European brown hare syndrome (EBHS) is a highly contagious and fatal viral disease mainly affecting European brown hares (Lepus europaeus) (Frölich and Lavazza, 2007; Capucci et al., 2021; Fitzner et al., 2022). The disease was known to occur in brown hares since the early 1980s and was diagnosed in free-ranging and farmed hare populations across Europe, including Italy, where it is endemic (Lavazza and Vecchi, 1989; Gavier-Widén and Mörner, 1991; Poli et al., 1991; Duff et al., 1994; Scicluna et al., 1994; Chiari et al., 2014). EBHS generally occurs in an acute form with a high mortality rate, between 90% to 100% in adult animals of naïve populations, strongly impacting on the population dynamics (Wirblich et al., 1994). Acutely affected hares often die suddenly of hepatic failure and necrosis without overt clinical signs (Gavier-Widén and Mörner, 1991; Zanni et al., 1993; Lavazza and Neimanis, 2021; World Organisation for Animal Health (WOAH), 2021). Diffuse hemorrhagic lesions and liver degeneration characterize the acute form of the disease. Hares younger than 2–3 months old are considered resistant to EBHS as they do not manifest clinical signs and mortality, but develop antibodies (Zanni et al., 1993; Edwards et al., 2000; Lavazza and Neimanis, 2021). In endemic areas, virus circulation is correlated to hare population density, animal age and season (Scicluna et al., 1994; Paci et al., 2011; Chiari et al., 2014). Where EBHS is endemic, mortality rates are usually lower due to pre-existing population immunity (World Organisation for Animal Health (WOAH), 2021).

EBHS is transmitted by the oral-faecal route through direct or indirect contact with infected sources. Carnivores, insects, birds, and humans can facilitate virus spread, and insects can act as mechanical vectors, but no reservoir hosts other than lagomorphs have been definitively identified. Indirect transmission occurs through contaminated fomites, including equipment, cages, clothes, vehicles, and utensils, likely occur. Infection via contaminated green forage and vegetation is also possible, and lagomorph carcasses are believed to contribute to the environmental persistence of the virus (World Organisation for Animal Health (WOAH), 2021).

The etiological agent, EBHS virus (EBHSV), belongs to the Lagovirus genus of the Caliciviridae family and has a single-stranded RNA of positive polarity, approximately 7.5 kb in length (Capucci et al., 1991; Poli et al., 1991; Wirblich et al., 1994). The genome contains two open reading frames (ORFs): ORF1 encodes a polyprotein, which is processed by virus-encoded 3C-like protease (3CLpro) for the release of mature non-structural proteins and the capsid protein VP60; ORF2 encodes a minor structural protein (VP10) (Le Gall et al., 1996). Phylogenetically, two lineages are described: group A, which persisted until 1989, when it apparently disappeared; group B, which appeared in the mid-1980s and contains the most recently detected strains (Lopes et al., 2014). Previous studies showed a high genetic homogeneity of EBHSV strains from different European countries despite their geographical occurrence and time of discovery. These strains are phylogenetically closely related (over 97% homology), likely confirming the slow evolutionary dynamics of this lagovirus species (Fitzner et al., 2021).

The Italian hare (Lepus corsicanus) is endemic to Central-Southern Italy and Sicily and is classified as Vulnerable according to the International Union for Conservation of Nature in its Red List (Randi and Riga, 2019). Currently, the most conspicuous populations live in protected areas (Buglione et al., 2020). L. corsicanus was included among the lagomorph species susceptible to EBHSV (Lavazza and Neimanis, 2021; World Organisation for Animal Health (WOAH), 2022). Sporadic disease cases and serological evidence of specific antibodies were repeatedly reported in free-ranging L. corsicanus from Northern Italy (Guberti et al., 2000; Capucci, personal communication). Nevertheless, extensive genetic identification and characterization of the EBHSV strains detected from Italian hares and genetic species identification of the positive hares have yet to be performed. In addition, L. corsicanus is also susceptible to RHDV2 (Camarda et al., 2014), a lagovirus which can cause mortality and lesions overlapping those caused by EBHSV, therefore requiring a differential diagnosis.

The present study aims to describe an outbreak of EBHS involving two free-ranging L. corsicanus in a protected area of Central Italy and to investigate the viral phylogenetic relationships to reference EBHSV strains identified previously in Italy and Europe.

EBHSV was first detected in European brown hares (L. europaeus), and this should be considered the elective host species. However, it is less frequently found also in mountain hares (L. timidus) (Lavazza and Vecchi, 1989; Gavier-Widén and Mörner, 1991; Frölich and Lavazza, 2007). The same EBHSV strains can indistinctly infect and cause disease in different hare species, even living in the same areas (Frölich and Lavazza, 2007).

This study describes the molecular characterization of an EBHSV strain detected in two Italian hares (L. corsicanus). These results reinforce and complement the already known data regarding the susceptibility of L. corsicanus to infection with EBHSV (Guberti et al., 2000) and extend the case history reports of this fatal disease in this species. On the other hand, EBHS was never reported in other European hare species, such as the Iberian hare (L. granatensis) and the Broom hare (L. castroviejoi), both present mainly in the Iberian Peninsula and in the Cape hare (L. capensis var. mediterraneus), present in Sardinia (Italy) (Puggioni et al., 2013; Velarde et al., 2017). In addition, field and experimental data demonstrated that the eastern cottontail (Sylvilagus floridanus) is susceptible to infection with EBHSV (but not RHDV), occasionally causing EBHS-like disease. So far, infection of the eastern cottontail with EBHSV is considered a spillover event, as this species is deemed a dead-end host, unless new evidence is collected to support an active role in the epidemiology of EBHSV (Lavazza et al., 2015).

In our study, gross pathological and histopathological findings in the two hares investigated were consistent with those described by other authors as typical of lagovirus diseases, mainly characterized by hemorrhages and tracheal mucosa congestion, degeneration of hepatic tissue with necrosis, splenomegaly, lung and kidney congestion (Marcato et al., 1991; Zanni et al., 1993; Gavier-Widén, 1994; Scicluna et al., 1994; Velarde et al., 2017). The good body condition of both animals and the presence of food in the stomach suggest a peracute or acute form of infection. The isolation of bacteria (Streptococcus gallolyticus and Acinetobacter lwofii) from one of the two hares is probably attributable to secondary infections or post-mortem dissemination, while the detected parasites represent a normal finding in free-living hares. Taken together, acute death, gross and histopathological lesions, positive ELISA and PCR results, in the absence of other evident causes of death, demonstrate that EBHS is the most probable cause of death for both animals. Furthermore, the diagnostic approach used allowed to confirm the presence of a lagovirus in the organs examined, identifying it as EBHSV (EBHSV_Rm23), while ruling out the possibility that it could be RHDV2. This latter virus, which emerged in 2010, is also capable of infecting several other species of hares and other lagomorphs and was repeatedly identified in recent years in Europe, Australia, and the Americas (Puggioni et al., 2013; Camarda et al., 2014; Hall et al., 2017; Velarde et al., 2017; Neimanis et al., 2018; Asin et al., 2021).

The phylogenetic analysis conducted, based on the VP60 sequences, showed that the OR096234 strain belongs to group B, clustering with the GII.1 (EBHSV) genotype, showing 98.86% nucleotide identity and 100% amino acid identity with a strain isolated in Germany in 2019 (Szillat et al., 2020). These findings are consistent with previous VP60 sequence analyses of European EBHSV strains (Lopes et al., 2014; Fitzner et al., 2021), with group B representing the totality of the strains detected recently in Europe, while group A viruses, which encompasses the oldest strains from the Scandinavian Peninsula, disappeared in the late 1980s.When dealing with lagovirus diseases, it is always difficult to trace the origin of the infection, due to the multiplicity of ways, especially indirect, by which the virus is transmitted. Likely, we can rule out that the virus can “persist” in an environment without giving any sign of mortality for years. In fact, to date, the existence of healthy carrier status among lagomorphs or asymptomatic hosts was never demonstrated, as we exclude the cottontail species presence in the CPE, considered as potential spillover species. On the other hand, the virus can also be indirectly transmitted by animated subjects (e.g., predators, both mammals and birds, insects, and humans) and fomites. Considering that in 2023 in Central Italy cases of EBHS were numerous, both in captive and in wild animals, we can consider one of these indirect modes of transmission as the probable source of infection, explaining the epidemiology of this outbreak. Although the entire CPE is fenced, this is unlikely to act as a complete physical separation, considering the epidemiology of lagoviruses and the host species, able to jump over 1.80 meters. It is therefore not possible to prevent contact between the species inside the reserve and those outside, and the possible passage of any EBHSV infection in a context where susceptible species are present. This is precisely the situation that is currently observed in Central-Southern Italy, at least if we refer to the numerous cases recently diagnosed in free-living brown hares (Lavazza, personal communication). It will be interesting to genetically compare our strain with those that have been recently causing cases in Italy, when those sequences will be made available. Our strain is related with a virus lineage consisting of Swedish and French strains discovered from 2008 to 2014, and in particular to newer German strains identified from 2019 to 2020 (Szillat et al., 2020), confirming the successful dispersal and persistence of this lineage throughout Europe (Lopes et al., 2014). High similarities between strains detected in different countries is quite a common evidence for most of the lagoviruses. This is basically due to the low EBHSV genetic variability, and its slow evolutionary dynamics compared with other lagovirus species, such as RHDV (Lopes et al., 2014; Fitzner et al., 2021). Precise definition of the origin of the virus is almost impossible, considering the several ways of transmission described for lagoviruses, however the hypothesis of indirect transmission is the most likely. Data available from some European countries collected during the last 50 years show a drastic decline in the abundance of free-living brown and mountain hares due to environmental and demographic reasons, heavy predation, anthropogenic factors such as poaching, and the occurrence of infectious diseases (Edwards et al., 2000; Smith et al., 2005). In Europe, a constant decline of the L. europaeus and L. timidus populations was indeed reported due to EBHSV outbreaks (Gavier-Widén and Mörner, 1991; Morisse et al., 1991; Edwards et al., 2000; Fitzner et al., 2021). A threat for hare populations is also represented by RHDV2, which was demonstrated to be able to overcome the interspecies barrier and cause a fatal disease with clinical symptoms similar to EBHS in various hare species, as well as infecting non-lagomorph species (World Organisation for Animal Health (WOAH), 2022). RHDV2 was already reported to infect L. corsicanus, although the virulence of RHDV2 is clearly reduced when compared with the far higher mortality observed in Oryctolagus cuniculus (Camarda et al., 2014).

Lepus corsicanus is classified as vulnerable according to the International Union for Conservation of Nature in its Red List (Randi and Riga, 2019). The distribution of this endemic species was subjected in the last decades to a substantial contraction accompanied by a significant reduction in the population density, mainly due to habitat alterations, low numbers and fragmented populations, and ecological competition with the sympatric European hare (Buglione et al., 2020; Freschi et al., 2022). The occurrence of EBHS in a protected enclosed area, such as the CPE, represents an important threat to the conservation of this vulnerable species that could possibly lead to the point of extinction of the local population. This event would have consequences on the maintenance of the balance and biodiversity in the local wildlife. Although control of the disease in such a situation is difficult, an effort to maintain a disease-free sub-population could be made by isolating some CPE zones or groups of hares combined with the implementation of a vaccination programme, using autovaccines (Drews et al., 2011; World Organisation for Animal Health (WOAH), 2021).

In this work, we described the anatomo-histopathological lesions and reported the genomic characterization of the EBHSV strain causing an outbreak involving two free-ranging Italian hares (L. corsicanus) in a protected area of Central Italy. The identified strain belonged the group B and was phylogenetically related to other EBHSV strains circulating in Europe in L. europaeus.

Our findings further confirm that EBHSV can cause fatal disease in the Italian hare, thus representing an important threat to the conservation of this vulnerable species, especially in populations kept in enclosed protected areas.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, OR096234.

AB, AC, CE, MD, MS, PC, and RN: conceptualization. AP, CA, CE, DS, FM, IR, MD, PC, RO, and VG: investigation. DC and GM: resources. MD and PC: data curation. AC, GP, IR, LA, MD, PC, and VG: writing-original draft. AB, AC, AL, CE, MD, MS, PC, and RN: writing-review and editing. MD, PC, RN, DC, GM, CE, VG, LA, AP, RO, CA, DS, GP, FM, AC, IR, AB, AL, and MS: visualization. AB, AL, and MS: supervision. AB, AC, AL, MS, PC, and RN: project administration. AC and DC: funding acquisition. All authors contributed to the article and approved the submitted version.