Nowhere to run, rabbit: the cold-war calculus of disease ecology.

During the cold war, Frank Fenner (protégé of Macfarlane Burnet and René Dubos) and Francis Ratcliffe (associate of A. J. Nicholson and student of Charles Elton) studied mathematically the coevolution of host resistance and parasite virulence when myxomatosis was unleashed on Australia's rabbit population. Later, Robert May called Fenner the "real hero" of disease ecology for his mathematical modeling of the epidemic. While Ratcliffe came from a tradition of animal ecology, Fenner developed an ecological orientation in World War II through his work on malaria control (with Ratcliffe and Ian Mackerras, among others)-that is, through studies of tropical medicine. This makes Fenner at least a partial exception to other senior disease ecologists in the region, most of whom learned their ecology from examining responses to agricultural challenges and animal husbandry problems in settler colonial society. Here I consider the local ecologies of knowledge in southeastern Australia during this period, and describe the particular cold-war intellectual niche that Fenner and Ratcliffe inhabited.

A case of low success of blind vaccination campaigns against myxomatosis and rabbit haemorrhagic disease on survival of adult European wild rabbits.

Vaccination campaigns against myxomatosis and rabbit haemorrhagic disease (RHD) are commonly used in translocation programs conducted for the purpose of recovering wild European rabbit populations in Iberian Mediterranean ecosystems. In most cases rabbits are vaccinated 'blind' (i.e. without assessing their prior immunological status) for economic and logistic reasons. However, there is conflicting evidence on the effectiveness of such an approach. We tested whether blind vaccination against myxomatosis and rabbit haemorrhagic disease improved rabbit survival in a rabbit translocation program where wild rabbits were kept in semi-natural conditions in three enclosures. We conducted nine capture sessions over two years (2008-2010) and used the information collected to compare the survival of vaccinated (n=511) versus unvaccinated (n=161) adult wild rabbits using capture-mark-recapture analysis. Average monthly survival was no different for vaccinated versus unvaccinated individuals, both in the period between release and first capture (short-term) and after the first capture onward (long-term). Rabbit survival was lower in the short term than in the long term regardless of whether rabbits were vaccinated or not. Lower survival in the short-term could be due to the stress induced by the translocation process itself (e.g. handling stress). However, we did not find any overall effect of vaccination on survival which could be explained by two non-exclusive reasons. First, interference of the vaccine with the natural antibodies in the donor population. Due to donor populations have high density of rabbits with, likely, high prevalence of antibodies as a result of previous natural exposure to these diseases. Second, the lack of severe outbreaks during the study period. Based on our findings we argue that blind vaccination of adult rabbits in translocation programs may be often mostly ineffective and unnecessarily costly. In particular, since outbreaks are hard to predict and vaccination of rabbits with natural antibodies is ineffective, it is crucial to assess the immunological status of the donor population before translocating adult rabbits.

Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms.

Despite the success of vaccination against myxoma virus, myxomatosis remains a problem on rabbit farms throughout Spain and Europe. In this study we set out to evaluate possible causes of myxoma virus (MYXV) vaccine failures addressing key issues with regard to pathogen, vaccine and vaccination strategies. This was done by genetically characterising MYXV field isolates from farm outbreaks, selecting a representative strain for which to assay its virulence and measuring the protective capability of a commercial vaccine against this strain. Finally, we compare methods (route) of vaccine administration under farm conditions and evaluate immune response in vaccinated rabbits. The data presented here show that the vaccine tested is capable of eliciting protection in rabbits that show high levels of seroconversion. However, the number of animals failing to seroconvert following subcutaneous vaccination may leave a large number of rabbits unprotected following vaccine administration. Successful vaccination requires the strict implication of workable, planned, on farm programs. Following this, analysis to confirm seroconversion rates may be advisable. Factors such as the wild rabbit reservoir, control of biting insects and good hygienic practices must be taken into consideration to prevent vaccine failures from occurring.

Cutaneous lesions in pet rabbits following subcutaneous administration of a novel bivalent vaccine against myxomatosis and rabbit haemorrhagic disease.

BACKGROUND: A novel bivalent vaccine to protect against myxomatosis and rabbit haemorrhagic disease is commercially available for pet rabbits. HYPOTHESIS/OBJECTIVES: To describe the appearance of cutaneous lesions arising in pet rabbits positive for myxoma virus (MV) by RT-PCR evaluation shortly after vaccination. ANIMALS: Four pet rabbits presenting with papular, crusting skin lesions ~10 days after vaccination. METHODS: Histological evaluation of formalin-fixed skin biopsies obtained from lesional skin (case 1). Real-time polymerase chain reaction (RT-PCR) evaluation of paraffin-embedded tissue from skin biopsies (case 1) and crusts obtained from the lesion surface (cases 2-4) for myxoma virus are reported as cycle threshold (Ct ) values. RESULTS: Lesions affecting the ear pinna, dorsal aspect of the nose, vulva and/or conjunctiva are reported. Histopathological findings included severe ulcerative, necrotizing dermatitis and intralesional cytoplasmic inclusion bodies in myxoma cells. DNA was amplified from all the paraffin-embedded skin biopsies (Ct  = 34-35) and crusts (Ct  = 20-24). CONCLUSIONS AND CLINICAL IMPORTANCE: Although a wild virus challenge cannot be definitively excluded, veterinarians and pet-owners should be aware that cutaneous lesions have been observed after vaccination with this novel vaccine in low numbers of rabbits.

Viral skin diseases of the rabbit.

This article describes the viral skin diseases affecting the domestic rabbit, the most important being myxomatosis. Transmission and pathogenesis, clinical signs, diagnosis, treatment, and control are described and the article will be of interest to veterinary practitioners who treat rabbits. Shope fibroma virus, Shope papilloma virus, and rabbitpox are also discussed.

Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells.

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease.

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.

The current status and future directions of myxoma virus, a master in immune evasion.

Myxoma virus (MYXV) gained importance throughout the twentieth century because of the use of the highly virulent Standard Laboratory Strain (SLS) by the Australian government in the attempt to control the feral Australian population of Oryctolagus cuniculus (European rabbit) and the subsequent illegal release of MYXV in Europe. In the European rabbit, MYXV causes a disease with an exceedingly high mortality rate, named myxomatosis, which is passively transmitted by biting arthropod vectors. MYXV still has a great impact on European rabbit populations around the world. In contrast, only a single cutaneous lesion, restricted to the point of inoculation, is seen in its natural long-term host, the South-American Sylvilagus brasiliensis and the North-American S. Bachmani. Apart from being detrimental for European rabbits, however, MYXV has also become of interest in human medicine in the last two decades for two reasons. Firstly, due to the strong immune suppressing effects of certain MYXV proteins, several secreted virus-encoded immunomodulators (e.g. Serp-1) are being developed to treat systemic inflammatory syndromes such as cardiovascular disease in humans. Secondly, due to the inherent ability of MYXV to infect a broad spectrum of human cancer cells, the live virus is also being developed as an oncolytic virotherapeutic to treat human cancer. In this review, an update will be given on the current status of MYXV in rabbits as well as its potential in human medicine in the twenty-first century.

Genome sequence of SG33 strain and recombination between wild-type and vaccine myxoma viruses.

Myxomatosis in Europe is the result of the release of a South America strain of myxoma virus in 1952. Several attenuated strains with origins in South America or California have since been used as vaccines in the rabbit industry. We sequenced the genome of the SG33 myxoma virus vaccine strain and compared it with those of other myxoma virus strains. We show that SG33 genome carries a large deletion in its right end. Furthermore, our data strongly suggest that the virus isolate from which SG33 is derived results from an in vivo recombination between a wild-type South America (Lausanne) strain and a California MSD-derived strain. These findings raise questions about the use of insufficiently attenuated virus in vaccination.

Molecular characterization of SG33 and Borghi vaccines used against myxomatosis.

Myxoma virus is a poxvirus responsible for myxomatosis in European Rabbits (Oryctolagus cuniculus). The entire genome of the myxoma virus has been sequenced, allowing a systemic survey of the functions of a large number of putative pathogenic factors that this virus expresses to subvert the immune and inflammatory pathways of infected rabbit hosts. In Italy, industrial rabbits are mostly vaccinated against myxomatosis using the attenuated myxoma virus strains Borghi or SG33. We have identified genetic markers specific for Borghi or SG33 vaccine strains and established a PCR-based assay that could be used to: (a) rapidly diagnose the presence of myxoma virus in infected organs; (b) discriminate between field strain-infected and vaccinated rabbits and (c) differentiate between Borghi or SG33 vaccine strain.

Vaccination strategies against myxomavirus infections: are we really doing the best?

Vaccination is the best way to control myxomatosis in both pet and production rabbits. Two types of myxomatosis vaccines are commercially available, namely, a vaccine prepared from the Shope fibroma virus (SFV) and one prepared from an attenuated myxoma virus (MV) strain, e.g., SG33. The first one is weakly immunogenic and provides only short-term protection whereas atypical reactions have been described with the second one. This short review describes the vaccine strains and provides some data on the host-virus relationship, resistance, and immunity in myxomatosis. In the last section, recommended myxomatosis vaccination schemes for production and pet animals are presented.

Myxomatosis in wild rabbit: design of control programs in Mediterranean ecosystems.

A cross-sectional study was carried out in natural wild rabbit (Oryctolagus cuniculus) populations from southern Spain to identify risk factors associated to myxoma virus infection. Blood samples from 619 wild rabbits were collected, and questionnaires which included variables related to host, disease, game management and environment were completed. A logistic regression analysis was conducted to investigate the associations between myxomatosis seropositivity (dependent variable) across 7 hunting estates and an extensive set of explanatory variables obtained from the questionnaires. The prevalence of antibodies against myxomatosis virus was 56.4% (95% CI: 52.5-60.3) and ranged between 21.4% (95% CI: 9.0-33.8) and 70.2% (95% CI: 58.3-82.1) among the different sampling areas. The logistic regression analysis showed that autumn (OR 9.0), high abundance of mosquitoes (OR 8.2), reproductive activity (OR 4.1), warren's insecticide treatment (OR 3.7), rabbit haemorrhagic disease (RHD) seropositivity (OR 2.6), high hunting pressure (OR 6.3) and sheep presence (OR 6.4) were associated with seropositivity to myxomatosis. Based on the results, diverse management measures for myxomatosis control are proposed.

Field experimental vaccination campaigns against myxomatosis and their effectiveness in the wild.

We conducted a field experiment in SW Spain to test the efficacy of a myxomatosis vaccine, a viral disease strongly affecting wild rabbit populations, by assessing individual survival and antibody seroprevalence of monthly live-trapped, vaccinated (N=466) and unvaccinated (N=558) juvenile wild rabbits, between April and October 2007. Eight percent of all juveniles caught from April to June showed maternal antibodies against myxomatosis, whereas all animals were seropositive to the disease after the outbreak. Juveniles vaccinated before the outbreak showed 17% higher survival (31% vs. 14%) and an increased mortality probability of 8% after the outbreak. Results suggest that only a costly and systematic vaccination performed before the annual myxomatosis outbreak, would improve the survival of juvenile rabbits, a premise not always accomplished that compromises its efficacy in the field.

Ultrastructural study of poxvirus causing myxomatosis in rabbits, in São Paulo and Santa Catarina, Brazil / Estudio ultraestructural del poxvirus causal de mixomatosis en conejos, en São Paulo y Santa Catarina, Brasil

The myxomatosis is a contagious worldwide disease caused by poxvirus which infects domestic and wild rabbits. In the present study we present two distinct outbreaks of myxomatosis when raising rabbits, one for commercial purpose of production of meat and skins and, another one for the commercialization of ornamental rabbits. The observed signs were ocular, auricular, nasal, testis lesions and many times scattered throughout the body of the animals. The lesions were characterized by formation of nodules that by palpation disclosed gummy or gelatinous aspect. At the transmission electron microscopy, all the skin and crust samples were analyzed by negative staining technique. A great number of particles with morphology similar to the poxvirus, some enveloped in a brick-shaped and irregular disposition of tubules on the external membrane, measuring 300x240 nm on the average were visualized. Ultra thin sections revealed the presence of intracytoplasmic inclusion bodies surrounded by membrane containing oval particles, measuring 270 x 130 nm, containing nucleus or an internal biconcave (dumbbell-shaped) core. Immature particles (empty), surrounded by membrane were also observed. In addition, intracytoplasmic electron dense inclusion bodies containing viral particles budding of dense amorphous material and intranuclear fibrillar or "digital" inclusions showing a regular striation and arranged in groups were found in the middle of granular material. The nuclei were deformed with densely condensed chromatin forming amorphous and electron dense inclusion bodies. In the immunocytochemistry technique, the antigen-antibody reaction was strongly marked by the particles of colloidal gold, emphasizing the viral particles. The techniques used in this study were important in the diagnosis of the affected animals.

La mixomatosis es una enfermedad contagiosa de distribución mundial, causada por poxvirus que infecta conejos domésticos y salvajes. En este estudio presentamos dos distintos surtos por mixomatosis que ocurrieron en producciones de conejos, una para fines comerciales de producción de carne y pieles y otra para el comercio de conejos domésticos. Las señales observadas fueron afecciones oculares, nasales, testiculares y, a veces, también distribuida por todo el cuerpo de los animales. Estas se caracterizaban por formación de nódulos que a la palpación tenían un aspecto gelatinoso o gomoso. En la microscopía electrónica de transmisión, por la técnica de contrastación negativa, se pudo observar en todas las muestras examinadas de piel y de costras, un gran número de partículas típicas de poxvirus, con envoltura y forma de ladrillo, mostrando disposición irregular de los túbulos sobre la membrana externa, midiendo 300 x 240 nm en el promedio. Cortes ultrafinos de fragmentos de piel y de costras revelaron la presencia de cuerpos de inclusión intracitoplasmáticas, envueltos por membrana y conteniendo partículas ovales, midiendo 270 x 130 nm, conteniendo núcleo o centro interno bicóncavo (forma de mancuernas). Partículas inmaduras (vacías) envueltas por membrana fueron observadas. También fueron analizados cuerpos de inclusión intracitoplasmáticos, electrodensos, conteniendo partículas virales brotando del material denso y amorfo. Fueron observadas inclusiones intranucleares fibrilares o "digitales" mostrando una estriación periódica y disposición en grupos en medio del material granular. Los núcleos estaban deformados con cromatina densamente condensada formando cuerpos de inclusiones electrodensas y amorfas. En la técnica de imunocitoquímica la reacción antígeno-anticuerpo fue intensamente marcada por las partículas de oro coloidal realzando fuertemente las partículas virales.

Vaccination of free-living juvenile wild rabbits (Oryctolagus cuniculus) against myxomatosis improved their survival.

For several decades, the populations of the European wild rabbit (Oryctolagus cuniculus) have declined, which is partly due to myxomatosis. Vaccination against this disease is expected to contribute to restoration of rabbit populations but the actual impact of myxomatosis is not well known and vaccination might have some negative effects. We analyzed the capture-mark-recapture data obtained in a 4-year field experiment (1991-1994) in a park near Paris, France wherein 300 out of 565 seronegative juvenile rabbits were vaccinated at first capture against myxomatosis with the nontransmissible Dervaximyxo SG33 vaccine. After accounting for weight at first capture, age-class (juvenile/adult), "trap-happiness" and season (spring/autumn) of the capture event, vaccinated rabbits had 1.8-fold greater odds of surviving than the unvaccinated rabbits. The average summer survival risk for vaccinated juveniles was 0.63 (+/-0.08 S.E.) whereas it was 0.48 (+/-0.08 S.E.) for unvaccinated juvenile rabbits.

Effects of vaccination against viral haemorrhagic disease and myxomatosis on long-term mortality rates of European wild rabbits.

The effects of vaccination against myxomatosis and viral haemorrhagic disease (VHD) on long-term mortality rates in European rabbits (Oryctolagus cuniculus) were studied from 1993 to 1996 by radiotracking a free-living population of wild rabbits. During the three months after immunisation, unvaccinated young rabbits weighing between 180 and 600 g were 13.6 times more likely to die than vaccinated young rabbits. In adult rabbits, vaccination did not significantly decrease mortality, mainly owing to the high proportion of rabbits which had previously been exposed to the antigens of both diseases. Compared with adult rabbits with natural antibodies to VHD, rabbits without these antibodies were 5.2 times more likely to die of VHD during annual outbreaks.

Occurrence of atypical myxomatosis in Central Europe: clinical and virological examinations.

An outbreak of the atypical form of myxomatosis struck a rabbit farm in Hungary. The animals had previously been vaccinated with a vaccine containing Shope rabbit fibroma virus strain. The disease appeared in winter when the presence of mosquitoes and fleas is not common. The virus was isolated from an eyelid specimen of a naturally infected rabbit. The surviving animals were observed for four weeks, blood samples were collected and, after euthanasia, organ specimens were also examined by morphological methods including pathology and electron microscopy. Serum samples were examined by virus neutralisation for antibodies. Genetic analysis of the isolated virus was carried out by polymerase chain reaction (PCR) and direct sequencing. The primers were designed on the basis of the major envelope gene (Env) of the Lausanne reference strain in the GenBank. The viral proteins were examined by SDS-PAGE. The isolated virus (ref. no.: BP04/2001) was able to infect the susceptible animals directly, by contact. The disease was characterised by respiratory symptoms of the upper tracheal tract, conjunctivitis and high mortality by the 11th-14th day. Aerogenic infection with strain BP04/2001 resulted in 100% morbidity among the susceptible animals. Sequencing of the amplified 400-bp-long DNA revealed 97% homology with the Env gene of the Lausanne strain, which proves that strain BP04/2001 is a variant of the Lausanne strain having been enzootic throughout Europe. The live vaccine strain used in Hungary against myxomatosis, which is also a Lausanne-derived strain, protected the animals. According to the protein analysis a protein of 200 kDa in size is not expressed in strain BP04/2001. This is the first report on atypical myxomatosis in Central Europe. The virus spreads by airborne transmission and may cause severe losses in the rabbit population.

First field trial of a transmissible recombinant vaccine against myxomatosis and rabbit hemorrhagic disease.

As a novel approach for immunisation of wild rabbits, we have recently developed a transmissible vaccine against myxomatosis and rabbit hemorrhagic disease (RHD) based on a recombinant myxoma virus (MV) expressing the RHDV capsid protein [J. Virol. 74 (2000) 1114]. The efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions. In this study, we report the first limited field trial of the candidate vaccine that was undertaken in an island of 34 Has containing a population of around 300 rabbits. Following administration by the subcutaneous route to 76 rabbits, the vaccine induced specific antibody responses against both myxomatosis and RHDV in all the inoculated rabbits. Furthermore, the recombinant virus exhibited a limited horizontal transmission capacity, promoting seroconversion of around 50% of the uninoculated rabbit population. No evidence of undesirable effects due to the recombinant virus field release was detected.

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease.

We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [Bárcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114-23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its field use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo.