Assessing micro-macroparasite selective pressures and anthropogenic disturbance as drivers of immune gene diversity in a Neotropical wild cat.

Anthropogenic environmental change is reducing available habitat for wild species, providing novel selection pressures such as infectious diseases and causing species to interact in new ways. The potential for emerging infectious diseases and zoonoses at the interface between humans, domestic animals, and wild species is a key global concern. In vertebrates, diversity at the major histocompatibility complex MHC is critical to disease resilience, and its study in wild populations provides insights into eco-evolutionary dynamics that human activities alter. In natural populations, variation at MHC loci is partly maintained by balancing selection, driven by pathogenic selective pressures. We hypothesize that MHC genetic diversity differs between guigna populations inhabiting human-dominated landscapes (higher pathogen pressures) versus more natural habitats (lower pathogen pressures). We predict that MHC diversity in guignas would be highest in human-dominated landscapes compared with continuous forest habitats. We also expected to find higher MHC diversity in guignas infected with micro and macro parasites (higher parasite load) versus non infected guignas. We characterized for the first time the genetic diversity at three MHC class I and II exons in 128 wild guignas (Leopardus guigna) across their distribution range in Chile (32-46° S) and Argentina, representing landscapes with varying levels of human disturbance. We integrated MHC sequence diversity with multiple measures of anthropogenic disturbance and both micro and macro parasite infection data. We also assessed signatures of positive selection acting on MHC genes. We found significantly higher MHC class I diversity in guignas inhabiting landscapes where houses were present, and with lower percentage of vegetation cover, and also in animals with more severe cardiorespiratory helminth infection (richness and intensity) and micro-macroparasite co-infection. This comprehensive, landscape-level assessment further enhances our knowledge on the evolutionary dynamics and adaptive potential of vertebrates in the face of emerging infectious disease threats and increasing anthropogenic impacts.

Cross-species transmission of retroviruses among domestic and wild felids in human-occupied landscapes in Chile.

Human transformation of natural habitats facilitates pathogen transmission between domestic and wild species. The guigna (Leopardus guigna), a small felid found in Chile, has experienced habitat loss and an increased probability of contact with domestic cats. Here, we describe the interspecific transmission of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) between domestic cats and guignas and assess its correlation with human landscape perturbation. Blood and tissue samples from 102 free-ranging guignas and 262 domestic cats were collected and analyzed by PCR and sequencing. Guigna and domestic cat FeLV and FIV prevalence were very similar. Phylogenetic analysis showed guigna FeLV and FIV sequences are positioned within worldwide domestic cat virus clades with high nucleotide similarity. Guigna FeLV infection was significantly associated with fragmented landscapes with resident domestic cats. There was little evidence of clinical signs of disease in guignas. Our results contribute to the understanding of the implications of landscape perturbation and emerging diseases.

Epidemiology and molecular characterization of Carnivore protoparvovirus-1 infection in the wild felid Leopardus guigna in Chile.

Landscape anthropization has been identified as one of the main drivers of pathogen emergence worldwide, facilitating pathogen spillover between domestic species and wildlife. The present study investigated Carnivore protoparvovirus-1 infection using molecular methods in 98 free-ranging wild guignas (Leopardus guigna) and 262 co-occurring owned, free-roaming rural domestic cats. We also assessed landscape anthropization variables as potential drivers of infection. Protoparvovirus DNA was detected in guignas across their entire distribution range, with observed prevalence of 13.3% (real-time PCR) and 9% (conventional PCR) in guignas, and 6.1% (conventional PCR) in cats. Prevalence in guigna did not vary depending on age, sex, study area or landscape variables. Prevalence was higher in juvenile cats (16.7%) than in adults (4.4%). Molecular characterization of the virus by amplification and sequencing of almost the entire vp2 gene (1,746 bp) from one guigna and five domestic cats was achieved, showing genetic similarities to canine parvovirus 2c (CPV-2c) (one guigna and one cat), feline panleukopenia virus (FPV) (one cat), CPV-2 (no subtype identified) (two cats), CPV-2a (one cat). The CVP-2c-like sequence found in a guigna clustered together with domestic cat and dog CPV-2c sequences from South America, suggesting possible spillover from a domestic to a wild species as the origin of infection in guigna. No clinical signs of disease were found in PCR-positive animals except for a CPV-2c-infected guigna, which had haemorrhagic diarrhoea and died a few days after arrival at a wildlife rescue centre. Our findings reveal widespread presence of Carnivore protoparvovirus-1 across the guigna distribution in Chile and suggest that virus transmission potentially occurs from domestic to wild carnivores, causing severe disease and death in susceptible wild guignas.

Gastrointestinal and cardiorespiratory endoparasites in the wild felid guigna (Leopardus guigna) in Chile: Richness increases with latitude and first records for the host species.

Guignas (Leopardus guigna) are small felids closely associated with native forest habitats. In fragmented landscapes, they use vegetation corridors and forest remnants to move across the landscape. In these contexts, guignas may increase contact probabilities with domestic animals, being therefore relevant to assess their pathogens and parasites. The aim of this study was to characterize the helminth fauna in the gastrointestinal tract and cardiorespiratory system of guignas from central and southern Chile. Between 2015 and 2018, 33 dead free-ranging guignas were found road-killed or were collected from wildlife rescue centers. Thirty-two gastrointestinal tracts and 32 cardiorespiratory organs were analyzed through direct analysis and artificial digestion. We found 81.8% (27/33) guignas were positive for helminth endoparasites (84.4% (27/32) positive for gastrointestinal parasites, 37.5% (12/32) positive for cardiorespiratory parasites). Fourteen parasites were identified (7 at genus level and 7 at species level), with Angiostrongylus sp., Molineus sp., Oslerus sp. and Troglostrongylus sp. as first records in guignas. The most prevalent parasites were the species Toxascaris leonina, Toxocara cati and Uncinaria stenocephala. Uncinaria stenocephala showed the highest intensity of infection. Multiparasitism was observed in 76% of the animals. Significant differences in richness of endoparasites and prevalence of cardiorespiratory parasites were found between geographic zones; higher values in the southern zone are possibly due to favorable environmental characteristics for endoparasite development. There were no statistically significant differences between sexes. All the parasites found in this study have been previously reported in domestic cats. These results are valuable to understand parasite transmission at the domestic-wildlife interface; the possibility of endoparasite transmission between domestic cats and guignas should be clarified with molecular analysis.

Antibiotic resistance genes as landscape anthropization indicators: Using a wild felid as sentinel in Chile.

Antimicrobial resistance is a global emerging public health issue whose presence and impact in wildlife are widely unknown. Antimicrobial resistance genes (ARGs) are considered environmental contaminants, suitable to evaluate the degree of anthropic impact on wildlife and the environment. We used a wild felid, the guigna (Leopardus guigna), as a sentinel for the presence of ARGs in anthropized and pristine areas across their entire distribution range in Chile. We evaluated fecal samples from 51 wild guignas, collected between 2009 and 2018. Real-time PCR essays were employed to detect and quantify 22 selected ARGs in their fecal microbiome. All animals (100%) were positive for at least one ARG. The most prevalent ARG families were those that confer resistance to tetracycline (88.2%) and beta-lactamase (68.9%), with tet(Q) (60.8%), tet(W) (60.8%), and blaTEM (66.7%) as the most prevalent ARGs. Multi-resistance profiles were observed in 43% of the guignas. Statistically significant differences were found between anthropized and pristine areas for tet(Q) (p = 0.014), tet(W) (p = 0.0037), tetracycline family (p = 0.027), multi-resistance profile prevalence (p = 0.043) and tet(W) quantification (p = 0.004). Two animals from anthropized landscapes were positive for mecA, a gene associated with Staphylococcus aureus and other staphylococci resistant to methicillin, while three animals from anthropized areas were positive for blaCTX-M, that encodes class A extended-spectrum beta-lactamase. Both genes have been identified in bacteria causing relevant nosocomial infections worldwide. This is the first study on ARGs in wild felids from Chile and the first detection of mecA in South American wild felids. We observed an association between the degree of landscape anthropization and ARG prevalence, confirming that ARGs are important indicators of wildlife exposure to human activity/presence, with a widespread distribution.

Molecular and serological survey of carnivore pathogens in free-roaming domestic cats of rural communities in southern Chile.

Owned, free-roaming domestic cats are abundant in the Chilean countryside, having high probability of contact with wildlife and potentially participating as reservoirs of zoonotic pathogens. In the present study, 131 cats from two remote study areas (Valdivia and Chiloe Island) in southern Chile were analyzed for infection/exposure to eight pathogens. Serum samples from 112 cats were tested for antigens against feline leukemia virus (FeLV antigen-ELISA) and antibodies against feline immunodeficiency virus (FIV-ELISA) and canine distemper virus (CDV-serum neutralization), yielded occurrence of 8.9, 1.7 and 0.8% respectively. The presence of DNA of five vector-borne pathogens, piroplasmids, Ehrlichia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp. was investigated in thirty cats. Overall observed occurrence was 6.6% (2/30) for both Anaplasma platys, and B. henselae, and 3.3% (1/30) for both Bartonella sp. and Theileria equi. Observed occurrence for all vector-borne pathogens in Valdivia area was significantly higher than in Chiloe Island (5/15 vs 0/15; P=0.04). Our results represent the first description of exposure to CDV and DNA detection of T. equi and A. platys in domestic cats in Chile. The results highlight the importance of performing pathogen screening in owned, free-roaming rural cats to evaluate their potential role as reservoirs of infection and vectors for disease transmission to wildlife.