Serological and molecular survey of canine distemper virus in red foxes (Vulpes vulpes): Exploring cut-off values and the use of protein A in ELISA tests.

The wide distribution and ecological plasticity of the red fox (Vulpes vulpes) make it a potential reservoir for many infectious diseases shared with domestic and wild carnivores. One of such diseases is canine distemper, which is caused by an RNA virus and its main domestic reservoir is the dog. However, other carnivores can also participate in its maintenance, as shown by the recent upsurge of reported cases in wildlife in many parts of the world, and by the fact that red foxes may act as true reservoirs for canine distemper virus (CDV). The lack of validated serological tests for wildlife or other non-target species may be a handicap for monitoring this virus. In this study, serological assays were compared in 147 red fox sera using a commercial ELISA validated for its use in dogs and a non-specific modified ELISA with Protein A peroxidase conjugate to detect bound antibodies. In addition, the presence of CDV RNA in brain, spleen, lung, and liver samples from 144 foxes was investigated by a RT-qPCR. Through the comparison of the results of both ELISAs and the use of a finite mixture model of the optical density values obtained by both techniques, we adjusted the cut-off point of the commercial ELISA to obtain the seroprevalence in foxes. The overall seroprevalence detected was 53.7% (79/147) and 57.1% (84/147) by the commercial and modified ELISA, respectively, with a moderate agreement according to Cohen's Kappa statistic (κ = 0.491, z = 5.97, p < 0.0001). CDV RNA was detected in 30 out of 144 foxes, which resulted in 20.8% of CDV-infected foxes. At individual level, the results obtained by relating the serological status and the presence/absence of RNA in different organs were explained in terms of the pathogenesis of the infection. Our results highlight the convenience of adjusting the cut-off point when using an ELISA assay developed in domestic dogs for its use in foxes. Moreover, Protein A is confirmed to be a good alternative to be used in red foxes, presenting a good reactivity towards its IgG.

Spatio-temporal patterns of sarcoptic mange in red deer and Iberian ibex in a multi-host natural park.

The reliance on multiple hosts to survive is what makes the management and control of multi-host infectious agents challenging. Sarcoptes scabiei causes sarcoptic mange in a wide range of mammal species with ungulates being an important host. Little is known about the role different ungulates play in sustaining endemic transmission of the disease and no study has yet to describe the long-term multi-host sarcoptic infestation dynamics in free-ranging wildlife. Here, we explore 24 years of sarcoptic mange infestation data for two Mediterranean ungulate species, red deer and Iberian ibex, living in the Sierras de Cazorla, Segura y Las Villas Natural Park of southern Spain. The temporal analysis showed a clear seasonal pattern of infestation in both ungulates with a peak in early spring and a decline throughout the summer. The spatial analysis, however, showed that caprinae rather than cervidae is the most competent host for sarcoptic mange spreading and persistence. Considering that few studies have described the spatio-temporal pattern of mange outbreaks for long periods of time, the information reported in this work aims to improve our understanding of sarcoptic mange epizootic in wild ruminant populations.

Risk factors of Leptospira infection in Mediterranean periurban micromammals.

Urbanization of natural areas can change abiotic factors, providing artificial sources of humidity in summer and decreasing variation of temperatures in winter. Our study aimed at document risk factors of infection in mammal reservoirs of pathogenic Leptospira in the human/wildlife interface of a large metropolitan area. We hypothesize that survival of Leptospira and thus their prevalence in animal reservoirs should be higher in residential areas than in natural habitats, especially after the hot, dry Mediterranean summers. We established the prevalence of Leptospira spp. and identified the serovars in 353 urine samples from micromammals (chiefly the wood mouse Apodemus sylvaticus, n = 266) using direct immunofluorescence and PCR. Animals were captured in spring and autumn, 2011-2012, in two natural parks and two adjacent residential areas in periurban Barcelona (NE Spain). Overall observed prevalence of infection was 11%, ranking between 8% and 13% in the better represented host species. We observed marked differences between seasons; the probability of finding a micromammal infected in spring was three times greater than in autumn (almost four times for wood mouse). Prevalence was not related with type of habitat, micromammal relative abundance or sex of the animal. Three Leptospira species were confirmed: Leptospira interrogans (47% of cases), Leptospira borgpetersenii (41%) and Leptospira kirschneri (12%). The serovars most commonly detected were those typically hosted by rodents, and serovars Ballum and Icterohemorrhagiae were the only ones found in autumn. People living in periurban Barcelona and those visiting the natural areas of the metropolitan area face hazard of infection with rodent-borne Leptospira, especially during spring.

Evaluation of Combination Drug Therapy for Treatment of Antibiotic-Resistant Inhalation Anthrax in a Murine Model.

Bacillus anthracis is considered a likely agent to be used as a bioweapon, and the use of a strain resistant to the first-line antimicrobial treatments is a concern. We determined treatment efficacies against a ciprofloxacin-resistant strain of B. anthracis (Cipr Ames) in a murine inhalational anthrax model. Ten groups of 46 BALB/c mice were exposed by inhalation to 7 to 35 times the 50% lethal dose (LD50) of B. anthracis Cipr Ames spores. Commencing at 36 h postexposure, groups were administered intraperitoneal doses of sterile water for injections (SWI) and ciprofloxacin alone (control groups), or ciprofloxacin combined with two antimicrobials, including meropenem-linezolid, meropenem-clindamycin, meropenem-rifampin, meropenem-doxycycline, penicillin-linezolid, penicillin-doxycycline, rifampin-linezolid, and rifampin-clindamycin, at appropriate dosing intervals (6 or 12 h) for the respective antibiotics. Ten mice per group were treated for 14 days and observed until day 28. The remaining animals were euthanized every 6 to 12 h, and blood, lungs, and spleens were collected for lethal factor (LF) and/or bacterial load determinations. All combination groups showed significant survival over the SWI and ciprofloxacin controls: meropenem-linezolid (P = 0.004), meropenem-clindamycin (P = 0.005), meropenem-rifampin (P = 0.012), meropenem-doxycycline (P = 0.032), penicillin-doxycycline (P = 0.012), penicillin-linezolid (P = 0.026), rifampin-linezolid (P = 0.001), and rifampin-clindamycin (P = 0.032). In controls, blood, lung, and spleen bacterial counts increased to terminal endpoints. In combination treatment groups, blood and spleen bacterial counts showed low/no colonies after 24-h treatments. The LF fell below the detection limits for all combination groups yet remained elevated in control groups. Combinations with linezolid had the greatest inhibitory effect on mean LF levels.

Wide exposure to Coxiella burnetii in ruminant and feline species living in a natural environment: zoonoses in a human-livestock-wildlife interface.

Assessment of the role of wild and domestic hosts as potential reservoirs of misdiagnosed zoonoses, such as Q fever by Coxiella burnetii, is an important public health issue today both for wildlife conservation and management of disease in human-livestock-wildlife interface. This study used ELISA, an indirect antibody, to research (2003-2013) C. burnetii infection in seven free-living wild and domestic ruminant species and in European wildcats (Felis silvestris). The animals studied were 0 European wildcats, 21 Spanish ibex (Capra pyrenaica), 314 red deer (Cervus elaphus), 556 fallow deer (Dama dama), 211 European mouflon (Ovis aries musimon), eight roe deer (Capreolus capreolus), 407 bovines (Bos taurus) and 3739 sheep (Ovis aries). All the animals shared the same habitat in the Serranía de Cuenca Natural Park (Castile-La Mancha, Spain). The study area is an example of human-domestic-wildlife interface where people and domestic animals live in close proximity to wildlife. Observed C. burnetii seropositive frequencies were: 33·3% European wildcats, 23·8% Spanish ibex, 22·5% domestic sheep 1·5% red deer, 1·4% European mouflon, 0·24% cattle, 0·18% fallow deer and 0% roe deer. The study found a wide C. burnetii prevalence of previous and present exposure in wild and domestic ruminant hosts in the Serranía de Cuenca Natural Park and reports the first evidence of C. burnetii exposure in free-living European wildcats.

Coinfection is an important factor in epidemiological studies: the first serosurvey of the aoudad (Ammotragus lervia).

Despite being considered an invasive ungulate outside its native range (North Africa), little information exists regarding the role of the aoudad (also called Barbary sheep, Ammotragus lervia) as a pathogen reservoir. Furthermore, in most epidemiological surveys the potential role of coinfections (e.g. a first infection may make the host more immuno-competent or susceptible against a second pathogen) as a risk factor is often neglected. In this study we first performed a serological survey for selected pathogens (Mycobacterium bovis, M. avium subsp. paratuberculosis, Chlamydophila abortus, bovine viral diarrhoea/border disease viruses (BVDV-BDV), Salmonella spp., Brucella melitensis and Toxoplasma gondii) on free (n = 66) and captive (n = 25) aoudad from south-east Spain. Then, by using Akaike's information criterion, we evaluated the importance of coinfection in two statistical models that included the effects of population, age, and sex. Our results show that neither free nor captive aoudad had antibodies against Brucella melitensis, Chlamydophila abortus, or BVDV-BDV. However, compared to other wild ungulates in Spain, aoudads have high prevalence of antibodies against M. bovis (free = 49.5%; captive = 8%), very high prevalence of antibodies against M. avium subsp. paratuberculosis (free = 19.4%; captive = 56%), and intermediate prevalence of antibodies against Salmonella spp. (free = 13.4%; captive = 0%) or T. gondii (free = 1.5%; captive = 24%). Although the additive effects of population and age were included in our set of selected models, coinfection was the most influential factor to detect antibodies response against mycobacterials and salmonella infections. The direction of this influence could be exclusion of disease between tuberculosis and paratuberculosis seroreactor animals, or enhanced susceptibility to disease between tuberculosis and salmonella seroreactor animals. In conclusion, we believe that wildlife managers must pay more attention to the potential risk posed by aoudads as hosts (and probably reservoirs) of paratuberculosis and tuberculosis mycobacterials, while epidemiologists should be more aware of coinfection as an important factor in epidemiological surveys, especially in wildlife populations where multiple infections are common.