Author(s) (*corresponding author)

Antonio Lavazza*, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy; antonio.lavazza@izsler.it

Mario Chiari, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy; mario.chiari@izsler.it

Reviewers 

Dolores Gavier-Widén, National Veterinary Institute, Uppsala, Sweden

Gudrun Wibbelt, Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, Germany

Last update

26.11.2015

Etiology

Affected species (wildlife, domestic animals, humans)

Epidemiological characteristics and disease course

EBHSV is transmitted directly or indirectly, mainly by oro-faecal and respiratory routes. Carnivores, insects, birds and humans can act as animate vectors, but no reservoir hosts other than hares have been yet identified. Indirect ways of transmission, including equipment, cages, clothes, vehicles and utensils likely occur, especially in outbreaks in farmed hares. Infection via consumed vegetation is also likely, with the virus being excreted and spread in the faeces of predators that have consumed infected hares.

EBHS is characterized by 100% morbidity in immunologically naïve susceptible populations, but mortality (varying rate, but often close to 50%) is observed only in adult hares. The clinical disease is not observed in hares younger than 2-3 months of age. These may contract the infection but do not usually develop clinical disease and show long-lasting protective immunity.

Mortality is highest in autumn when the population is most dense and the young of the year become susceptible. After the initial epidemic peak, the infection tends to become endemic. Areas where EBHS is endemic appear to maintain a stable hare population in which most hares are immune and mortality rates are low.

An epidemiological model that considers the peculiar characteristics of EBHS (juvenile resistance to the disease till 60-90dd of age, high environmental resistance of the virus and quick diffusion of infection) has shown that the impact of the infection on the population dynamic can be dramatic when the hare density is low (<8 hares/km²). In fact, in such case, there are lower possibilities that young hares that are naturally resistant to the disease become infected and develop long-lasting immunity. The mortality can therefore be reduced and attain endemic stability by increasing the host density (over 15 adults/km²). This situation in high density areas supports the infection of young animals when resistant to the disease and the development of immunity and thus the high (60-90%) seroprevalences of populations.

Clinical signs

Gross lesions

Histological lesions

Liver: Necrotic hepatitis. Diffuse acute coagulative hepato-cellular necrosis of hepatic periportal and midzonal areas (sometimes the whole lobule may be affected) along with formation of acidophilic bodies. In the sub-acute or chronic clinical course, lesions in the liver include: less extensive necrosis, more pronounced fatty change, more inflammatory changes, and proliferation of bile ducts.

Spleen: Congestion and oedema of red pulp. Often follicular depletion, but sometimes follicular hyperplasia. 25% of affected animals have hyaline-like changes in the cords and sinuses and splenic cellular depletion.

Respiratory: Tracheal hyperaemia and sometimes submucosal haemorrhages. Congestion, oedema and haemorrhages in the lungs.

Kidney: nephrosis with tubular degeneration and necrosis in about one third of the cases. Proteinaceous casts in the tubules.

Brain: cerebellar Purkinje cells and cerebral neurons exhibit granular and vacuolar degeneration.

Differential diagnosis

Criteria for diagnosis

Recommended diagnostic method(s) and preferred samples 

Identification of the virus in liver homogenate or other tissue can be done using haemagglutination test (with human O red cells), electron microscopy (EM), immune EM using convalescent anti-EBHSV serum or specific anti-EBHS MAbs, histopathology and immunohistochemistry (with hyperimmune serum or specific anti-EBHS MAbs, ELISA and PCR.

The adoption of an EBHSV-specific sandwich ELISA technique using a high-titre positive anti-EBHSV hare serum as catcher and cross-reacting RHDV MAbs or specific EBHSV MAbs as tracer, is highly recommended, notably for the simplicity of implementation and the low cost. Such sandwich ELISA in association with a panel of MAbs is also useful for antigenic characterisation of different viral strains.

RT-PCR and real-time RT-PCR appear to be very sensitive methods for the detection of EBHSV and are at least 104-fold more sensitive than ELISA. They are also more convenient and rapid than other tests. Even if they are now often used, RT-PCR methods are not strictly necessary for routine diagnosis.

EBHSV can also be identified in diagnostic samples by EM examination of liver homogenate in veterinary laboratories equipped with this device. In particular, to increase the sensibility, the immune-EM method using convalescent anti-EBHSV serum or specific anti-EBHS MAbs can be used to identify EBHSV.

Due to the significant antigenic differences existing between RHDV and EBHSV, the serological techniques, which use RHDV as antigen, are not recommended for the serological diagnosis of EBHSV. However, a direct ELISA method could be employed for the detection of positive and negative EBHSV hare sera; in fact, the adsorption of RHDV on to the solid phase of an ELISA microplate exposes cross-reactive internal antigenic determinants. This method is sensitive but has a very low specificity. Thus, to detect specific anti-EBHSV antibodies, a specific competition-ELISA for EBHSV using specific antigen and antisera or MAbs should be used.

APHAEA protocol

Direct diagnosis: PCR is the universal method able to ascertain viral positivity in short time with high sensitivity.

Indirect diagnosis: the detection of specific antibodies against EBHSV is achieved by cELISA that should be considered the elective method at population level.

Laboratories that can be contacted for diagnostic support

O.I.E.Laboratory reference for Rabbit haemorrhagic disease: Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna Via Bianchi 9, 25124 Brescia – Italy

antonio.lavazza@izsler.it; lorenzo.capucci@izsler.it;

Anses Ploufragan-Plouzane Laboratory, Unit of Virology, immunology and parasitology in poultry and rabbits. 41, rue de Beaucemaine - BP 53 - 22440 PLOUFRAGAN – France

Ghislaine.LEGALL-RECULE@anses.fr

Recommended literature

Capucci L, Lavazza A, 2012. Rabbit Haemorrhagic Disease. Chapter 2.6.2 (version adopted in may 2010) OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7th ed. OIE., Paris, pp 941-955.. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.06.02_RHD.pdf

Chiari M, Ferrari N, Giardiello D, Avisani D, Zanoni M, Alborali LG, Guberti V, Capucci L, Lavazza A. European Brown Hare Syndrome (EBHS): effect of density and the temporal dynamics of infection in wild European brown hares (Lepus europaeus). European Journal of Wildlife Research 09/2014; 60(6). DOI:10.1007/s10344-014-0856-6

Frölich K, Lavazza A, 2007. European Brown Hare Syndrome. In: Lagomorph Biology, Alves PC, Ferrand N, Hacklaender K, Springer Berlin, pp. 253-262.

Lavazza A, Cavadini P, Barbieri I, Tizzani P, Pinheiro A, Abrantes J, Esteves PJ, Grilli G, Gioia E, Zanoni M, Meneguz PG, Guitton J-S, Marchandeau S, Chiari M, Capucci L, 2015 Susceptibility of the eastern cottontail (Sylvilagus floridanus) to European brown hare syndrome (EBHS) virus and sporadic occurrence of EBHS-like disease suggests an active role for the lagomorph in the disease’s epidemiology. Veterinary Research (2015) 46:13 DOI 10.1186/s13567-015-0149-4

Le Gall G, Huguet S, Vende P, Vautherot JF,  Rasschaert D, 1996. European brown hare syndrome virus: molecular cloning and sequencing of the genome. Journal of General Virology 77:1693-1697.