Wild rabbits are Leishmania infantum reservoirs in southeastern Spain.

OBJECTIVE: We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. RESULTS: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3-554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. CONCLUSIONS: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates.

Occurrence of Anaplasma spp. in wild lagomorphs from Southern Spain: Molecular detection of new Anaplasma bovis lineages.

Wild lagomorphs can act as reservoirs of several pathogens of public and animal health concern. However, the number of studies assessing the presence of Anaplasma spp. in these species is scarce. The aim of the present study was to molecularly identify Anaplasma spp. in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) from Southern Spain and assess their epidemiological role in the maintenance of the bacterium. During 2017-2021, spleen samples of 394 wild rabbits and 145 Iberian hares were collected. Anaplasma DNA was detected using different PCR assays (16S rRNA and groEL) and phylogenetic analyses were carried out by Bayesian approach. The possible influence of lagomorph species, age and sex on the prevalence of Anaplasma spp. was evaluated by a multiple logistic regression model. The 9.4% of the rabbits were positive to Anaplasma bovis, but all the hares were negative. No significant differences were found in Anaplasma spp. prevalence regarding to age or sex. This is the first report of A. bovis in lagomorphs from Europe. The phylogenetic analysis of A. bovis confirms the existence of different clusters suggesting the existence of several lineages. In addition, a high divergence of nucleotide identity was observed within the lineage 4, which could result in the under-detection of some strains when using A. bovis-specific PCR, hindering its detection and characterization. Since this analysis is based on a limited number of nucleotide bases and sequences, more studies are needed for further characterize A. bovis, as well as its relationship with other Anaplasma spp.

Hepatic Coccidiosis in Wild Rabbits in Greece: Parasite Detection on Liver Imprints and the Associated Biochemical Profile.

(1) Background: Rabbit hepatic coccidiosis, caused by Eimeria stiedae, is a devastating disease with high morbidity and mortality rates. The disease is well described in rabbits, but little is known about E. stiedae infection in wild rabbits. In this study, we investigated the presence of E. stiedae infection in wild rabbits from the island of Lemnos, Greece, where this species is overpopulated, and the effects of infection on common hepatic biomarkers. (2) Methods: We used liver impression smears to detect the coccidian oocysts, and we defined the liver biochemical profile of the infected individuals. (3) Results: Overall, 13.3% of the liver imprints examined were positive for the presence of coccidial oocysts. The activities of liver enzymes, that is, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamyltransferase (GGT), as well as globulins (GLOB), were increased while the concentrations of albumins (ALB), total proteins (TP) and the albumin to globulin (A/G) ratio were decreased in the infected individuals compared to the non-infected ones. (4) Conclusions: This study adds to the current knowledge on the pathogens affecting wild rabbits and those circulating in this population on the island of Lemnos, Greece. Moreover, we showed that E. stiedae infection exerts pathological effects on the hepatocyte integrity and liver function of wild rabbits, as reflected by the abnormal values of liver injury and dysfunction biomarkers.

Molecular detection of Ehrlichia spp. in ticks parasitizing wild lagomorphs from Spain: characterization of a novel Ehrlichia species.

BACKGROUND: Several species belonging to the genus Ehrlichia are considered pathogenic for animals and humans. Although wildlife are known to play an important role in the epidemiology of these bacteria, information on the role of wild lagomorphs in their sylvatic cycle is limited. Thus, the objective of the present study was to assess the occurrence of Ehrlichia spp. in ticks collected from wild lagomorphs in Spanish Mediterranean ecosystems. METHODS: A total of 1122 pooled ticks (254 pools) collected from 506 wild rabbits (Oryctolagus cuniculus) and 29 Iberian hares (Lepus granatensis) were analysed using a nested PCR assay targeting the partial groEL gene. Ehrlichia spp.-positive samples were further subjected to a second PCR assay targeting 16S rRNA. RESULTS: Three (1.2%) tick pools comprising Rhipicephalus pusillus collected from nine wild rabbits were positive for Ehrlichia spp. All the Ehrlichia DNA sequences were identical, and use of sequence and phylogenetic analyses allowed us to identify a novel Ehrlichia species. CONCLUSIONS: We provide evidence that a novel Ehrlichia species, named herein as 'Candidatus Ehrlichia andalusi', which may be of concern for animal and public health, is circulating in R. pusillus in Spanish Mediterranean ecosystems. Further studies are warranted to assess the epidemiology, pathogenicity and zoonotic potential of this Ehrlichia species.

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.

Role of wild rabbits as reservoirs of leishmaniasis in a non-epidemic Mediterranean hot spot in Spain.

There is limited information regarding the role of wild mammals in the transmission dynamics of Leishmania infantum. A potential human leishmaniasis hot spot was detected in southern Spain that could not be explained solely by canine leishmaniasis prevalence. The aim of this work was to analyse the involvement of wild rabbits as the main factor affecting this Mediterranean hot spot. A survey of wild rabbits, dogs and sand flies was conducted in the human cases environment. A nearby region without clinical leishmaniasis cases was used as reference control. 51 wild rabbits shot by hunters were analysed by molecular techniques. 1100 sand flies were captured and morphologically identified. Blood collected from patients' relatives/ neighbours (n = 9) and dogs (n = 66) was used for molecular analysis and serology. In Mediterranean leishmaniasis hot spots such as Montefrío municipality (average incidence of 16.8 human cases per 100,000 inhabitants/year), wild rabbits (n = 40) support high L. infantum infection rates (100%) and heavy parasite burdens (average value: 503 parasites/mg) in apparently normal ear skin directly accessible to sand flies, enabling the existence of heavily parasitized Phlebotomus perniciosus females (12.5% prevalence). The prevalence of infection and median parasite load were very low among rabbits captured in Huéscar (n = 11), a human clinical leishmaniasis-free area for the last 18 years. P. perniciosus was the most abundant Phlebotomus species in all the domestic/peridomestic microhabitats sampled, both indoors and outdoors. Accordingly, leishmaniasis is clustering in space and time at this local scale represented by Montefrío due to the proximity of two competent host reservoirs (dogs and heavily parasitized wild rabbits) associated with overlapping sylvatic and domestic transmission cycles through the main vector, P. perniciosus. We highlight the usefulness of determining the prevalence of infection and parasite burden in wild rabbits as a control leishmaniasis measure with the advantage that the use of the ear offers.

Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain.

The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set off alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of different genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Identification of Alpha and Beta Coronavirus in Wildlife Species in France: Bats, Rodents, Rabbits, and Hedgehogs.

Coronaviruses are closely monitored in the context of emerging diseases and, as illustrated with Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-coronavirus (MERS-CoV), are known to cross the species barrier and eventually to move from wildlife to humans. Knowledge of the diversity of coronaviruses in wildlife is therefore essential to better understand and prevent emergence events. This study explored the presence of coronaviruses in four wild mammal orders in France: Bats, rodents, lagomorphs, and hedgehogs. Betacoronavirus and Alphacoronavirus genera were identified. The results obtained suggest the circulation of potentially evolving virus strains, with the potential to cross the species barrier.

The effect of excluding ungulates on the abundance of ixodid ticks on wild rabbit (Oryctolagus cuniculus).

Tick abundance depends heavily on host density, so reducing access to the host should reduce tick populations in a determined area. In this study we compared the Parasitic Index (PI = average number of ticks per rabbit) of two wild rabbit populations separated 16 years ago by a fence keeping ungulates from moving freely. Two areas were selected (closed and open) wherein 50 wild rabbits per area were sampled for ticks. The PI in the open area (PI = 989.62) was significantly higher than in the closed area (PI = 515.40). Hyalomma lusitanicum Koch was globally the most abundant species, followed by Rhipicephalus pusillus Gil Collado, Rhipicephalus bursa Canestrini and Fanzago, Haemaphysalis hispanica Gil Collado, Rhipicephalus sanguineus s.l. Latreille and Ixodes ventalloi Gil Collado. Differences between areas varied depending on the species. The number of H. hispanica, R. bursa and R. pusillus were significantly more abundant on rabbits in the closed area, whereas H. lusitanicum predominated in the open area. Ungulates in the open area may have played an important role as the main or alternative host for ticks and/or drawing some tick species away from rabbits. In the closed area other reasons such as inter-specific competition could have influenced the tick abundance. These results show a clear reduction in tick abundance for at least 16 years as well as influenced species distribution.

Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016-2017).

We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.

Genetic variability and infective ability of the rabbit trypanosome, Trypanosoma nabiasi Railliet 1895, in southern Spain.

Trypanosomes are widespread haemoflagellate protozoans, commonly found in all groups of vertebrates and usually transmitted by arthropods. Non-pathogenic species are those that cause little or no apparent negative effects in the host and it is accepted that Trypanosoma nabiasi is the species that infects the domestic and wild rabbit, Oryctolagus cuniculus. Knowledge about genetic variability, in vitro cultivation and infectivity of this parasite is very scarce, so the aim of this study was to provide an insight on them. The parasite was detected in all the type of samples of 121 wild rabbits. Epimastigotes were visualized and isolated from all the organ cultures types except from skin, and twenty-six strains were isolated and grown in mass. Epimastigote infectivity was assessed in vitro and in vivo. Amastigotes were obtained in infected macrophages from cultured epimastigotes. Furthermore, trypomastigotes were found in the peripheral bloodstream of an experimentally infected naïve domestic rabbit with cultured epimastigotes at the fourth day after infection. The rising titre of antibodies led to the disappearance of the parasite from blood. In addition, this study reports the existence of two T. nabiasi genetic lineages in southern Spain. Phylogenetic analysis places T. nabiasi in the same clade as T. lewisi and other rodent trypanosomes of the subgenus Herpetosoma.

Seasonal dynamics of ixodid ticks on wild rabbits Oryctolagus cuniculus (Leporidae) from Central Spain.

Rabbit Hemorrhagic Disease and Myxomatosis caused a decline in the rabbit population in the second half of the last century. Despite some recovery, the risk of vector-borne disease is present and thus the importance of controlling vector populations. In the current study, we describe the ixodid tick fauna in wild rabbit in a natural reserve in Ciudad Real (Central Spain) during the course of two 3-year periods (2007-2009 and 2012-2014). Of all the ticks collected on average 72.5 % were larvae, 24.4 % nymphs and 3.1 % adults, although the percentage varied monthly. Seven tick species were identified: Hyalomma lusitanicum Koch (Parasitic indicator [PI] = number of ticks per examined rabbit = 96.47), Rhipicephalus pusillus Gil Collado (PI = 47.37), Haemaphysalis hispanica Gil Collado (PI = 12.15), Ixodes ventalloi Gil Collado (PI = 0.65), R. bursa Canestrini and Fanzago (PI = 0.18), R. sanguineus Latreille (PI = 0.11), Dermacentor marginatus Sulzer (PI = 0.01). In spring and summer, most abundant were larvae of H. lusitanicum, followed by immature stages of R. pusillus and Ha. hispanica. In autumn, the main tick species were nymphs of I. ventalloi whereas in winter adults of Ha. hispanica were more numerous. Rhipicephalus pusillus was present all year long, although not always in high percentage. PI of other species (R. bursa, R. sanguineus and D. marginatus) were too low to be representative. The seasonal dynamics of ticks on wild rabbit defined in this study could be useful to design species-specific control strategies.

High rates of Leishmania infantum and Trypanosoma nabiasi infection in wild rabbits (Oryctolagus cuniculus) in sympatric and syntrophic conditions in an endemic canine leishmaniasis area: epidemiological consequences.

Leishmania infantum infection has been reported in various host species, both domestic and wild, in some cases with high prevalence rates. However, until the recent discovery of infected hares, no studies had provided clear evidence of any significant reservoir other than domestic dogs. Our focus was on another lagomorph, Oryctolagus cuniculus or wild rabbit. This species is native to the Iberian Peninsula and its presence and abundance gave rise to the name of Spain. In an endemic area for canine leishmaniasis in the southeast of Spain, 150 rabbits were captured over a period of three years. Samples of blood, bone marrow, liver, spleen, heart and skin were taken and analysed through parasitological, serological and molecular techniques in order to detect Leishmania and Trypanosoma. 20.7% of the rabbits were infected with L. infantum and 82.4% with Trypanosoma nabiasi, and 14.8% of mixed infections were detected. Both parasites were found in all the animal organs analysed, a factor which, along with the presence of serological cross-reactions, must be taken into account in epidemiological studies on leishmaniasis. O. cuniculus is an abundant and gregarious species, with a long enough average lifespan to ensure L. infantum transmission. The presence of the parasite in the skin and blood of these rabbits with no acute manifestation of disease ensures its contact with the vector, which finds in their warrens a suitable biotope to inhabit. The rabbit therefore seems to meet the most of conditions for being considered a reservoir host of L. infantum.