Antibiotic Resistance in the Apennine Wolf (Canis lupus italicus): Implications for Wildlife and Human Health.

The Apennine wolf (Canis lupus italicus) is a subspecies of gray wolf that is widespread throughout Italy. Due to hunting and habitat loss, their population declined dramatically in the late 19th and early 20th centuries, but conservation efforts improved to restore the species to an estimated population of 3300 individuals. The presence of antibiotic-resistant bacteria in Apennine Wolf may pose a risk to its health and survival, as well as the health of other animals in its environment. In this study, we investigated the antibiotic resistance profiles of bacteria collected from Apennine wolves admitted to the Wildlife Research Center of Maiella National Park (Italy) in 2022. A total of 12 bacteria collected from four wolves were isolated and tested for susceptibility to antibiotics used in veterinary medicine and to critically important antibiotics for human health by means of the Vitek 2 system. All isolates were resistant to at least one antibiotic, and six bacteria were multidrug resistant to critically important antibiotics (third-generation cephalosporins, carbapenems and fluoroquinolones). The results of this pilot study have allowed for the characterization of resistant profiles in Escherichia coli, Enterococcus faecalis and other bacterial species not previously reported in Apennine wolves. Our findings provide important insights into antibiotic resistance in wildlife and its potential implications for the conservation of biodiversity and public health.

Antibiotic-Resistant Bacteria Dissemination in the Wildlife, Livestock, and Water of Maiella National Park, Italy.

Antimicrobial resistance (AMR) is a global health concern that has been linked to humans, animals, and the environment. The One Health approach highlights the connection between humans, animals, and the environment and suggests that a multidisciplinary approached be used in studies investigating AMR. The present study was carried out to identify and characterize the antimicrobial resistance profiles of bacteria isolated from wildlife and livestock feces as well as from surface water samples in Maiella National Park, Italy. Ecological and georeferenced data were used to select two sampling locations, one where wildlife was caught within livestock grazing areas (sympatric group) and one where wildlife was caught outside of livestock grazing areas (non-sympatric group). Ninety-nine bacterial isolates from 132 feces samples and seven isolates from five water samples were collected between October and December 2019. The specimens were examined for species identification, antibiotic susceptibility and molecular detection of antibiotic resistance. Forty isolates were identified as Escherichia coli, forty-eight as Enterococcus spp., eight as Streptococcus spp. and ten as other gram-negative bacteria. Phenotypic antibiotic resistance to at least one antimicrobial agent, including some antibiotics that play a critical role in human medicine, was detected in 36/106 (33.9%, 95% CI: 25-43) isolates and multidrug resistance was detected in 9/106 isolates (8.49%, 95% CI: 3.9-15.5). In addition, genes associated with antibiotic resistance were identified in 61/106 (57.55%, 95% CI: 47.5-67) isolates. The samples from sympatric areas were 2.11 (95% CI: 1.2-3.5) times more likely to contain resistant bacterial isolates than the samples from non-sympatric areas. These data suggest that drug resistant bacteria may be transmitted in areas where wildlife and livestock cohabitate. This emphasizes the need for further investigations focusing on the interactions between humans, wildlife, and the environment, the results of which can aid in the early detection of emerging AMR profiles and possible transmission routes.

Welfare and Clinical Assessment on Physical Captures Followed by Anesthesia in Apennine Chamois (Rupicapra pyrenaica ornata).

The Apennine chamois (Rupicapra pyrenaica ornata) is one of the rarest subspecies in Italy, listed in Annexes II and IV of the Habitats Directive and currently included as a vulnerable subspecies in the International Union for Conservation of Nature (IUCN) Red List. The Maiella National Park population has recently been defined as a source population for reintroduction into other parks. Since collective captures allow for better selection of target animals for the establishment of new colonies, the aim of this study is to evaluate the physiological conditions and animal welfare in free-ranging Apennine chamois after collective physical capture followed by chemical immobilization with medetomidine 0.054 mg ± 0.007, ketamine 2.14 mg ± 0.28, and acepromazine 0.043 mg ± 0.006. Twenty-one Apennine chamois (18 females and 3 males) were captured and translocated for conservation purposes. The effects of capture and anesthesia were evaluated using clinical variables, hematology, serum biochemistry, and venous blood gas analysis, the latter being used in the field for the first time in chamois capture. A risk of metabolic acidosis and capture myopathy was identified, although it did not compromise the survival of 19 chamois, which adapted to novel environments and founded new colonies, as verified through GPS radiocollars. The protocol applied in this study represents an innovative approach to assessing animal physiology and welfare in collective mountain ungulate captures, useful for improving management activities for conservation purposes.

Whole Genome Sequencing of Escherichia coli and Enterococcus spp. in wildlife-livestock interface: a pilot study.

OBJECTIVES: This pilot study provides a multidisciplinary investigation to monitor livestock-wildlife interface. Ecological data, microbiological investigations, and whole genome sequencing were used to characterize eight bacterial isolates obtained from sympatric domestic and wild ruminants in Maiella National Park (Italy) in terms of genetic patterns of antimicrobial resistance. METHODS: Using selective culturing of fresh fecal samples of monitored and georeferenced populations of Apennine chamois, goats, red deer, and sheep, Escherichia coli, Enterococcus faecium, and Enterococcus faecalis isolates were isolated and subjected to minimum inhibitory concentration determination and whole genome sequencing. RESULTS: The analyzed isolates showed phenotypic and genotypic resistance to tetracycline and critically important antibiotics such as linezolid and carbapenems. Virulence genes related to biofilm regulation and Shiga toxins were also detected. Furthermore, serotypes related to nosocomial infections, harbouring plasmids recognized as important mobile resistance gene transmitters, were identified. CONCLUSIONS: This multidisciplinary pilot study represents a promising initial step to identify the environmental drivers and the transmission routes of antimicrobial resistance and virulence factors, providing new data on bacteria from rare and endangered species such as Apennine chamois.

Microbial community and antimicrobial resistance in fecal samples from wild and domestic ruminants in Maiella National Park, Italy.

This study aimed to provide new insights about antimicrobial resistance genes abundance and microbial communities of wild and domestic ruminants in wildlife-livestock interface. In total, 88 fecal samples were recovered from Apennine chamois, red deer, goat, cattle and sheep, and were collected in pools. The populations under study were selected based on ecological data useful to define sympatric and non-sympatric populations. Samples were screened for commonly used in farms under study or critically important antimicrobial resistance genes (aadA2, TetA, TetB, TetK, TetM, mcr-1). The microbial community composition was found to be different based on the species and land use of animals under study. Indeed, it was mostly characterized by phyla Firmicutes in bovine, Bacteroidota in chamois and Proteobacteria in red deer. Additionally, positive correlations between antibiotic resistance genes and microbial taxa (e.g., Tet genes correlated with Firmicutes and Patescibacteria) were described. Of the antimicrobials investigated, the abundance of mcr-1 gene suggests the importance of monitoring the wildlife in order to detect the emerging resistance genes contamination in environment. This study provides new data that highlight the importance of multidisciplinary and uncultured study in order to describe the spreading of antimicrobial resistance and related contamination in the environment.

Evidence of Linezolid Resistance and Virulence Factors in Enterococcus spp. Isolates from Wild and Domestic Ruminants, Italy.

The aim of this study was to evaluate the resistance patterns against selected critically and highly important antibiotics (quinupristin/dalfopristin, vancomycin, and linezolid) in 48 Enterococcus isolates obtained from wild (red deer and Apennine chamois) and domestic (cattle, sheep, and goats) ruminants living with varying degrees of sympatry in the protected area of Maiella National Park (central Italy). According to CLSI breakpoints, 9 out of 48 isolates (18.8%) showed resistance to at least one antibiotic. One Apennine chamois isolate was resistant to all tested antibiotics. The PCR screening of related resistance genes highlighted the occurrence of msrC or cfrD in seven Enterococcus resistant isolates. In addition, msrC and vanC genes were amplified in susceptible isolates. Specific sequences of virulence genes (gelE, ace, efa, asa1, and esp) related to pathogenic enterococci in humans were amplified in 21/48 isolates (43.75%), belonging mostly to wild animals (15/21; 71.42%). This is the first report of linezolid-resistant enterococci harboring virulence genes in Italian wildlife with special regard to the red deer and Apennine chamois species. The results allow us to evaluate the potential role of wild animals as indicators of antibiotic resistance in environments with different levels of anthropic pressure.

Resistance Patterns, mcr-4 and OXA-48 Genes, and Virulence Factors of Escherichia coli from Apennine Chamois Living in Sympatry with Domestic Species, Italy.

The aim of this study was to determine and characterize potential resistance mechanisms against selected Critically Important Antibiotics in Escherichia coli isolates collected from wild and domestic ruminants living in the Maiella National Park, in Central Italy. A total of 38 isolates were obtained from red deer, Apennine chamois, cattle, sheep, and goats grazing in lands with different levels of anthropic pressure. Antimicrobial susceptibility was determined by Minimal Inhibitory Concentration testing, showing phenotypic resistance to colistin, meropenem, or ceftazidime in 9 isolates along with one bacterial strain being resistant to three of the tested antibiotics. In addition, the biomolecular assays allowed the amplification of the genes conferring the colistin (mcr-4), the carbapenems (OXA-48), penicillins and cephalosporins (TEM, SHV, CMY-1, CMY-2) resistance. In order to describe the potential pathogenicity of isolates under study, virulence genes related to Shiga toxin-producing (STEC) and enteropathogenic (EPEC) pathovars were identified. This study is the first report of mcr-4 and OXA-48 genes in resistant E. coli harboring virulence genes in Italian wildlife, with special regard to Apennine chamois and red deer species. The multidisciplinary approach used in this study can improve the early detection of emerging antibiotic resistance determinants in human-animal-environment interfaces by means of wildlife monitoring.

Infectious Diseases and Wildlife Conservation Medicine: The Case of the Canine Distemper in European Wolf Population.

Canine distemper is a contagious infectious disease, caused by canine distemper virus (CDV) belonging to Morbillivirus genus, Paramyxoviridae family, representing a serious threat for domestic and wild carnivores [...].

Detection of selected pathogens in Apennine wolf (Canis lupus italicus) by a non-invasive GPS-based telemetry sampling of two packs from Majella National Park, Italy.

In this study, a multi-pathogens survey was conducted to verify the sanitary status of two Italian wolf packs of Majella National Park. Twenty fecal samples (10/pack) were collected using a sampling protocol, based on the combining data from radio-collared wolves with geographic information system (GIS) analysis, allowing to mark off the home range of packs and to recover group-specific and high-quality specimens. Virological screening against the most prevalent canine viruses (protoparvovirus, distemper virus, adenoviruses, and coronaviruses) was carried out by molecular methods, while parasites were detected by means of copromicroscopic and molecular analysis. Canine parvovirus type 2b (CPV-2b) is the most prevalent virus in both packs (7/20), followed by canine adenovirus type 2 (CAdV-2), while no sequences of canine distemper virus and coronaviruses were detected. The sequence analysis of the viruses demonstrated the domestic origin of the infection, highlighting the importance of vaccination of local dogs in order to reduce the risk of exposure of wildlife to these pathogens. Fourteen samples resulted positive for parasites. Capillaria aerophila (sin. Eucoleus aerophilus), Ancylostoma/Uncinaria, Trichuris vulpis eggs, Sarcocystis spp., Cystoisospora canis, and Angiostrongylus vasorum larvae were identified. Echinococcus granulosus sensu stricto (ovine genotype G1) and Giardia duodenalis(canid-specific Assemblage C) were also characterized, providing insights into the wolves' diet and their effects on environmental contamination. The sampling protocol applied in this study, based on a multidisciplinary approach, represents an innovative tool for the survey of Apennine wolf, able to integrate sanitary data with the ecological and demographic features of this population.

Helminth infections in faecal samples of Apennine wolf (Canis lupus italicus) and Marsican brown bear (Ursus arctos marsicanus) in two protected national parks of central Italy

This article reports the results of a copromicroscopic and molecular investigation carried out on faecal samples of wolves (n=37) and brown bears (n=80) collected in two protected national parks of central Italy (Abruzzo Region). Twenty-three (62.2%) samples from wolves were positive for parasite eggs. Eight (34.78%) samples scored positive for single infections, i.e. E. aerophilus (21.74%), Ancylostoma/Uncinaria (4.34%), Trichuris vulpis (4.34%), T. canis (4.34%). Polyspecific infections were found in 15 samples (65.21%), these being the most frequent association: E. aerophilus and Ancylostoma/Uncinaria. Thirty-seven (46.25%) out of the 80 faecal samples from bears were positive for parasite eggs. Fourteen (37.83%) samples were positive for B. transfuga, and six (16.21%) of them also contained Ancylostoma/Uncinaria, one (2.7%) E. aerophilus and one (2.7%) both E. aerophilus and Ancylostoma/Uncinaria. Of the other samples, 19 (51.35%) were positive for Ancylostoma/Uncinaria, two (5.4%) for E. aerophilus and two (5.4%) for both. Molecular analysis found the roundworm and capillariid eggs found in wolves and bear samples to be Toxocara canis, Baylisascaris transfuga and Eucoleus aerophilus (syn. Capillaria aerophila). Considering the high prevalence of zoonotic intestinal helminths detected in this study, it is important to improve the knowledge and awareness of the general public and park operators regarding the potential health risk associated with infections in wildlife.