Oral supplementation with postbiotics modulates the immune response produced by myxomatosis vaccination in wild rabbits.

Rabbits (Oryctolagus cuniculus) are vitally important species in the Iberian Peninsula ecosystem. However, since 1950, there has been a significant population decline, with major repercussions. This situation is mainly due to the presence of infectious diseases, such as myxomatosis, which is expanding and is characterized by severe and fatal clinical manifestations. Current control measures, mainly those based on vaccinations, are ineffective. Therefore, new strategies need to be developed and implemented. This study aimed to evaluate whether supplementation with postbiotic products modulates the immune response in wild rabbits vaccinated against myxomatosis. For this purpose, two groups of rabbits were established: a control group fed with standard feed ad libitum from weaning (28 days) until two months of age, and a treated group, which was fed under the same conditions but supplemented with postbiotics (3 kg/Tm). All the studied rabbits were vaccinated against this disease during weaning. In addition, a blood samples were obtained from all animals immediately before vaccination and 30 days later, which allowed us to evaluate the level of antibodies against myxomatosis virus (ELISA detection) and the relative expression of gene encoding to cytokines related to the immune response (IL6, TNFα and IFNγ), at both times of the experience. Weight and length measurements were also taken at both times to calculate body index and mean daily gain (MDG). No statistically significant differences in growth parameters were observed. There were also no differences in the serological response among groups. However, a relative underexpression of gene codifying to TNFα (p-value = 0.03683) and a higher expression on IFNγ (p-value = 0.045) were observed in the treated group. This modulation in cytokines could lead to less severe lesions in wild rabbit naturally infected with myxomatosis virus.

Epidemiologic, clinicopathologic, and diagnostic findings in pet rabbits with myxomatosis caused by the California MSW strain of myxoma virus: 11 cases (2022-2023).

OBJECTIVE: To determine epidemiologic features of naturally occurring myxomatosis in domestic rabbits in California and to characterize clinicopathologic and diagnostic findings. ANIMALS: 11 client-owned rabbits, Oryctolagus cuniculus subsp domesticus. CLINICAL PRESENTATION: A prospective study of pet rabbits with myxomatosis seen at an exotic animal specialty clinic in Santa Cruz county, California, was conducted between January 1, 2022, and December 31, 2023. Rabbits were included in the study if they had bilateral blepharedema and were PCR positive for myxoma virus. RESULTS: All infected rabbits had spent time outdoors. Common clinical signs included bilateral blepharedema (11/11), anogenital edema (10/11), rectal temperature ≥ 39.7 °C (5/9), and sudden death (4/11). Eyelid biopsies from all rabbits (11/11) were positive for myxoma virus by qualitative PCR followed by Sanger sequencing (100% nucleotide identity to strain MSW, also known as California/San Francisco 1950 [Genbank accession KF148065]). Most rabbits had keratinocytes containing eosinophilic intracytoplasmic viral inclusions in biopsies of edematous skin (8/11) and lymphocyte necrosis in the spleen (10/11). Immunohistochemistry identified myxoma virus in samples of skin, heart, lung, ileum, spleen, and lymph node. CLINICAL RELEVANCE: Clinical signs of myxomatosis caused by the MSW strain of myxoma virus are distinctive but subtle. Cases occur regularly in the Santa Cruz and San Jose regions of California. As infection with this virus is almost 100% fatal and no vaccine is available in the US, owners of domestic rabbits in endemic areas should keep their pets indoors or behind mosquito screens. Myxomatosis is a reportable disease in the US, and the appropriate state or federal agencies should be contacted when outbreaks occur.

Effect of Myxoma Virus Species Jump on Iberian Hare Populations.

The myxoma virus species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis) has raised concerns. We assess the decline suffered by Iberian hare populations on the Iberian Peninsula and discuss the association between the effect of myxomatosis and the average abundance index, which we estimated by using hunting bags.

Quantifying resistance to myxomatosis in wild rabbits produces novel evolutionary insights.

Wild rabbits in Australia developed genetic resistance to the myxoma virus, which was introduced as a biological control agent. However, little is known about the rate at which this evolutionary change occurred. We collated data from challenge trials that estimated rabbit resistance to myxomatosis in Australia and expressed resistance on a continuous scale, enabling trends in its development to be assessed over 45 years up to 1995. Resistance initially increased rapidly, followed by a plateau lasting ten years, before a second rapid increase occurred associated with the introduction of European rabbit fleas as myxoma virus vectors. By contrast, in the United Kingdom, where rabbit flea vectors were already present when the myxoma virus initially spread, resistance developed more slowly. No estimates of rabbit resistance to myxomatosis have been made for almost 30 years, despite other highly lethal rabbit pathogens becoming established worldwide. Continued testing of wild-caught rabbits in Australia to determine current levels of resistance to myxomatosis is recommended to assess its current effectiveness for managing pest rabbits. Given the economic and environmental significance of invasive rabbits, it would be remiss to manage such biological resources and ecosystem services poorly.

Divergent Evolutionary Pathways of Myxoma Virus in Australia: Virulence Phenotypes in Susceptible and Partially Resistant Rabbits Indicate Possible Selection for Transmissibility.

To characterize the ongoing evolution of myxoma virus in Australian rabbits, we used experimental infections of laboratory rabbits to determine the virulence and disease phenotypes of recent virus isolates. The viruses, collected between 2012 and 2015, fell into three lineages, one of which, lineage c, experienced a punctuated increase in evolutionary rate. All viruses were capable of causing acute death with aspects of neutropenic septicemia, characterized by minimal signs of myxomatosis, the occurrence of pulmonary edema and bacteria invasions throughout internal organs, but with no inflammatory response. For the viruses of highest virulence all rabbits usually died at this point. In more attenuated viruses, some rabbits died acutely, while others developed an amyxomatous phenotype. Rabbits that survived for longer periods developed greatly swollen cutaneous tissues with very high virus titers. This was particularly true of lineage c viruses. Unexpectedly, we identified a line of laboratory rabbits with some innate resistance to myxomatosis and used these in direct comparisons with the fully susceptible rabbit line. Importantly, the same disease phenotype occurred in both susceptible and resistant rabbits, although virulence was shifted toward more attenuated grades in resistant animals. We propose that selection against inflammation at cutaneous sites prolongs virus replication and enhances transmission, leading to the amyxomatous phenotype. In some virus backgrounds this creates an immunosuppressive state that predisposes to high virulence and acute death. The alterations in disease pathogenesis, particularly the overwhelming bacterial invasions that characterize the modern viruses, suggest that their virulence grades are not directly comparable with earlier studies. IMPORTANCE The evolution of the myxoma virus (MYXV) following its release as a biological control for European rabbits in Australia is the textbook example of the coevolution of virus virulence and host resistance. However, most of our knowledge of MYXV evolution only covers the first few decades of its spread in Australia and often with little direct connection between how changes in virus phenotype relate to those in the underlying virus genotype. By conducting detailed experimental infections of recent isolates of MYXV in different lines of laboratory rabbits, we examined the ongoing evolution of MYXV disease phenotypes. Our results reveal a wide range of phenotypes, including an amyxomatous type, as well as the impact of invasive bacteria, that in part depended on the level of rabbit host resistance. These results provide a unique insight into the complex virus and host factors that combine to shape disease phenotype and viral evolution.

Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway.

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB­dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short­half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

O coelho é a saúva: a proposta brasileira e o uso do vírus do mixoma (MYXV) contra a praga de coelhos na Austrália, 1896-1952 / Rabbits and leaf-cutting ants: the Brazilian plan to use the myxoma virus (MYXV) against rabbit plagues in Australia, 1896-1952

Resumo O artigo analisa a singularidade dos processos históricos, científicos e políticos que vão da descoberta da doença que passou a ser conhecida como mixomatose infecciosa, causada pelo vírus do mixoma (MYXV), à sua aplicação no controle de uma praga de coelhos na Austrália. A narrativa segue especialmente as pesquisas de Henrique de Beaurepaire Aragão, pesquisador do Instituto Oswaldo Cruz, e posteriormente os esforços da cientista Jean Macnamara para promover pesquisas e implementar o MYXV na Austrália. Foram consultadas notas de pesquisa de cientistas, documentos oficiais que registraram o desenvolvimento dos experimentos, bem como periódicos. Nesse processo, foi considerado o desenvolvimento histórico do campo de estudos da virologia e controle biológico.

Abstract This article analyzes the singularity of historical, scientific, and political processes from the discovery of the disease caused by the myxoma virus (MYXV) that came to be known as infectious myxomatosis to the application of this virus against a plague of rabbits in Australia. This narrative focuses on research by Henrique de Beaurepaire Aragão, a researcher at the Oswaldo Cruz Institute, and later efforts by the scientist Jean Macnamara to promote studies and implement MYXV in Australia. The scientists' research notes were consulted, along with official documents recording the experiments and periodicals. In this process, the historical development of virology and biological controls as a field of study was also considered.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated.

[Rabbits and leaf-cutting ants: the Brazilian plan to use the myxoma virus (MYXV) against rabbit plagues in Australia, 1896-1952]. / O coelho é a saúva: a proposta brasileira e o uso do vírus do mixoma (MYXV) contra a praga de coelhos na Austrália, 1896-1952.

This article analyzes the singularity of historical, scientific, and political processes from the discovery of the disease caused by the myxoma virus (MYXV) that came to be known as infectious myxomatosis to the application of this virus against a plague of rabbits in Australia. This narrative focuses on research by Henrique de Beaurepaire Aragão, a researcher at the Oswaldo Cruz Institute, and later efforts by the scientist Jean Macnamara to promote studies and implement MYXV in Australia. The scientists' research notes were consulted, along with official documents recording the experiments and periodicals. In this process, the historical development of virology and biological controls as a field of study was also considered.

O artigo analisa a singularidade dos processos históricos, científicos e políticos que vão da descoberta da doença que passou a ser conhecida como mixomatose infecciosa, causada pelo vírus do mixoma (MYXV), à sua aplicação no controle de uma praga de coelhos na Austrália. A narrativa segue especialmente as pesquisas de Henrique de Beaurepaire Aragão, pesquisador do Instituto Oswaldo Cruz, e posteriormente os esforços da cientista Jean Macnamara para promover pesquisas e implementar o MYXV na Austrália. Foram consultadas notas de pesquisa de cientistas, documentos oficiais que registraram o desenvolvimento dos experimentos, bem como periódicos. Nesse processo, foi considerado o desenvolvimento histórico do campo de estudos da virologia e controle biológico.

Viruses for Landscape-Scale Therapy: Biological Control of Rabbits in Australia.

Viral diseases, whether of animals or humans, are normally considered as problems to be managed. However, in Australia, two viruses have been used as landscape-scale therapeutics to control European rabbits (Oryctolagus cuniculus), the preeminent invasive vertebrate pest species. Rabbits have caused major environmental and agricultural losses and contributed to extinction of native species. It was not until the introduction of Myxoma virus that effective control of this pest was obtained at a continental scale. Subsequent coevolution of rabbit and virus saw a gradual reduction in the effectiveness of biological control that was partially ameliorated by the introduction of the European rabbit flea to act as an additional vector for the virus. In 1995, a completely different virus, Rabbit hemorrhagic disease virus (RHDV), escaped from testing and spread through the Australian rabbit population and again significantly reduced rabbit numbers and environmental impacts. The evolutionary pressures on this virus appear to be producing quite different outcomes to those that occurred with myxoma virus and the emergence and invasion of a novel genotype of RHDV in 2014 have further augmented control. Molecular studies on myxoma virus have demonstrated multiple proteins that manipulate the host innate and adaptive immune response; however the molecular basis of virus attenuation and reversion to virulence are not yet understood.

Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus).

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains' ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.

Seasonality and risk factors for myxomatosis in pet rabbits in Great Britain.

Myxomatosis is a highly contagious, frequently fatal viral disease affecting both wild and domesticated European rabbits across many areas of the world. Here we used electronic health records (EHRs) collected from pet rabbits attending a sentinel voluntary network of 191 veterinary practices across Great Britain (GB) between March 2014 and June 2019 to identify new features of this disease's epidemiology. From a total of 89,408 rabbit consultations, text mining verified by domain experts identified 207 (0.23 %) cases where myxomatosis was the only differential diagnosis recorded by the attending practitioner. Cases occurred in all months but February and were distributed across the country. Consistent with studies in wild rabbits, the majority of cases occurred between August and November. However, there was also evidence for considerable variation between years. A nested case control study identified important risk factors for myxomatosis within this pet animal population including season, sex, age, vaccination status and distance to likely wild rabbit habitats. Female entire rabbits were twice as likely to be a case (odds ratio (OR) 1.98, 95 % confidence interval (CI) 1.26-3.13, p = 0.003), suggesting a novel role for behaviour in driving transmission from wild to domesticated rabbits. Vaccination had the largest protective effect with vaccinated rabbits being 8.3 times less likely to be a case than unvaccinated rabbits (OR = 0.12, 95 % CI 0.06-0.21, p = <0.001).

Detection of viral DNA of myxoma virus using a validated PCR method with an internal amplification control.

Recognition of myxomatosis is usually based on clinical symptoms, but amyxomatous cases of the disease require the use of laboratory methods. Nowadays PCR assays are routinely employed for detection of MYXV DNA, but none of them have had their diagnostic usefulness conclusively confirmed through validation. The aim of the study was the development and validation of a PCR with an internal amplification control (IAC) for intravital and postmortem detection of viral DNA of myxoma virus. To avoid false negative results a chimeric internal amplification control (IAC) was prepared and incorporated into the PCR and amplified by the same primer set as the target DNA (M071L). The optimal concentration of particular ingredients in the PCR mixture (including IAC concentration and volume of DNA sample) was determined. To minimize the risk of amplicon carry-over contamination, uracil N-glycosylase was added to the reaction. Before proper validation the robustness of the IAC-PCR was verified. Validation of the method encompassed the following parameters: the analytical and diagnostic specificity (ASp, DSp) and sensitivity (ASe, DSe) of the assay, repeatability, and intra-laboratory reproducibility. The assay LOD was established at 2 TCIU of the virus particles/0.2 ml tissue homogenate with a 100% capacity to detect different MYXV strains (ASp). The method was characterized by good DSp of 0.955 (0.839-0.999 CI) and DSe of 0.976 (0.914-1.00 CI). In addition, it was repeatable and reproducible and confirmed its suitability for the detection of MYXV in clinical material. The IAC-PCR developed meets OIE validation requirements for virological methods and can be used in diagnostic or epidemiological studies of rabbit myxomatosis.

Parallel adaptation of rabbit populations to myxoma virus.

In the 1950s the myxoma virus was released into European rabbit populations in Australia and Europe, decimating populations and resulting in the rapid evolution of resistance. We investigated the genetic basis of resistance by comparing the exomes of rabbits collected before and after the pandemic. We found a strong pattern of parallel evolution, with selection on standing genetic variation favoring the same alleles in Australia, France, and the United Kingdom. Many of these changes occurred in immunity-related genes, supporting a polygenic basis of resistance. We experimentally validated the role of several genes in viral replication and showed that selection acting on an interferon protein has increased the protein's antiviral effect.

Detecting European Rabbit ( Oryctolagus cuniculus) Disease Outbreaks by Monitoring Digital Media.

Digital media and digital search tools offer simple and effective means to monitor for pathogens and disease outbreaks in target organisms. Using tools such as Rich Site Summary feeds, and Google News and Google Scholar specific key word searches, international digital media were actively monitored from 2012 to 2016 for pathogens and disease outbreaks in the taxonomic order Lagomorpha, with a specific focus on the European rabbit ( Oryctolagus cuniculus). The primary objective was identifying pathogens for assessment as potential new biocontrol agents for Australia's pest populations of the European rabbit. A number of pathogens were detected in digital media reports. Additional benefits arose in the regular provision of case reports and research on myxomatosis and rabbit haemorrhagic disease virus that assisted with current research.

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.