asas an an important important meat meat and and fur fur resource. resource. Artificial Artificial selection selection of ofsmall small breeds breeds of thisof this species species (Oryctolagus (Oryctolagus cuniculus cuniculus), weighing), weighing less less than than 2 kg, 2 kg, also also generated gener- atedan increasingly an increasingly popular popular pet-rabbit pet-rabbit trade. trade. Pet rabbits Pet rabbits can becan affected be affected by the by same the same diseases dis- easesfound found in the in rabbit the rabbit industry industry or wild or wild rabbit ra populations.bbit populations. However, Howeve ther, indoorthe indoor lifestyle life- styledecreases decreases the risk the of risk contact of contact with commonwith common rabbit rabbit diseases diseases such as such myxomatosis as myxomatosis and rabbit and rabbithaemorrhagic haemorrhagic disease disease (RHD). (RHD). RHD is a highly contagious infectious diseas diseasee caused by a virus from the Caliciviridae family, genus Lagovirus, which targets hepatocytes and cells of the mononuclear phagocytic system (e.g., Kuppfer cells and alveolar macropha macrophages)ges) [[2,3].2,3]. The disease was first first identifiedidentified inin 1984 in China and quickly spread to other continents,continents, evolving into different genotypes (GI.1a (RHDVa (RHDVa or or G6), G6), GI.1b GI.1b (G1), (G1), GI.c GI.c (G2) (G2) and and GI.1d GI.1d (G3–G5)) (G3–G5)) [4,5]. [4, 5Along]. Along with with myxoma- myxo- tosis,matosis, this this disease disease led ledto an to abrupt an abrupt decrease decrease in the in theEuropean European rabbit rabbit wild wild populations populations [6]. [6]. InIn 2010, a distinct virus (both genetic and antigenically) related to RHDV emerged in France [7]. [7]. This This new new virus, virus, referred referred to as RHDV2 or RHDVb, induced a disease very similar toto that that caused caused by by RHDV RHDV strains strains and and it it became became also also known known as as LagovirusLagovirus europaeus europaeus GI.2GI.2 [5]. [5 ]. More recently, natural recombinant RHDV2 strains were identifiedidentified containing the structural proteins of RHDV2 (VP60 and mi minornor protein-encoding genes), genes), but but the the non- non- structural proteins from either non-pathogenic calicivirus (NP-CV GI.4 and GI.3)). Fur- Fur- thermore, the the non-structural proteins proteins from from th thee RHDV strains (GI.1b) were also found in RHDV2 recombinants [8]. [8]. Figure 11 representsrepresents thethe genomicgenomic organizationorganization of of RHDV2 RHDV2 RNA. RNA. Figure 1. SimplifiedSimplified schematic representation ofof thethe genomicgenomic RNARNA ofof RHDV2.RHDV2. NucleotideNucleotide positionsposi- tionswere were calculated calculated from from sequence sequence KF442964.2. KF442964.2. The The genome genome includes includes two two ORFs. ORFs. ORF1ORF1 encodes a a polyprotein that that is is cleaved cleaved to to form form the the non-structur non-structuralal proteins proteins p16, p16, p23, p23, the thehelicase, helicase, p29, p29,VPg, VPg, a proteasea protease and and the the viral viral polymerase, polymerase, and and the the major major structural structural protein protein VP60. VP60. ORF ORF 2 2encodes encodes a aminor minor structural protein VP10. Different clinicalclinical presentationspresentations and and disease disease progression progression can can occur occur with with RHDV2 RHDV2[9– 12[9–] 12]and and those those variations variations can becan associated be associated with strain.with strain. The incubation The incubation period period of RHD of ranges RHD rangesbetween between 1 and 1 3 and days. 3 days. In peracute In peracute forms, forms, sudden sudden death death may may occur occur within within 1212 hours after infection. In In acute acute and and subacute subacute forms forms of of RHD, RHD, different different clinical clinical signs signs can can be be ob- ob- served, suchsuch asas anorexia, anorexia, mucosal mucosal congestion, congestion, neurologic neurologic signs, signs, cyanosis, cyanosis, dyspnoea, dyspnoea, foamy foamyhaemorrhagic haemorrhagic epistaxis, epistaxis, ocular haemorrhageocular haemorrhage and others and [ 13others]. In chronically [13]. In chronically infected rab-in- fectedbits, the rabbits, onset the of anorexia,onset of anorexia, lethargy lethargy and jaundice and jaundice usually usually precedes precedes death indeath about in about 1 to 3 1weeks to 3 weeks