Seasonality of Coxiella burnetii among Wild Rabbits (Oryctolagus cuniculus) and the Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem

(1) Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs. Presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.

Distribution and Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV genomes were amplified and sequenced from 48 positive samples. Furthermore, we tested 380 serum samples from 11 locations across Australia for antibodies against HEV. HEV was detected in rabbits from all states and territories, except the Northern Territory. Seroprevalence varied between locations (from 0% to 22%), demonstrating that HEV is widely distributed in rabbit populations across Australia. Phylogenetic analyses showed that Australian HEV sequences are genetically diverse and that HEV was likely introduced into Australia independently on several occasions. In summary, this study broadens our understanding of the genetic diversity of rabbit HEV globally and shows that the virus is endemic in both domestic and wild rabbit populations in Australia.

Wild Rabbit Exposure to Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi Evidenced by Serum and Aqueous Humor Antibody Detection

Wild rabbits (Oryctolagus cuniculus) can be important sentinel species for the presence of zoonotic pathogens. Therefore, we collected blood samples from wild rabbits harvested by hunters during the hunting season 2019–2020 on the island of Lemnos, to determine exposure of wild rabbits to the zoonotic pathogens Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi, as well as aqueous humor to assess its diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios. Antibodies against these pathogens were detected by Indirect Immunofluorescence Antibody (IFA) assay. Out of the 72 wild rabbits included in the study, 4.2%, 5.5%, 18% and 9.7% were seropositive to L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively. Although less frequently, antibodies were also detected in aqueous humor of wild rabbits. The antibody detection in aqueous humor presented 100% specificity but decreased sensitivity compared to serum suggesting that aqueous humor could be successfully used in epidemiological studies to confirm exposure at the population level but has little diagnostic value at the individual level. This is the first report on the seropositivity of wild rabbits to A. phagocytophilum and B. caballi and the detection of antibodies against A. phagocytopylum, L. infantum, T. gondii and B. caballi in the aqueous humor.

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.

What Are the Keys to the Adaptive Success of European Wild Rabbit (Oryctolagus cuniculus) in the Iberian Peninsula?

The European wild rabbit (Oryctolagus cuniculus) plays an important ecological role in the ecosystems of the Iberian Peninsula. Recently, rabbit populations have drastically reduced, so the species is now considered endangered. However, in some places, this animal is considered a pest. This is the conservation paradox of the 21st century: the wild rabbit is both an invasive alien and an endangered native species. The authors of this review aimed to understand the keys to the adaptive success of European rabbits, addressing all aspects of their biology in order to provide the keys to the ecological management of this species. Aspects including nutrition, genetics, immunity interactions with the environment, behaviour, and conflict with human activities were reviewed. Ultimately, rabbits are resilient and adaptable. The main adaptations that explain the rabbit’s adaptive success are its nutrition (wide adaptation to food and good nutritional use of caecotrophy), immune system (powerful and developed), and other aspects related to genetics and behaviour. Rabbits’ relationship with humans has led them to colonise other places where they have become pests. Despite these adaptations, populations in native places have been drastically reduced in recent years. Since it serves as a bastion of the Mediterranean ecosystem, a specific conservation program for this species must be carried out. Therefore, a study of the rabbit’s response to diseases and nutrition (especially protein), as well as the interaction between them, is of special interest.

Age and Infectious Dose Significantly Affect Disease Progression after RHDV2 Infection in Naïve Domestic Rabbits

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.