Leporipoxvirus infections

DEBRA BOURNE

Wildlife Information Network, Twycross Zoo — East Midland Zoological Society, Atherstone, UK

Viruses within the genus L eporipoxvirus cause disease in rabbits and hares, including most importantly myxomato­sis, caused by Myxoma virus, but also hare fibromatosis, caused by Hare fibroma virus.

MYXOMATOSIS

AETIOLOGY

In native species of rabbit hosts in the New World (Sylvi- lagus brasiliensis and Sylvilagus bachmani), myxoma viruses (Brazilian strain in Sylvilagus brasiliansis, Californian strain in Sylvilagus bachmani) cause only a mild, self- limiting cutaneous fibroma. However, in Oryctolagus cuniculus, the European wild rabbit (including the domestic rabbit), it causes a severe, often-fatal illness characterized by blepha­roconjunctivitis and oedema around the head, particularly the eyes, and the perineal area, together with listlessness and fever; later (if the rabbit has not died in the first 48 hours) the classical subcutaneous swellings develop. The severity of disease varies with the strain of the virus and with host resistance. Other leporipoxviruses affecting lep- orids are Hare fibroma virus (see below), Rabbit fibroma virus (Shope’s fibroma virus) in North America, which is cross-protective with myxomatosis, and African hare fibroma virus (not necessarily distinct from Hare fibroma virus).

EPIDEMIOLOGY

Originating in the New World, myxoma viruses are now endemic in Europe and Australia as well as North and South America. Both in Australia and Europe, Brazilian strains of the virus were introduced. Within Europe, myxoma virus was first introduced onto a private estate in France in 1951. Spread was rapid, and by the end of the following year it was found across France, Belgium, The Netherlands, Luxembourg, Germany, Spain and England.

Initially, myxomatosis killed more than 99% of infected Oryctolagus cuniculus. However, over time, wild rabbits developed a degree of immunity, and less virulent strains of virus predominated over the most virulent strains, although as rabbits have become more resistant, somewhat more virulent strains of the virus have become more preva­lent. Death rates in infected wild rabbits have decreased; nevertheless outbreaks continue to occur and studies on rabbits in Britain and Spain show that myxomatosis con­tinues to regulate rabbit populations¹-¹⁷’¹⁸). Strains of mod­erate virulence are most likely to be transmitted, as these have the longest periods of time for which infectious titres of virus are present in the skin.

In France, there is a definite seasonal pattern, with epi­demics occurring in summer associated with high vector mosquito populations. In the UK, early reports found infected rabbits in all months of the year, but it is generally accepted that there is a major peak in prevalence in autumn, associated with the high rabbit populations fol­lowing the breeding season, and a minor peak in spring (when fleas become more active). In Spain, myxomatosis is endemic with epizootics associated with recruitment of young, susceptible rabbits to the population; in a study in north-eastern Spain, a delay in breeding one year was fol­lowed by a delay in the annual myxomatosis outbreak⁽¹⁸⁾.

The main host and species affected in Europe is the European rabbit. Individual hares (Lepus europaeus (Euro­pean brown hare) and Lepus timidus (mountain hare)) can become infected and occasionally develop clinical disease. Reduction in mortality associated with myxomatosis in free-living Oryctolagus cuniculus in Europe (and in Aus­tralia) appears to be associated with natural selection for innate resistance in the wild rabbits, as well as emergence of reduced-virulence strains of the virus.

Within Oryctolagus cuniculus, susceptibility decreases with age; very young rabbit kittens die only 5—6 days after intradermal inoculation, with few external signs of disease (eyelid margins may be thickened), whereas adults survive longer (9—11 days post-inoculation) with development of obvious clinical signs: swelling of the head, closure of the eyes, conjunctival discharge, oculonasal discharge and development of multiple skin nodules. Susceptibility of kittens is reduced if they have passive immunity transmit­ted by their immune mother, and kittens with passive immunity survive longer if they do become infected⁽¹⁹⁾. In a study in a wild population in north-eastern Spain, clini­cal cases were seen only in young rabbits. All older rabbits maintained high antibody levels, while prevalence of anti­bodies in young rabbits was low initially, gradually increas­ing during the year⁽¹⁸⁾. There is no sex difference in susceptibility.

For rabbits infected with moderately virulent strains of myxoma virus, severity of disease is reduced and survival is higher in high environmental temperatures than in low temperatures (experimentally in Australia, survival was highest in rabbits housed at 37-39°C, lowest in those kept at 0-4° C). This appears to be associated with reduced spread of the virus from the primary lesion to the lymph nodes and reduced systemic spread of the virus. With highly virulent strains, mortality is high in all environmen­tal conditions¹-²⁰).

Transmission of myxoma virus by arthropod vectors is mechanical, occurring when a blood-feeding arthropod feeding on affected skin areas of an infected host (e.g. the eyelids, ear base), where virus is present in high concentra­tions, then feeds on susceptible individuals. A wide variety of parasitic arthropods - mosquitoes, fleas, simuliidae, biting flies, mites, lice etc. - can act as mechanical vectors. There is no evidence that the virus replicates in mosquitoes or in any other arthropod.

PATHOGENESIS, PATHOLOGY AND IMMUNITY

Following skin inoculation of susceptible rabbits, initially the virus replicates locally in MHC-II (major histocompat­ibility complex) dendritic-like cells, within which it moves to the local draining lymph node within 24 hours, and replicates there mainly in T lymphocytes, high titres being found by 3 to 4 days post-infection. It then disseminates in leucocytes to the epidermis of the skin, mucocutaneous junctions, and to the lungs, spleen, testes and other internal organs. At this time, clinical signs are restricted to the primary lesion and slight reddening of the eyelids. Replica­tion in the testes and epididymis results in orchitis, epidi­dymitis and reduced fertility of surviving male rabbits. Myxoma virus produces several immunomodulatory pro­teins, resulting in apoptosis of lymphocytes within lymph nodes, and systemic immunosuppression. In resistant rabbits, although initial replication occurs in the skin at the site of inoculation, titres of virus in the lymph node draining the inoculation site are much lower than in fully susceptible individuals, and virus replication in tissues distal to this lymph node is controlled⁽²²⁾.

The primary lesion, forming at the site of infection, is a subcutaneous mass, developing about 3—5 days after virus inoculation. Subcutaneous swellings are mucoid, not fibromatous. With amyxomatous myxomatosis in experi­mentally inoculated New Zealand white rabbits, external lesions included conjunctivitis, diffuse ear swelling, some­times flat swellings on the ears or nose, sometimes with crust formation, but there were few gross internal lesions. Gross lung lesions, which were not always present, involved light to dark red consolidated areas of the anteroventral lobes. In some rabbits, splenomegaly was noted⁽²⁴⁾.

Microscopically, within the subcutaneous masses, large stellate mesenchymal cells proliferate within a seromuci- nous homogenous matrix; a few inflammatory cells are present. Endothelial cells show hypertrophy and prolifera­tion; in severe disease, this results in narrowing of the lumen of capillaries and necrosis of the myxomatous lesions. In the epidermis overlying the masses, hyperplasia or degeneration may be noted. In the affected epidermis, as well as epidermal cells of the conjunctiva, intracytoplas- mic inclusions may be found. Additional findings may include lymphoid depletion of the spleen (common), alve­olar epithelial proliferation, hypertrophy and hyperplasia of reticulum cells in the lymph nodes and the spleen, focal necrosis, haemorrhage and proliferative vasculitis.

Antibodies can be detected starting from 8—13 days and remaining (as detected with complement fixation (CF)) for 6-8 months if there is no further contact with the virus.

CLINICAL SIGNS AND TREATMENT

Clinical signs include a primary subcutaneous mass as described above, blepharoconjunctivitis and oedema around the head, particularly the eyes (the swollen eyelids may be completely closed, with an obvious seropurulent discharge), and oedema of the perineal area, together with listlessness and fever.

In the amyxomatous form of the disease, which has been described in wild rabbits in France, Spain and Belgium, mainly respiratory signs develop, with few, small skin nodules. In New Zealand white rabbits experimentally infected with strains isolated from natural amyxomatous infections, blepharoconjunctivitis leading to closure of the eyes was the most prominent clinical sign, sometimes with diffuse swelling of the ears; one strain also produced acute respiratory distress in the rabbits, and abundant sero­mucous nasal discharge was noted.

Regarding treatment, for individual wild rabbits pre­sented to rehabilitation centres, maintenance at moder­ately high temperature (28°C) may be beneficial, together with general nursing care, non-steroidal anti-inflammatory drugs, and antibiotics to reduce the risk of secondary bacterial infections. Use of corticosteroids is contraindi­cated, due to their immunosuppressive effects. Opioid analgesics do not appear to reduce signs of pain in affected rabbits. However, the prognosis is poor and euthanasia is often preferable on welfare grounds.

DIAGNOSIS

Diagnosis of myxomatosis is based on the typical clinical signs of the disease. In atypical cases, virus isolation and further identification are required. Examination of a portion of lesions using negative-staining electron micros­copy reveals the typical poxvirus particles (but does not distinguish from Shope’s fibroma virus). A portion of lesion (particularly the eyelids) is used for infection of appropriate cell culture such as rabbit kidney cells (primary culture or established cell line), with a typical cytopathic effect gener­ally developing after 24—48 hours of incubation at 37°C (up to 7 days may be required for some strains).

In the agar gel immunodiffusion (AGID) test with stand­ard antiserum, several, usually three or less, lines of precipi­tation appear with myxoma virus but only one line if Shope’s fibroma virus is present (heterologous reaction)¹-²¹). The fluorescent antibody test can also be used on frozen tissue sections. PCR is also useful for rapid diagnosis.

In addition to CF, the indirect fluorescent antibody test, AGID and ELISA may be used for serology; ELISA is the most sensitive⁽²¹⁾.

MANAGEMENT, CONTROL AND REGULATIONS

When myxomatosis first occurred in England, control was attempted by local rabbit population elimination, but failed. In much of Europe, in situations in which wild rabbits were considered pests, control measures were not thought necessary. Experimentally in the UK, where the virus is transmitted mainly by rabbit fleas, insecticide treatment to eliminate the fleas also reduced myxomatosis in the treated population. Vaccines developed for use in domestic rabbits are effective in reducing mortality when wild rabbits (particularly juveniles) are vaccinated, but their use has been limited by the need to catch and hand- inject rabbits(²⁵). It has been suggested that vaccination campaigns have little effect and are not cost- effective¹-²⁶). Recombinant vaccines against both myxoma virus and rabbit haemorrhagic disease (RHD) (based on naturally attenuated myxoma virus, also expressing RHD major capsid protein VP60), with limited rabbit-to-rabbit trans­missibility, have been developed. Laboratory trials have demonstrated safety and efficacy, with safety and positive serological results in a limited field trial⁽²⁷⁾. Insecticide treatment of rabbit warrens may also be useful⁽²³⁾.

Myxomatosis is a very serious disease of domestic rabbits as well as wild rabbits, and it is one of three diseases of leporids notifiable to the World Organisation for Animal Health (OIE).

PUBLIC HEALTH CONCERN

Leporipox viruses do not infect humans.

SIGNIFICANCE AND IMPLICATIONS FOR ANIMAL HEALTH

In many parts of Europe, reductions in wild rabbit popula­tions due to myxomatosis when it was first introduced were considered to be beneficial, reducing crop losses. However, there have been widespread, complex and long­term effects on ecological communities. Of particular importance is the effect on European rabbits in their origi­nal habitats in the Iberian Peninsula. Here, rabbits are an important staple prey for a variety of mammalian and avian predators, including endangered species such as Iberian lynx (Lynx pardinus) and Spanish imperial eagles (Aquila adalberti). Reductions in rabbit populations due to myxomatosis and, more recently, RHD, have had severe negative consequences for several predators in Spain, exac­erbated in some cases by increased persecution of predators by rabbit hunting interests¹-²⁸).

HARE FIBROMA VIRUS INFECTION

This Leporipoxvirus infection causes hare fibromatosis, a disease that has been reported uncommonly in European brown hares in France, Germany, Italy and the UK. This disease was first recognized in the early 1900s, decades before the introduction of myxomatosis into Europe. Unlike myxomatosis in the European rabbit, but similar to that disease in its natural hosts, mortality is low, with most affected individuals showing spontaneous recovery. Morbidity may be high; during an outbreak on a hare farm in Italy in 2001, about 25% of the population was affected(²⁹). Affected individuals can act as sources of virus for a month or more and transmission is thought to occur mechanically via arthropods but may also occur through direct contact, with skin micro-lesions or trauma predis­posing to infection. Outbreaks in wild hares have occurred in late summer and autumn only(^29-31).

Lesions are solid skin tumours, 1—3 cm diameter, single or multiple, mainly on the ears, the head (particularly the eyelids) or the legs, but also found on the flank. The tumours spontaneously reduce in size and crust over after 4 to 6 weeks, and may spontaneously detach; at this stage, dry crusts or bleeding scars may be seen. Gross pathology reveals nodular swellings, with the base up to 3 cm in diameter and sometimes forming finger-like projections 4—5 cm long. Histologically, connective tissue proliferation is prominent, containing large spindle-shaped or star­shaped fibroblasts with large nuclei and in the cytoplasm, periodic acid-Schiff (PAS)-positive inclusions. Vascular hyperplasia is present, the new blood vessels having dilated walls and containing large numbers of red blood cells. Electron microscopy reveals typical poxvirus particles with randomly arranged surface tubules, both in crusted mate­rial and more abundantly in homogenized material from a nodular tumour. In inoculated embryonated chicken eggs, focal 1—2 mm yellowish-white pocks developed on the cho­rioallantoic membrane (CAM) after three passages, with poxvirus particles visible in material from the CAM^(31)(29).