Scientific and common names of the six hare species found in Europe. The recognizable subspecies for Lepus europaeus (16 subspecies), Lepus timidus (16 subspecies), Lepus granatensis (3 subspecies) and Lepus capensis (13 subspecies) are shown.
Although there are around 40 species of hares in the world divided into three different genera (Lepus, Caprolagus, and Pronolagus), only six species inhabit Europe, all belonging to genus Lepus. The conservation status of these six species was recently revised in the International Conservation Union (IUCN) Red List of Threatened Species. Lepus castroviejoi and L. corsicanus were attributed the status of “vulnerable”. The other four species, L. europaeus, L. timidus, L. capensis, and L. granatensis, were considered of “least concern” although a declining trend was recognized for the last two species’ wild populations. Here we review the major threats to the hare species in Europe, with emphasis on infectious diseases. Furthermore, we present the sanitary data regarding the Iberian hare populations from Portugal, which were severely affected by the emergence of a naturally occurring recombinant myxoma virus (MYXV), first reported in mid-2018. The recent detection in 2019 of a leporid herpesvirus (LeHV-5), which pathogenicity appears to be exacerbated in MYXV-infected hares, brings additional concerns to the health and conservation of the Iberian hare.
- hare species
- Iberian hare
- Lepus granatensis
- viral diseases
- myxoma virus
- rabbit hemorrhagic disease virus
- leporid herpesvirus
1.1 Geographic distribution in Europe
The Lagomorpha order (belonging to the Mammalia class) includes the Ochotonidae family, with one sole genus designated
Like the other hare species in the world, the six hare species found in Europe are small herbivorous mammals belonging to the order Lagomorpha, family Leporidae, and genus
These hare species, however, have different geographical distributions (Figure 1). The Iberian hare (
|Liebre ibérica (Spanish)|
|No subspecies||Broom hare|
|No subspecies. Recently considered to be a distinct species from ||Corsican hare|
Southern Europe provides, therefore, suitable habitats for the largest number of hare species. Morphologically, the six species of hare that inhabit Europe are distinguishable and follow the Bergmann rule, which establishes a direct relationship between the adults of medium size and the colder environments, for a given taxonomic group with wide geographical distribution .
1.2 Favorite habitats
The Iberian hare occupies a wide variety of habitats , namely, coastal dunes, wet mountain forests, and dry areas . Like the other species found in Europe, such as the European hare, generally it does not need open water to sustain its metabolism . Besides the open fields, the greater species densities are registered in intensive agricultural areas [12, 13] such as olive tree, sunflower fields, and vineyards .
1.3 Morphological characteristics
The Iberian hare is smaller than the other sympatric species, namely, the European hare and Broom hare, with mean body weight ranging from 2.0 to 2.6 kg . Females are bigger than the males . The Iberian hare has an extensive white ventral area that extends partially to the forefeet and hindfeet. This species has an evident contrast between the fur color of the back (ochraceous brown/gray-brown) and the belly (white). It has large brown eyes and long ears (with dark extremities) as a heat dissipation mechanism. The tail is also black on the dorsal surface and white on the ventral side (Figure 3). The hind limbs are longer than the front ones . These characteristics added to a cleft lip and second pair of incisors in the upper jaw allow for the differentiation of leporids from rodents.
1.4 Natural behavior
Hares are solitary, as they do not have a social organization nor inhabit burrows . However, they can gather in groups following complex age-dependent patterns, mostly during feeding time, hence reducing predation risk and increasing feeding efficiency , or at the time of mating . They do not have a territorial behavior, unlike other lagomorphs such as the wild rabbit .
Hares are active primarily during twilight and at night, though in summer they may be observed during the day . During daylight, they seek refuge at the surface, in depressions a few inches deep, dug into the ground or in foxes and marmot’s burrows .
The Iberian hare is highly specialized in camouflage and when chased by predators is capable of rapid escape, reaching around 70 km/hour . It has a relatively lighter skeleton and larger heart than rabbits, which is only found in the fast-running species .
Reproductive parameters and breeding activities depend on the hare species and environmental conditions. A study on the reproductive strategies of genus
The European brown hare, best studied due to its extensive geographic distribution, is a polyestrous seasonal breeder . During the breeding peak, in the spring, mating leads to agglomerations of solitary hares, the so-called “March madness”. This species produces an average of 3–5 litter per year . The mean litter size appears to be dependent on the region occupied, ranging between 2.0 and 2.7, with a maximum of 6 . European hares newborns have an average weight of around 100g and are fully furred, born with eyes open, and able to walk. Weaning occurs around 4–5 weeks when juvenile weight reaches around 1 kg. The European hare is fertile at around 4–5 months of age reaching maximum weight at 8 months. When in continental climates, the reproduction in the year of birth is frequent . This species has an age expectancy of 8–12 years .
The principal breeding season of Mountain hare occurs from February to September. The gestation period is about 42 days, but this inter-birth interval can be of 36 days in case of superfetation . The species has a mean litter size between 1.9 and 2.1 with a maximum of 5 leverets.
The reproduction period of Iberian hare occurs throughout the year, although there is a certain seasonality in its reproductive activity, peaking in March and April with a minimum in autumn . The onset of sexual activity is not season dependent but rather depends on the size of the animals . The Iberian hare reproductive strategy, of continuous procreation , is concordant with smaller litters and longer breeding seasons . Gestation period is also around 42 days. The seasonal trend in the population of young depends on the percentage of pregnant females and litter size. The most frequent gestations involve one or two fetuses; however, litter size may range from one to seven leverets, the largest litter size reported in the wild [11, 27]. The mean annual litter size was estimated in 2.1 leverets per litter . Based on embryo counts, the mean litter size was 1.58 (range 1–4). Annual changes in the environment impact on the reproduction of the Iberian hare causing seasonal variations . Newborn Iberian hares are also fully furred, born with eyes open, and able to walk  (Figure 4). These characteristics differentiate hares from other lagomorphs . The mean weight of newborn leverets in captivity is 128.6 g (range 123–140 g) .
1.6 Ecological relevance
Hares have an ecological substantial importance as prey of several species like the golden eagle (
1.7 Importance as a small game species
As a game species, the Iberian hare is much appreciated by Portuguese and Spanish hunters (Figure 5). In Portugal, hare hunting is permitted from September to February by different modalities, namely, “salto” and “batida” (the name of two hunting processes in the Portuguese territory), and also standing, coursing, and falconry (Article 93, Decree-Law No. 202/2004 of 18 August); the last two are only allowed between January and February. The ability of high-speed endurance running is used in hare coursing (Figure 6), a modality that has led to the selection of the greyhound breed.
Although there is no hare population census-supported data from Portugal, the trends from the National Gamebag Census indicate a reduction in the Iberian hare populations in the recent decades that has accompanied the decline in the wild rabbit populations . This decrease resulted from the combined and cumulative effect of several environmental factors that simultaneously affected the wild rabbit and the Iberian hare, along with the emergence of infectious diseases, namely, myxomatosis and rabbit hemorrhagic disease (RHD).
2. The top threats to hares in Europe
Most hare species have been subjected to a multitude of threats, which consequently have led to the reduction of the wild populations.
The International Union for Conservation of Nature (IUCN) has recognized as threatening factors to hares in Europe, the loss of habitats due to changes in the agriculture practices and development of urban areas, overhunting, poaching, trapping, agriculture pollution, human intrusions, and disturbances due to recreation activities. The impact of invasive non-native or problematic native species with consequent competition and hybridism has also been pointed out as treats to hare species.
The fragility of the hare species is also related to its own idiosyncrasies, such as the biology of its reproduction. The relatively small size of the litter and the characteristics of the shelters constructed above ground expose the juveniles to much higher predation rates than do rabbits.
Juvenile mortality is considered the most critical factor in the population dynamics. The hares’ abundance is directly related to female breeding success and to juvenile survival rates , both directly dependent on habitat suitability . The Iberian hare population dynamics is also greatly affected by food availability . The highest juvenile mortality is observed after the maximum reproductive intensity period . This mortality is due to, among other factors, agricultural landing, diseases, and predation . Juvenile mortality of the European hare may reach 90% . Data on the Iberian hare suggest that nearly 60% of the young die, corresponding to an increase of 40% in the population numbers . More recently, prenatal mortality was estimated between 18% and 21% [24, 26]. One study refers to a minimum annual survival rate in young of 27.91% .
Below we detail some of the main threats to hares in Europe.
2.1 Habitat loss
Many factors have cumulatively led to ecosystem modifications and to the deterioration of the hares’ preferential habitats. The changes in agricultural practices, namely, by the cultivation of annual and perennial non-timber crops, have played a major role in habitat reduction. In addition, the expansion of urban and industrial areas as well as of roads and railroads brought limitations and barriers to the natural habitats and movements of wild species [1, 3, 5, 6, 7, 8].
A meta-study involving 12 European countries concluded that the primary cause of the European hare decline since 1910 was agricultural intensification . The average density of the European hare in the original habitats was two individual per 100 ha, although densities of 275/100 ha have already been recorded in territories with favourable conditions . The territory range is directly dependent of the agricultural intensity and can be less of 20 ha (well-structured habitat) or up to 300 ha in habitats subject to intensive agriculture .
In the case of the Iberian hare, climate changes and reforestation of old cultures with the densification of open scrubland areas have been contributing to lower habitat suitability .
2.2.1 Management of wildlife diseases
Management strategies for wildlife disease encompass the prevention of introduction and spread of new diseases and control or eradication of an existing disease.
However, management of hares’ diseases is hampered for several reasons. The high resistance of some pathogens in the environment, where they may persist infectious for long periods, the lack of identification of putative species’ reservoirs, and their dissemination via arthropod vectors, such as the European brown hare syndrome virus (EBHSV), RHDV, RHDV2, and MYXV, potentiate new infections and make control very difficult.
Viral dissemination of leporid diseases has often been linked to anthropogenic activities [37, 38]. Changes in human activities, such as the introduction of cleaning and disinfection practices after hunting, the proper disposal of animal by-products, the periodic deworming of dogs, and the restriction of hare translocations to prevent disease spreading, can be easily implemented and impact positively on the recovery of the populations.
Immunization of wild populations, through oral bait vaccines, has been successful in the control of a few diseases such as rabies  and Aujeszky disease . However, immunization is best suited for microparasitic exogenous infections with a low reproductive rate and in populations which have a low turnover , which is not the case of wild leporids. Several attempts to produce vaccines against RHDV for wild rabbits have been made in the past [42, 43].
Disease surveillance programs of wild animal populations are particularly crucial to obtain data regarding the animal health. The frequent movements of people through territories previously not occupied by man and the recurrent translocation of animals for hunting and conservation purposes increase the contact between wild and domestic animals and humans.
2.2.2 Importance of a conclusive diagnosis in decision-making
Despite its limitations, the implementation of appropriate infection control measures must always be supported by a conclusive diagnosis, for which laboratory testing is essential.
The clinical evaluation of sick animals for diagnosis and research purposes is particularly difficult in wild species, where samples usually reach the laboratories as cadavers or organ samples. In these cases, histopathology provides the only possible bridge to understand the physiopathology of the disease. However, many factors compromise the rigor of the histopathological evaluation, such as an advanced degree of autolysis, which is common in wild species, and the consequences of sample freezing, often done due to transportation and logistic difficulties. Nonetheless, an exhaustive and systematic necropsy is the basis for a successful and complete laboratory diagnosis.
Isolation of pathogens, namely, of viruses by multiplication in sensitive cell lines, facilitates diagnosis and research. However, some pathogens of leporids are not cultivable
Molecular methods, in use for many decades, provide the possibility of testing many samples in a few hours with high specificity and sensibility.
Serologic examination of wild species allows the assessment of the previous contact of a population with a particular pathogen (herd immunity).
2.2.3 Relevant hare pathogens
Until recently, unlike rabbits, infectious diseases were not considered a major threat to hares, except for the European brown hare syndrome (EBHS) .
Pathogens that infect hares may have an impact at individual or population level. Some are also zoonotic causing disease in humans.
Staphylococcosis (caused by the bacterium
Examples of diseases that may have an impact at the population level are pasteurellosis, outbreaks of which, despite residing within the upper respiratory tract of most animals, can kill up to 80% of the population. Hares are also particularly vulnerable to coccidial infections. It is believed that Coccidia play an important role in leveret’s mortality rate but it also affects adults. Several species of
The gastrointestinal nematode,
The typical examples of the zoonotic disease group are tularemia, a bacterial disease caused by
Brucellosis is another important zoonosis that can infect hares. In this species, it is caused by
Some pathogens, such as
There is little information on the prevalence and species diversity of cestode infections in rabbits because of their low pathogenicity and the limited opportunities available to diagnose infection . In the European brown hare, the prevalence of
A combination of pathological-, parasitological-, and molecular-based techniques is usually employed for diagnostic purposes .
The presence of mature cysticerci within the abdominal cavity, the most common clinical presentation, can be observed during postmortem examination [77, 78, 79]. Yellowish-white parasitic cysts (2–18 mm) are observed in diverse locations, namely, in the peritoneum and liver surface. In massive infections, cysts may be found in the thorax, affecting the mediastinal space and pulmonary parenchyma, scrotum, small and large intestine, and renal capsule (Figure 7). Moderate to severe hepatomegaly may be observed.
Vesicles are covered by a membrane (thickness about 1 mm) containing clear liquid and an invaginated scolex of white cysticerci [80, 81]. In some cases, the migratory path of the larvae in the liver can be visualized as lighter colored areas. A host reaction to the parasite, with moderate interstitial lymphocytic hepatitis is observed. Liver parenchyma around protoscolex sections of cysts is usually surrounded by a fibrous reaction or granulomatous inflammation with multinucleated giant cells, macrophages, plasma cells, and many eosinophils (Figure 8). Multifocal to disseminated interstitial chronic hepatitis with diffuse biliary stasis may be observed .
At the parasitological examination, cysticerci appear as small, transparent, fluid-filled cysts with a broad anterior and narrow, tail-like posterior. Examination by light microscopy using staining techniques can reveal features consistent with described morphologies of
The molecular diagnosis of
18.104.22.168 European brown hare syndrome
European brown hare syndrome (EBHS) is caused by a single-stranded RNA non-enveloped virus (EBHSV) belonging to the
EBHS causes severe necrotic hepatitis in both wild and captive hares (European brown hares and mountain hares) [46, 93, 95]. The infection has significant similarities to RHD in its epidemiology, symptomatology, and pathology [93, 96], being characterized by rapid progression, mild nervous symptoms (including depression, muscular tremors, and incoordination), presence of sero-haemorrhagic liquid at the nostrils, congestion and extensive haemorrhages on the lungs and on serosa and mucosa, severe necrotic hepatitis, and congestion of the spleen and kidneys [90, 92, 93, 96].
Severe congestion of the trachea may also be present. Splenomegaly and dark red-black discoloration of the spleen, kidney congestion, and hepatomegaly may also be present. Liver is usually friable and discolored [93, 97]. Death occurs within 3 days after the onset of clinical signs, and mortality rates are extremely high and can reach 100% [90, 92].
Microscopically, necrosis may affect the whole hepatic lobule or be confined to peripheral or periportal areas. Hepatocytes nucleus appear swollen and lytic or may have completely disappeared. Coagulation necrosis can occur. Periportal and midzonal hepatocellular necrosis is the most consistent histopathologic finding. Nuclear degradation appears to be rare in EBHS contrary to what happens in RHD. The hepatocytes frequently contain fine iron granules.
Other findings are marked hyperemia irregularly distributed over the hepatic lobule and disruption of sinusoids with hemorrhage. In the necrotic and hemorrhagic areas, neutrophil infiltration and mesenchymal cell proliferation are observed.
Moderate dilatation of segments of the proximal and distal tubules with flattened epithelium and focal hydropic degeneration of the proximal tubules may be present. The tubules can contain pale eosinophilic protein casts of a slightly granular appearance. Congestion of spleen red pulp has been described. White follicles can appear depleted of lymphocytes, with karyorrhexis or pyknosis of B and T cells. Follicular hyperplasia can also occur [93, 97].
As with other lagoviruses, EBHSV cannot be isolated or propagated in rabbit and hare primary cell cultures or in cell lines (RK-13, PK-15, FL) [46, 98, 99]. The virus can be detected usually in the liver, which contains high viral loads, by electron microscopy, which shows 30–35 nm virions indistinguishable from those of RHDV [90, 100]. Other methods for virus detection include the hemagglutination (HA) test, using human type “A” or “O” red blood cells [46, 100], enzyme-linked immunosorbent assay (ELISA)-based methods [46, 90, 96, 98], and RT-PCR. Bascuñana et al.  developed two conventional RT-PCR assays for the detection and differentiation of RHDV and EBHSV, both able to detect as few as 10 copies of cloned viral genomic fragments, with no cross-amplification between the two viruses. The system can be used for amplification of VP60 genomic sequences from fresh and fixed tissues. Primers were selected from similar regions of the VP60 genes to amplify a fragment of 316 nucleotides from the genome of RHDV and a region of 265 nucleotides from the genome of EBHSV. In addition, Le Gall-Reculé et al.  developed an immunocapture (IC)-RT-PCR for EBHSV diagnosis that can be carried out directly with the liver exudate. Viral particles present in the sample are captured by specific antibodies immobilized on a microtitration plate. After enzymatic disruption of virus-antibody complexes, viral RNA is released and subjected to RT-PCR. The assay combines the rapidity of an ELISA test (because immunocapture and the RT reaction are carried out in the same microtiter plate) and the sensitivity of PCR, being suitable for the processing of large numbers of samples and phylogenetic studies. In 2011, Zexiao et al.  also developed a RT-PCR for the detection of EBHSV with good specificity and sensitivity, able to detect about 50 copies of cloned viral genomic fragments (pGM-T-EBHSV), with no amplifications for RHDV.
22.214.171.124 Rabbit hemorrhagic disease
RHD is caused by two types of lagoviruses, namely, RHDV that emerged in China in 1986 in domestic rabbits  and RHDV2 (RHDVb or GI.2), a virus genetically related to but distinct from RHDV, that emerged in France in 2010 in rabbits . RHDV2 quickly replaced the circulating strains of RHDV in most European countries, both in the wild and domestic populations. In the Iberian hare, Lopes et al.  reported RHDV only in two animals during a retrospective study. However, in the last decade, several cases of RHDV2 disease have been reported in the European hare in France (2013) , Spain (2014), and Italy (2012) , in the United Kingdom (2018 and 2019) , in Australia (2015) , in Sweden (2016 and 2017) , and in the Netherlands (2017) (https://www.dwhc.nl/en/haas-rhdv-2-nederland/). RHDV2 was also reported in the Cape hare on the island of Sardinia (2011)  and in the Italian hare, in Sicily (2012) . More recently (in 2019), cases of RHDV2 infection have been reported in the Mountain hare in Scotland  and Ireland (https://www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapFullEventReport&reportid=31386).
However, RHDV2 infections have never been described in the Iberian hare.
The clinical evolution of RHD can be peracute, acute, subacute or chronic. The clinical manifestations are mainly present in the acute form of infection though in the subacute form, similar but milder signs can be observed. In the peracute form, usually there are no clinical signs of disease . Chronic and subacute forms are more frequent in RHDV2 infections , which also differ from RHDV in affecting rabbit kits as young as 11 days old that develop disease [104, 109, 110].
The incubation period may vary between 1 and 5 days. Death may occur 12–36 hours after the onset of fever (>40°C). The clinical signs observed include prostration, apathy, convulsion, ataxia, paralysis, opisthotonos, paddling, anorexia, dullness, groans and cries, dyspnea, frothy and bloody nasal discharge, and cyanosis of mucous membranes. Subacute cases may present with malaise, mild anorexia, apathy, weight loss, and jaundice .
RHDV2-infected hares exhibited clinical signs and lesions similar to those induced by EBHSV [64, 65, 66]. Epistaxis and a RHD-like disease was reported in the Italian hare  and in the Sardinian Cape hare .
The macroscopic findings in hares affected by RHDV2 include extensor rigidity, epistaxis, and hyperemia of the tracheal mucosa, where a foamy bloody mucous can be found. The liver can be pale or congested (light brown to orange-pink) or presenting a reticular enhancement pattern suggestive of zonal vacuolar hepatocellular degeneration and necrosis. Moderated to severe congestion or petechial hemorrhages (or multifocal to coalescing hemorrhage) can also be observed in the lungs and kidneys.
Microscopic lesions may comprise generalized hepatic necrosis, including coagulative necrosis and multifocal areas of lytic necrosis. Acidophilic bodies may be observed in the hepatocytes. Other findings may include fatty degeneration of hepatocytes and periportal mononuclear infiltration. Degeneration and necrosis of kidney proximal tubules cells or acute tubular nephrosis may also be registered. In some cases, it is possible to observe moderated lymphocytolysis in the spleen white pulp, and moderate to severe fibrin deposition and necrosis of the red pulp can be observed [62, 63, 64, 66, 67].
Different molecular assays for the detection of RHDV have been described since the late 1990s, including conventional RT-PCR assays [99, 111, 112, 113], immunocapture RT-PCR , real-time multiplex RT-PCR , and more recently, loop-mediated isothermal amplification  and SYBR green-based real-time PCR . None is designed to specifically detect RHDV2 strains.
For the amplification of RHDV2, Le Gall-Reculé et al.  described specific primers which amplify a 794-bp sequence located in the C-terminal of the gene encoding the VP60 of RHDV2.
Duarte et al.  developed a specific real-time TaqMan RT-PCR for the detection of RHDV2. The system was designed to amplify a 127-nucleotide-long RNA region located within the
More recently, Dalton et al.  developed a diagnostic real-time RT-PCR for the detection of RHDVb strains targeting a 91-bp amplicon within the VP60 protein that covers nucleotides (nts) 6190–6280. The RT-PCR is carried out as a duplex using the endogenous amplification control of the beta-actin gene from a commercial kit (EXOPOL S.L.). The same authors also designed a conventional RT-PCR for the differentiation of RHDV2 strains from RHDV2 recombinants by subsequent sequencing of the amplicon. Primers were designed to cover a 449-nt region from the 3′ region of the RHDV polymerase (3D) to the sg promoter region. Degenerate primers were designed to bind at positions 4837–4856 and 5266–5286 in the RHDV2 genome .
Myxomatosis, caused by a double-stranded DNA enveloped virus belonging to the family
The hare myxoma virus (MYXV) is a recombinant with additional genetic material in comparison with the MYXV from rabbits. The inclusion of this supplementary material led to the disruption of one gene and to the duplication of four others. While the origin of this recombinant is not yet completely understood, some authors have suggested that the extra genes originated from a capri- or cervipoxvirus [69, 70], and others propose the translocation of genes from the rabbit myxoma genome itself .
In rabbits, when the typical signs and skin lesions develop, the clinical diagnosis is possible. These signs may include blepharitis, blepharoconjunctivitis, anorexia, listlessness, fever, and depression. In the dermatrophic form of myxomatosis, disseminated cutaneous and mucocutaneous lesions, accompanied by oedema, are usually present in the eyelids, nose, lips, ears, and genitals (vulva, or scrotum). Emaciation and dyspnea followed by death may occur within a few weeks [127, 128]. However, in peracute forms, sudden death may occur with no clinical signs of disease or unspecific signals such as lethargy, loss of appetite, fever, and eventually swelling of the eyelids . The clinical diagnosis of the atypical amyxomatous forms of myxomatosis, characterized by minor cutaneous signs and intense respiratory distress, may be complicated as well [127, 129, 130, 131].
In hares, as for rabbits, the clinical diagnosis relies on the observation of the typical skin lesions, namely, small nodules and oedema, usually present in the eyelids, nose, and genitalia. The typical myxomas observed in the ears, eyelid, or other areas of the skin in rabbits are rarely observed in hares (Figure 9). Blepharoconjunctivitis and mucopurulent eye discharge are a common feature [68, 69, 126].
MYXV-infected hares can show good body condition, contrary to what is common in rabbits with myxomatosis.
Histopathological findings include epidermal hyperplasia and balonization of the epidermal keratocytes. Eosinophilic cytoplasmic inclusion bodies may be observed in the keratinocytes. In the dermis, an abundant basophilic myxoid matrix admixed with oedematous areas with inflammatory infiltrates of macrophages, lymphocytes, and polymorphonuclear cells may also be seen (Figure 10). A severe depletion of lymphocytes may be noted in the spleen [68, 132, 133]. As for rabbits, the clinical diagnosis of the atypical form of the disease in hares may be limited.
Molecular methods provide the possibility to detect a reduced number of viral DNA copies in multiple tissue samples such as nasal and conjunctival swabs, skin oedema, myxomas, crusts, lungs, and semen. The highly conserved regions of the MYXV genome are used for primer design for PCR-based assays, allowing for detection of the circulating virus strains. Several conventional  and real-time PCR methods [135, 136, 137] were described and can be used to detect MYXV.
The conventional PCR system by Cavadini et al.  is based on the MYXV genes
Dalton et al.  developed a long-range PCR-RFLP method directed towards amplification of genomic MYXV DNA from the left and right terminal inverted repeat regions (TIRs) with subsequent differentiation of virus strains by RFLP. Two sets of primers were designed covering the entire TIRs and flanking sequences (
Quantitative PCR systems (qPCR) allow the determination of viral copy number in the tested sample. In the PCR-based method developed by Albini et al. , primers and probe were designed to amplify a 147-bp fragment of the serpin (
The TaqMan qPCR by Belsham et al.  that targets a
A highly sensitive qPCR assay targeting nucleotide sequences within the MYXV gene
For many years, the laboratorial diagnosis of myxomatosis was based on the isolation of the virus in cell culture. Isolation of MYXV can be accomplished in primary cultures of rabbit kidney (RK) cells or in cell lines (RK-13, Vero). This method is recommended by the OIE, particularly for the diagnosis of amyxomatous form of the disease. In this case, antemortem diagnosis can be done using nasal and conjunctival swabs .
The virus multiplies in the cytoplasm of the infected cells [141, 142]. For the classical MYXV strains from rabbits, a cytopathic effect (CPE) may develop in 24–48 hours or take up to 1 week, depending on the virulence of the strain . CPE is usually characterized by the formation of syncytia and roundup and contraction of the infected cells. Later, when all cells are affected, the cell monolayer detaches completely.
Viral isolation of the Iberian hare recombinant MYXV strains is more difficult since CPE is less obvious, differing from the one induced by the rabbit MYXV strains.
126.96.36.199 Leporid herpesvirus
Herpesvirus DNA was detected in hares with myxomatosis, where, in most cases, herpetic-like skin vesicles were present in the nostrils and lips along with necrosis of the genitalia, most evident in males affecting the penile glans but also observed in females (Figure 11) .
However, LeHV-5 DNA was also detected in apparently healthy hares, testing negative to MYXV, probably representing the latent stage of infection.
The penile and foreskin epithelia of some LeHV-5 positive hares was mostly necrotic and replaced by a thick band of necrotic cells, heterophils, and red blood cells. Severe heterophilic infiltrations of the stroma, in either a diffuse pattern or multifocal aggregates, are common.
Proliferation of pleomorphic spindle cells is observed, with some nuclei almost completely filled with slightly eosinophilic inclusion bodies, resembling Cowdry nuclear inclusions . Coalescent intraepidermal and subepidermal vesicopustules filled with fibrin and necrotic cell debris and multifocal detachment of the eyelids, lips, and foreskin epidermis were seen (Figure 12) . In the dermis, multifocal hemorrhages, intense infiltration by heterophils, and necrotic cells with accumulation of chromatin debris were present .
Clinical diagnosis of this disease is difficult and may pass unnoticed. During lytic replication (reactivation phase), small vesicles may be observed in the mucous membranes and skin, which may still be intact, or after erosion, mainly in the lips and nostrils. Necrosis of the genitalia may not be present. The conclusive diagnosis is achieved by PCR and sequencing or by electron microscopy.
2.3 Excessive predation
Predation can have a major impact on hare populations since, unlike the wild rabbit, hares generally do not burrow, except when subjected to high persecution pressure and without alternative escape, and are therefore more exposed to this phenomenon. The hares’ natural predators include large birds of prey and wild canids and felids . In fact, the Iberian hare is the preferred prey of some Iberian vertebrate predators, such as the Eurasian eagle owl (
Some carnivorous bird species that can prey on hares have shown a progressive increase on the Iberian Peninsula. This is the case of the white stork (
The wild boar (
2.4 Hunting pressure
Extrinsic factors threatening the conservation of hare populations include reduced size and quality of the habitat, which is particularly critical for hare species whose territories are restricted to very limited geographical areas, such as the northern Castroviejoi broom hare, in Spain, and the Italian hare, in Italy. In these small territories, excessive hunting pressure can pose a serious threat to the preservation of these species [5, 7].
In addition, the imbalance of the complex Iberian trophic chains resulting, among other aspects, from the reduction of wild rabbit populations, increases predation on hares by predators that preferentially feed on wild rabbit [31, 32].
2.5 Other causes
The mechanization of cereal harvesting was recognized as a cause of direct mortality of juvenile hares by their exposure on the soil surface. Also, the effect of pollution resulting from agricultural chemicals, agricultural and forest effluents , and road traffic pose additional threats to the species .
3. Sanitary surveillance of the Iberian hare in Portugal
In Portugal, an action plan to control rabbit viral hemorrhagic disease was set in place in August 2017 by a nine-institution partnership, following the Dispatch 4757/17 of May 31 of the Ministry of Agriculture. This partnership is comprised of the National Institute of Agrarian and Veterinary Research (INIAV. PI), the General Directorate of Food and Veterinary (DGAV), the Institute for Nature Conservation and Forests (ICNF), the Research Centre in Biodiversity and Genetic Resources (CIBIO), the Institute for Experimental Biology and Technology (iBET), the Portuguese first sector hunting organizations (FENCAÇA, ANPC, CNCP), and the National Order of Veterinary Doctors (OMV). Since its implementation from August 2017 to the end of January 2020, 1507 wild leporids originating from almost 50 hunting reserves where sampled and systematically tested for the presence of RHDV, RHDV2, and MYXV to assess the population health during the hunting seasons. Of these, 89.93% (1099/1222) were wild rabbits, and 10.07% (123/1222) were hares. Furthermore, during the same period, 285 animals found dead in the field in mainland Portugal were also screened, of which 77.54% (221/285) corresponded to wild rabbits and 22.46% (64/285) were hares.
Hunting associations are authorized by permits emitted by the National Forest Authority, Institute for Nature Conservation and Forests (ICNF).
Below we present the virological examinations regarding the Iberian hare. Despite RHDV2 being detected in several hare species, none of the 187 hares investigated in Portugal between August 2017 and the end of January 2020 were positive, confirming the lack of susceptibility of Iberian hare to this virus.
As in domestic and wild rabbits, where RHDV has no longer been detected in Portugal since the emergence of RHDV2 in 2012, RHDV-RNA was not detected in Iberian hares .
MYXV was first detected in Portugal in October 2018 in an adult hare, found dead in a hunting reserve, located in the district of Évora, Alentejo Region, in the south mainland. The virological diagnosis was made by the National Reference Laboratory for Animal Diseases (INIAV, Oeiras, Portugal) in October 2018.
The first evidence of Iberian hare mortality by myxomatosis was noticed in early summer of 2018, in Spain. The affected hares showed clinical signs compatible with myxomatosis and were found dead or moribund in hunting reserves in the municipalities of Montalbán and La Rambla (Córdoba), in the south. The diagnosis was confirmed by the Central Veterinary Laboratory in Algete, Madrid, Spain in July 2018 (OIE report).
Before the detection of the first MYXV case in the Iberian hare in Portugal, within the scope of the national surveillance action plan as referred above, 91 hares where sampled and screened for the presence of MYXV, between August 2017 and end of October 2018. None of the animals tested positive, suggesting that the virus was not circulating in the populations sampled. After the first case, by the end of October 2018 until the end of January 2020, 107 Iberian hares were tested for MYXV, 58.88% (63/107) of which were found dead or moribund and 41.12% (44/107) were legally hunted. In the first group, 82.54% (52/63) animals were positive for myxomatosis, reflecting the significance of this infection as a cause of death in Iberian hares. The percentage of positivity in the sample of hares found dead by trimester showed an increasing trend, suggesting the progressive spread of the disease (Figure 14).
In the group of 44 hunted animals, only 4 were positive (9.1%). Most of these samples (
Since the non-recombinant rabbit MYXV is endemic in the entire national territory, our results suggest that the Iberian hare is not susceptible to the rabbit strains. However, serological data obtained from Iberian hares shows the presence of antibodies against RHDV2 and myxoma virus indicating the ability of these viruses to induce an immune response .
The progressive loss of habitat due to the deep changes in land use, excessive predation, and hunting poses serious threats to the conservation of hare species in Europe.
The development of effective strategies that trigger the continuous implementation of good practices is urgent to ensure the preservation of wild populations and to promote their recovery in the most affected areas. Such measures should safeguard the persistence/existence of favorable habitats for the species, particularly threatened in the geographical areas where their natural territories are reduced or in areas where farmers adopted super-intensive production methods.
In Portugal and in Spain, government research institutions, academics and field agents joined efforts through projects +Coelho and MIXolepus, respectively, to evaluate and counteract the effects of this emergent virus in Iberian hare.
At a time when the conservation status of the wild rabbit has recently been revised from “least concern” to “endangered,” it seems inevitable that, in a near future, the status of some hare species, namely, the Iberian hare, will also be revised.
Apprehension on the preservation and sustainability of wild leporid populations in the Iberia is aligned with the concerns of many other wild species. According to the World Wildlife Fund (WWF) “Living Planet Report 2018” report, global wildlife populations have declined by an average of 60% over the past 40 years, demonstrating that the planetary biodiversity is threatened.
Infectious diseases, particularly those of viral etiology, have been gaining importance as disrupting factors for the stability of rabbits and hares. The potential evolution of the viral
The authors thank all the hunters who participated in the collection of biological samples, represented by João Carvalho (ANPC), Jacinto Amaro (FENCAÇA), and Fernando Castanheira Pinto (CNCP). Funding was supported by Fundo Florestal Permanente (Government of Portugal) within the scope of the action plan for the control of rabbit viral hemorrhagic disease (+Coelho 2 project nº 2018044300001 - Desenvolvimento e implementação de medidas práticas impulsionadoras da recuperação dos leporídeos silvestres em Portugal - financed by the Fundo Florestal Permanente (FFP), Dispatch nº4757/2017 of 31st May) by the Fundação para a Ciência e Tecnologia (FCT) (grant SFRH/BD/137067/2018) and by the Interdisciplinary Research Centre on Animal Health, Faculty of Veterinary Medicine, University of Lisbon (CIISA, FMV-UL) (Project UID/CVT/00276/2013).
Conflict of interest
The authors declare no conflict of interest.