Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Molecular surveillance of potential SARS-CoV-2 reservoir hosts in wildlife rehabilitation centers.

BACKGROUND: The COVID-19 pandemic, caused by SARS-CoV-2 infection, has become the most devastating zoonotic event in recent times, with negative impacts on both human and animal welfare as well as on the global economy. Although SARS-CoV-2 is considered a human virus, it likely emerged from animals, and it can infect both domestic and wild animals. This constitutes a risk for human and animal health including wildlife with evidence of SARS-CoV-2 horizontal transmission back and forth between humans and wild animals. AIM: Molecular surveillance in different wildlife rehabilitation centers and wildlife associated institutions in Chile, which are critical points of animal-human interaction and wildlife conservation, especially since the aim of wildlife rehabilitation centers is to reintroduce animals to their original habitat. MATERIALS AND METHODS: The survey was conducted in six WRCs and three wildlife associated institutions. A total of 185 samples were obtained from 83 individuals belonging to 15 different species, including vulnerable and endangered species. Each specimen was sampled with two different swabs: one oropharyngeal or nasopharyngeal according to the nostril diameter, and/or a second rectal sample. RNA was extracted from the samples and two different molecular assays were performed: first, a conventional RT-PCR with pan-coronavirus primers and a second SARS-CoV-2 qPCR targeting the N and S genes. RESULTS: All 185 samples were negative for SARS-CoV-2. CLINICAL RELEVANCE: This study constitutes the first report on the surveillance of SARS-CoV-2 from wildlife treated in rehabilitation centers in Chile, and supports the biosafety procedures adopted in those centers.

Blood Transfusion from a Magellanic Great Horned Owl (Bubo virginianus magellanicus) to a Barn Owl (Tyto alba): A Successful Case of Xenotransfusion.

This report describes successful transspecies blood transfusion from a Magellanic horned owl (Bubo virginianus magellanicus) to a barn owl (Tyto alba). The barn owl was admitted to a wildlife rehabilitation center with severe anemia (packed cell volume [PCV] = 6.7%) from suspected anticoagulant rodenticide poisoning. Procedures performed included patient stabilization, pharmacological treatment, and persistent monitoring following the blood transfusion. The patient's PCV was measured daily, increasing progressively, and attaining a normal value for the species (PCV > 40%) on the eighth day posttransfusion. With no possibility of obtaining a same-species donor and because of the serious condition of the patient and unfavorable prognosis, a decision was made to perform the xenotransfusion. The result was a successful recovery and ultimately the release of the owl into its natural habitat. We concluded that xenotransfusion for avian species should be considered in cases with no possibility of obtaining a homologous donor.

The systematics of Tachymenini (Serpentes, Dipsadidae): an updated classification based on molecular and morphological evidence

Despite the recent advances in the systematics of snakes, the diversity of several Neotropical groups of species remains poorly understood. The lack of studies focused on the phylogenetic relationship within most of the 20 tribes of Dipsadidae precludes a better understanding of the evolution of this diverse family. Here, we present a comprehensive phylogenetic analysis of Tachymenini, a heterogeneous tribe that comprises 36 viviparous species of dipsadids, grouped in seven genera based on morphological similarities. The tribe is widely distributed throughout South America presenting very distinctive phenotypes, habitats, and behaviours. The phylogenetic relationship among tachymenins is a well-recognized challenge regarding the systematics of the South American dipsadids. The similar morphotype of some generalist species, combined with the very derived morphology of some strict specialists, creates a complexity of traits that has prevented the comprehension of the systematics of the group. To address such a challenge, we combine molecular (six loci) and morphological (70 characters) datasets in an integrative phylogenetic approach. The resultant phylogenetic trees indicate, with strong support, that three of the seven current recognized genera (Tachymenis, Tomodon, and Thamnodynastes) are non-monophyletic and, consequently, we propose a new systematic arrangement for Tachymenini. We revalidate two genera, Dryophylax and Mesotes, and we describe three additional monotypic genera, Apographon gen. n., Tachymenoides gen. n., and Zonateres gen. n. to accommodate Tomodon orestes, Tachymenis affinis, and Thamnodynastes lanei, respectively. We also include Tomodon ocellatus and Pseudotomodon trigonatus in Tachymenis and describe a new genus, Galvarinus gen. n., to accommodate the Tachymenis chilensis species group. Furthermore, we also provide an evolutionary scenario for the speciation events based on a time-calibrated tree, commenting on the diversification and origin of the tribe, and on the probable existence of undescribed species of Mesotes and Dryophylax.

Survey of Zoonotic Bacterial Pathogens in Native Foxes in Central Chile: First Record of Brucella canis Exposure.

Brucella abortus, B. canis, and pathogenic Leptospira are zoonotic pathogens that infect humans, as well as domestic and wild animals. In wild canids, they may affect their fertility and reproduction, threatening their conservation. Wild canids play a crucial role in the environment as meso- and top-predators and environmental sentinels for zoonotic pathogens. In Chile, three species of wild canids are present, and due to changes in land use and environmental dynamics, it is of utmost relevance to determine the role of these species in the epidemiology of brucellosis and leptospirosis. This study aimed to detect the exposure to B. abortus, B. canis, and pathogenic Leptospira by serologic, bacteriologic, and molecular techniques in native foxes from rehabilitation and exhibition centers in Central Chile. Forty-six blood samples were obtained from Lycalopex culpaeus and L. griseus, detecting 10.9% of seropositivity to B. canis and 7.7% to L. Javanica. No seropositivity was seen for B. abortus. Exposure was not registered by culture and qPCR in any of the sampled animals. Our findings are the first register of exposure to any Brucella species in wild canids in Chile and highlight the need to establish surveillance programs of these emerging pathogens.

Antimicrobial resistance in wildlife and in the built environment in a wildlife rehabilitation center.

Injured and orphaned wildlife are often brought to Wildlife Rehabilitation Centers (WRC) to be cared for by professionals to ultimately be released back to their natural habitats. In these centers, animals may spend months and frequently receive prolonged antibiotic therapy. Therefore, WRC may play a role in the emergence and dissemination of antimicrobial resistance (AMR). The goal of this study was to investigate the presence and antibiotic resistance profiles of Gram-negative bacteria with reduced susceptibility to cephalosporins in both the wildlife admitted to a WRC and in the WRC built environment in Chile. A cross-sectional study was conducted sampling animals undergoing rehabilitation (n = 64) and the WRC environment (n = 160). Isolated bacterial species were identified with MALDI-TOF, and antimicrobial susceptibility determined using the disk diffusion method. Enterobacteriaceae and Pseudomonadaceae were the dominant bacterial families among the environmental (n = 78) and animal (n = 31) isolates. For Enterobacteriaceae, isolates of the most abundant species (E. coli) were classified into 20 antibiotic resistance profiles, with eight of those isolates being resistant to more than nine antibiotics, including imipenem. Isolates of the Pseudomonadaceae family identified 11 isolates with resistance to antibiotics such as carbapenems and quinolones. Even though a cluster analysis based on antibiotic resistance patterns did not show a clear overlap between environmental and animal isolates, it is important to highlight the identification of isolates resistant to carbapenems, which is very relevant from a public health perspective. Further, numerous antibiotic resistance profiles were observed in different bacterial species, indicating not only environmental contamination with a wide diversity of bacteria, but also a wide diversity of resistant bacteria in animals at the WRC. The approach taken by sampling animals and their hospital environment can be useful in understanding AMR dynamics in wildlife rehabilitation settings, as well as the potential dissemination of AMR into the natural environment.

Specialist versus generalist parasites: the interactions between host diversity, environment and geographic barriers in avian malaria.

The specialist versus generalist strategies of hemoparasites in relation to their avian host, as well as environmental factors, can influence their prevalence, diversity and distribution. In this paper we investigated the influence of avian host species, as well as the environmental and geographical factors, on the strategies of Haemoproteus and Plasmodium hemoparasites. We determined prevalence and diversity by targeting their cytochrome b (Cytb) in a total of 2,590 passerine samples from 138 localities of Central and South America, and analysed biogeographic patterns and host-parasite relationships. We found a total prevalence of 23.2%. Haemoproteus presented a higher prevalence (15.3%) than Plasmodium (4.3%), as well as a higher diversity and host specificity. We determined that Plasmodium and Haemoproteus prevalences correlated positively with host diversity (Shannon index) and were significantly influenced by bird diversity, demonstrating a possible "amplification effect". We found an effect of locality and the avian family for prevalences of Haemoproteus and Plasmodium. These results suggest that Haemoproteus is more specialist than Plasmodium and could be mostly influenced by its avian host and the Andes Mountains.

Widespread Infection with Hemotropic Mycoplasmas in Free-Ranging Dogs and Wild Foxes Across Six Bioclimatic Regions of Chile.

Blood samples of 626 rural dogs, 140 Andean foxes (Lycalopex culpaeus), and 83 South American grey foxes (L. griseus) from six bioregions of Chile spanning 3000 km were screened for Mycoplasma DNA by conventional PCR and sequencing. Risk factors of infection were inferred using Generalized Linear Mixed Models and genetic structure by network analyses. Overall, Mycoplasma haemocanis/Mycoplasma haemofelis (Mhc/Mhf) and Candidatus Mycoplasma haematoparvum (CMhp) observed prevalence was 23.8% and 12.8% in dogs, 20.1% and 7.2% in Andean foxes, and 26.5% and 8.4% in grey foxes, respectively. Both hemoplasmas were confirmed in all the bioregions, with higher prevalence in those where ticks from the Rhipicephalus sanguineus species group were absent. Candidatus M. haematominutum and a Mycoplasma sp. previously found in South American carnivores were detected in one fox each. Although the most prevalent Mhc/Mhf and CMhp sequence types were shared between dogs and foxes, network analysis revealed genetic structure of Mhc/Mhf between hosts in some regions. Male sex was associated with a higher risk of Mhc/Mhf and CMhp infection in dogs, and adult age with CMhp infection, suggesting that direct transmission is relevant. No risk factor was identified in foxes. Our study provides novel information about canine hemoplasmas with relevance in distribution, transmission routes, and cross-species transmission.

Salmonella in Raptors and Aquatic Wild Birds in Chile.

Salmonella enterica is one of the main causes of gastrointestinal disease worldwide. Wild birds are capable of harboring a variety of Salmonella serovars, which could have an important role in the epidemiology of salmonellosis in humans and production animals. We tested 519 fecal samples from raptors and aquatic birds from different regions of central (three rehabilitation centers for wildlife and the coastal area) and southern areas of Chile for Salmonella. All samples were obtained in 2015 and 2017, covering all four seasons. Salmonella was isolated from 12 of the 519 samples (2%) analyzed, from two carnivorous birds, four birds with generalist habits, and six waterfowl. Among the isolates obtained, one showed resistance to gentamicin, and one showed a multidrug-resistance phenotype, with resistance to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, streptomycin, gentamicin, kanamycin, trimethoprim-sulfamethoxazole, and tetracycline. These results demonstrated the importance of characterizing Salmonella in wild birds because previous studies have shown genetic and phenotypic evidence suggesting interspecies transmission of Salmonella enterica that is resistant to antimicrobials between humans and wild and domestic birds.

Antimicrobial resistance genes in Andean foxes inhabiting anthropized landscapes in central Chile.

Antimicrobial resistance (AMR) is considered an emerging public health problem. Greater AMR development rate is associated with "antibiotic-using" environments. Wildlife thriving in anthropized landscapes could be good indicators of the burden of AMR and antibiotic resistance genes (ARGs) in these areas. The aim of this study was to determine the presence and load of ARGs in fecal swabs of wild Andean foxes (Lycalopex culpaeus) from anthropized landscapes of central Chile. DNA was extracted from samples of 72 foxes; 22 ARGs encoding resistance against 8 antibiotic groups were evaluated using qPCR. Eighteen of the 22 ARGs were found and tet(Q) (65.3%; 15/72 of the samples) was the most common gene detected. Almost half of the foxes presented a 'multiresistant microbiome' (i.e. at least three ARG encoding resistance to different groups of antimicrobials). Prevalence of tet(Q) was higher in the cold-humid season than in the warm-dry season, but not for other genes. Up to 15 and 13 ARGs were detected in the fecal samples from two additional foxes that were kept 6 and 11 days, respectively, in a clinical environment (Wildlife Rescue Center) and received antibiotic treatment. Some of the ARGs detected (e.g. mecA and blaCTX-M) in the present study are of particular concern from the public health perspective. Wild foxes seem to be good sentinels for ARG environmental burden in highly anthropized environments of central Chile.

Xenopus laevis and Emerging Amphibian Pathogens in Chile.

Amphibians face an extinction crisis with no precedence. Two emerging infectious diseases, ranaviral disease caused by viruses within the genus Ranavirus and chytridiomycosis due to Batrachochytrium dendrobatidis (Bd), have been linked with amphibian mass mortalities and population declines in many regions of the globe. The African clawed frog (Xenopus laevis) has been indicated as a vector for the spread of these pathogens. Since the 1970s, this species has been invasive in central Chile. We collected X. laevis and dead native amphibians in Chile between 2011 and 2013. We conducted post-mortem examinations and molecular tests for Ranavirus and Bd. Eight of 187 individuals (4.3 %) tested positive for Ranavirus: seven X. laevis and a giant Chilean frog (Calyptocephallela gayi). All positive cases were from the original area of X. laevis invasion. Bd was found to be more prevalent (14.4 %) and widespread than Ranavirus, and all X. laevis Bd-positive animals presented low to moderate levels of infection. Sequencing of a partial Ranavirus gene revealed 100 % sequence identity with Frog Virus 3. This is the first report of Ranavirus in Chile, and these preliminary results are consistent with a role for X. laevis as an infection reservoir for both Ranavirus and Bd.

Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins.

The evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans-species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long-term survival of the species.

Molecular Epidemiology of Avian Malaria in Wild Breeding Colonies of Humboldt and Magellanic Penguins in South America.

Avian malaria is a disease caused by species of the genera Haemoproteus, Leucocytozoon, and Plasmodium. It affects hundreds of bird species, causing varied clinical signs depending on the susceptibility of the host species. Although high mortality has been reported in captive penguins, limited epidemiological studies have been conducted in wild colonies, and isolated records of avian malaria have been reported mostly from individuals referred to rehabilitation centers. For this epidemiological study, we obtained blood samples from 501 adult Humboldt and 360 adult Magellanic penguins from 13 colonies throughout South America. To identify malaria parasitaemia, we amplified the mtDNA cytochrome b for all three parasite genera. Avian malaria was absent in most of the analyzed colonies, with exception of the Punta San Juan Humboldt penguin colony, in Peru, where we detected at least two new Haemoproteus lineages in three positive samples, resulting in a prevalence of 0.6% for the species. The low prevalence of avian malaria detected in wild penguins could be due to two possible causes: A low incidence, with high morbidity and mortality in wild penguins or alternatively, penguins sampled in the chronic stage of the disease (during which parasitaemia in peripheral blood samples is unlikely) would be detected as false negatives.