Introduction

Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), two very closely related but phylogenetically distinct viruses belonging to the genus Lagovirus, family Caliciviridae, cause rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS).

European brown hare syndrome was first observed in the 1980s in Sweden as an emerging disease of European brown hares Lepus europaeus (Pallas, 1778) clinically manifested by liver damage of unknown origin. Pesticides, mycotoxins, elevated selenium levels and new rapeseed varieties with low glucosinolate content were considered as possible causes. But no associations with the disease were found (Gavier-Widén and Mörner 1993).

It was not until 1989 that the first RHDV-like viruses were discovered by electron microscopy. EBHS is a contagious disease with a high mortality rate. In the acute form, adult mortality ranges from 90–100%, which significantly affects population dynamics (Lavazza and Vecchi 1989; Wirblich et al. 1994).

The first record of RHD dates back to 1984 in Jiangsu Province, China. It was found in Angora rabbits of commercial breeding imported from Germany. In less than a year, RHD caused mortality of more than 140 million domesticated rabbits in China and spread to over 50,000 km2 (Liu et al. 1984; Xu 1991). In Europe RHDV was first isolated from rabbits in Italy in 1986 from where it spread to other European countries (Cancellotti and Renzi 1991). The first outbreaks of RHD in Czechoslovakia dates back to 1987 (Rodák et al. 1991). It is a highly contagious disease accompanied by a pronounced hepatitis with a fatal course. It is most pathogenic in rabbits older than eight weeks, due to age-related differences in enterocyte receptor expression. The viruses that cause RHD are genetically variable. The classical RHDV has five genogroups (Xu and Chen 1989).

In 1996, another antigenically distinct variant of the G6 virus, termed rabbit hemorrhagic disease virus a (RHDVa), was discovered in Italy and Germany and gradually spread (Capucci et al. 1998; Schirrmeier et al. 1999). In 2010 in France, even vaccinated fattening animals suddenly started dying of RHD symptoms. Here, Le-Gall-Reculé et al. (2011) demonstrated a new antigenic variant of RHDV that differed from the previous ones mainly because it did not induce cross-immunity with previously known RHDV variants and was therefore designated as rabbit hemorrhagic disease virus type 2 (RHDV2). This new antigenic variant has spread quite rapidly in some European countries as Finland, Sweden, Poland (Le-Gall-Reculé et al. 2011).

In November 2012, a dead European brown hare with signs of congested trachea, visceral hemorrhage, mild splenomegaly and pale and friable liver was found in Italy near the town of Cavergana in Lombardy region. In February 2014, two dead European brown hares with pathological findings of epistaxis, rounded lung margins and multiple hemorrhages, pale liver with generalized pattern were found in Spain near the town of Oristà in Catalonia. In both cases, EBHSV was suspected, which was later refuted. Subsequently, RHDV2 was isolated from the carcasses. It was for the first time when interspecies transmission of RHDV2 on L. europaeus has been confirmed (Velarde et al. 2017).

Material and methods

All blood serum samples of European brown hares were tested by serological methods: hemagglutination inhibition test (HIT) for detection of antibodies to RHDVa and RHDV2; enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to EBHSV, RHDVa and RHDV2. The presence of antibodies to EBHSV was not detected by HIT because the antigen was not available.

Serum samples were obtained by drawing blood from the hearts of harvested hares without any signs of a clinical disease. From the Slovak Republic, we managed to collect 200 samples from 19 locations between 2016 and 2019 (Table 3). In 2022, 75 samples of hare sera from the Czech Republic were obtained from four locations (Table 3).

All sera were tested by HIT on the presence of antibodies to RHDVa and RHDV2 according to Terrestrial Manual 2023 by WOAH (WOAH 2023). RHDVa and RHDV2 antigens were obtained according to the above mentioned methodology from the livers of rabbits diagnosed in our laboratory. Human erythrocytes of blood group 0, obtained from the Transfusion and Tissue Department at the University Hospital Brno. After measuring of haematocrit, we diluted human erythrocytes to a concentration of 2%, which we found to be the most ideal for evaluation. Hare serum samples were heat inactivated, purified twice with kaolin and centrifuged. And in the last step sera were purified with 50% suspension of human erythrocytes for 60 min at 4 °C to remove non-specific inhibitors. After centrifugation, hare sera were diluted in a microtiter plate and titrated with antigen at eight hemagglutination units. After 30 min of incubation, 2% human erythrocytes were added and after 30 min, the results could be read.

Another serological method used was enzyme-linked immunosorbent assay (ELISA). To detect EBHSV, RHDVa and RHDV2 antibodies, we used a commercial ELISA kit (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy). Each ELISA kit contained a negative and positive controls, antibodies, antigen and specific monoclonal antibodies (MAbs) conjugated with horseradish peroxidase. ELISA testing was performed according to the manufacturer's instructions.

Results

A total of 275 hare sera from 23 locations in the Czech Republic and Slovakia were tested. EBHSV antibodies were detected in 64 individuals (27.27%). Antibodies to RHDVa were detected in two hares (0.73% prevalence) and antibodies to RHDV2 were detected in two other hares (0.73% prevalence).

Tables 1 and 2 summarise the results of European brown hares blood serum tests during 2016–2022 in the Czech Republic and the Slovak Republic. Table 3 shows in detail the seropositivity of examined hares from particular localities in Czech Republic and Slovak Republic. Only at six localities (Čechynce, Čermany, Hul, Kuzmice, Oponice and Vlkov) no antibodies were detected in tested hare sera. In addition to antibodies against EBHSV, antibodies against RHDVa were also detected in examined hares from Lehnice and Veľký Cetín. Except for antibodies against EBHSV, antibodies against RHDV2 were also detected in examined hare sera collected in Bedřichovice and Šlapanice. These two localities are geographically located in close proximity.

Table 1 Results from serologic detection of antibodies in blood sera from the Slovak Republic
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Table 2 Results of serologic detection of antibodies in hares sera from the Czech Republic
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Table 3 Seropositivity of examined hares from particular localities
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The map (Fig. 1) shows in colour the locations of animals with specific antibodies to EBHSV, RHDVa and RHDV2 in the L. europaeus on 23 study sites in the Czech Republic and Slovak Republic between 2016 and 2022.

Fig. 1
figure 1

Map showing the origin of positive hares with antibodies to EBHSV, RHDVa and RHDV2

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Discussion

Among mammals, the European brown hare in particular has experienced a dramatic decline in many European countries. Population densities have been declining across Europe since the second half of the 1970s (Edwards et al. 2000). For example, in the Czech Republic, 32,100 hares were harvested in 2020 and 13,015 hares were harvested in the Slovak Republic in 2022. For comparison, in 1973, 1,214,000 hares were harvested in former Czechoslovakia (Vodňanský 2021; Czech Statistical Office 2022; Enviroportal.sk 2022).

Factors such as agricultural intensification and mechanization associated with the degradation of biodiversity in the farmland are considered to be the main causes of the population decline of the European brown hare (Edwards et al. 2000). Brown hares are also exposed to contamination from specific environmental pollutants, such as heavy metals, which accumulate in the organisms of hares at a higher rate than in other animals (Beuković et al. 2022, 2023). Predators, especially the red fox Vulpes vulpes (Linnaeus, 1758), collisions with vehicles and infectious diseases are the main reasons given for the mortality of European brown hares (Lindström et al. 1994; Lamarque et al. 1996; Haerer et al. 2001).

Lamarque et al. 1996 found that more than 50% of hares found dead in France between 1986 and 1994 had died on infectious diseases, primarily EBHSV infection, then pseudotuberculosis, pasteurellosis and coccidiosis. The juvenile hares are highly susceptible to EBHS once protection by maternal antibodies is lost and they produce the highest levels of mortality between September and December (Edwards et al. 2000).

This study presents the results of serological screening of circulating lagoviruses in L. europaeus in different localities of the Czech Republic and Slovakia. A limiting factor of the study is relatively low number of screened individuals across sampling localities and the termination of active monitoring of tularemia in the Czech Republic in 2019. The prevalence of detected antibodies to EBHSV was 28% (n = 75) on the Czech localities, while on the Slovak localities the prevalence ranged from 9 to 33% (n = 200) in each year.

Frölich et al. (2003), who examined sera from 321 hares in Schleswig–Holstein, Germany, between 1997 and 1999, obtained similar results. Antibodies to EBHSV were detected by ELISA in 92 sera, corresponding to 29% prevalence (n = 321). In Italy, 331 hares sera were tested by ELISA method between 1989 and 1991, of which 146 were found to have antibodies to EBHSV, i.e. 44% (n = 331) (Scicluna et al. 1994).

In hares originating from two localities in the Slovak Republic (Lehnice and Veľký Cetín), antibodies to RHDVa were detected. So far, RHDV has only been detected in the Cape hare Lepus capensis mediterraneus (Wagner, 1841) and in the Iberian hare L. granatensis (Rosenhauer, 1856) (Lopes et al. 2014). Our results showed that interspecies transmission of RHDVa to the European brown hare is possible. However, we do not know how RHDVa is able to cross the species boundary.

Antibodies to RHDV2 in examined hares were detected in two localities in the Czech Republic (Bedřichovice and Šlapanice). Our results correspond with the study by Velarde et al. (2017), who also detected antibodies against RHDV2 in hares in Spain and Italy. Previously, antibodies to RHDV2 have been detected in the Cape hare in Sardinia and in Iberian hare in Sicily, which, however, was in direct contact with an infected rabbit (Puggioni et al. 2013; Camarda et al. 2014).

Similar research was conducted in Poland, where ELISA methods were used for detection of antibodies to EBHSV and RHDV2. A total of 113 European brown hare sera were tested and antibodies to EBHSV were detected in 99 samples, but the presence of antibodies to RHDV2 was not confirmed (Fitzner et al. 2022).

Conclusion

The prevalence of antibodies to EBHSV in 2016–2019 ranged from 9–33% when investigating blood sera from hares originating from SR sites. In two cases, antibodies to RHDVa were detected, while antibodies to RHDV2 were not detected in the sera of hares in either case.

In the investigated sites in the Czech Republic in 2022, the prevalence of antibodies to EBHSV was detected in 28% of hares and in two cases antibodies to RHDV2 were detected, while in neither case were antibodies to RHDVa detected in the sera.

This study improved our knowledge about circulation of RHDV in Central European ecosystem and its ability to cross interspecies barriers. However, from an epizootiological point of view, it is likely European brown hare is not a significant source of RHD infection for domesticated rabbits.