Occurrence and Quantification of Antimicrobial Resistance Genes in the Gastrointestinal Microbiome of Two Wild Seabird Species With Contrasting Behaviors.

Antimicrobial resistance genes (ARGs) are environmental pollutants and anthropization indicators. We evaluated human interference in the marine ecosystem through the ocurrence and quantification (real-time PCRs) of 21 plasmid-mediated ARGs in enema samples of 25 wild seabirds, upon admission into rehabilitation: kelp gull (Larus dominicanus, n = 14) and Magellanic penguin (Spheniscus magellanicus, n = 11). Overall, higher resistance values were observed in kelp gulls (non-migratory coastal synanthropic) in comparison with Magellanic penguins (migratory pelagic non-synanthropic). There were significant differences between species (respectively, kelp gull and Magellanic penguin): ARGs occurrence (bla TEM [p = 0.032]; tetM [p = 0.015]; tetA [p = 0.003]; and sulII [p = 0.007]), mean number of ARGs per sample (p = 0.031), ARGs mean load percentage (aadA [p = 0.045], tetA [p = 0.031], tetM [p = 0.016], bla TEM [p = 0.032], sulII [p = 0.008]), percentage of genes conferring resistance to an antimicrobial class (betalactams [p = 0.036] and sulfonamides [p = 0.033]), mean number of genes conferring resistance to one or more antimicrobial classes (p = 0.024]), percentage of multiresistant microbiomes (p = 0.032), and clustering (p = 0.006). These differences are likely due to these species' contrasting biology and ecology - key factors in the epidemiology of ARGs in seabirds. Additionally, this is the first report of mecA in seabirds in the Americas. Further studies are necessary to clarify the occurrence and diversity of ARGs in seabirds, and their role as potential sources of infection and dispersal within the One Health chain of ARGs.

Neoplasms and novel gammaherpesviruses in critically endangered captive European minks (Mustela lutreola).

The European mink (Mustela lutreola) is a riparian mustelid, considered one of the most endangered carnivores in the world. Alpha, beta and gammaherpesviruses described in mustelids have been occasionally associated with different pathological processes. However, there is no information about the herpesviruses species infecting European minks. In this study, 141 samples of swabs (oral, conjunctival, anal), faeces and tissues from 23 animals were analysed for herpesvirus (HV) using a pan-HV-PCR assay. Two different, potentially novel, gammaherpesvirus species were identified in 12 samples from four animals (17.3%), and tentatively named Mustelid gammaherpesvirus-2 (MUGHV-2) and MuGHV-3. Gross examination was performed on dead minks (n = 11), while histopathology was performed using available samples from HV-positive individuals (n = 2), identifying several neoplasms, including B-cell lymphoma (identified by immunohistochemistry) with intralesional syncytia and intranuclear inclusion bodies characteristic of HV (n = 1), pulmonary adenocarcinoma (n = 1), and biliary (n = 1) and preputial (n = 1) cystadenomas, as well as other lesions (e.g., axonal vacuolar degeneration [n = 2] and neuritis [n = 1]). Viral particles, consistent with HVs, were observed by electron microscopy in the mink with neural lymphoma and inclusion bodies. This is the first description of neoplasms and concurrent gammaherpesvirus infection in European minks. The pathological, ultrastructural and PCR findings (MuGHV-2) in the European mink with lymphoma strongly suggest a potential role for this novel gammaherpesvirus in its pathogenesis, as it has been reported in other HV-infected species with lymphoma. The occurrence of neural lymphoma with intralesional syncytia and herpesviral inclusions is, however, unique among mammals. Further research is warranted to elucidate the potential oncogenic properties of gammaherpesviruses in European mink and their epidemiology in the wild population.

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.

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.

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.

Carnivore Parvovirus Ecology in the Serengeti Ecosystem: Vaccine Strains Circulating and New Host Species Identified.

Carnivore parvoviruses infect wild and domestic carnivores, and cross-species transmission is believed to occur. However, viral dynamics are not well understood, nor are the consequences for wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multihost system such as the Serengeti ecosystem and identify potential threats for wildlife conservation, we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separate routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP) and, second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized; new host-virus associations involving wild dogs, jackals, and hyenas were discovered; and our results suggest that mutations in the fragment of the vp2 gene were not required for infection of different carnivore species. In domestic dogs, 6 sequences belonged to the CPV-2a strain, while 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.IMPORTANCE Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. This study furthers the understanding of carnivore parvovirus epidemiology, suggesting that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP), with new host species identified, and that canine parvoviruses are present in the dog population living around the SNP. Little evidence of transmission of canine parvoviruses into wild carnivore species was found; however, the detection of vaccine-derived virus (described here for the first time to be circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.

Novel herpesviruses in riverine and marine cetaceans from South America.

Herpesvirus (HV) infections in cetaceans are frequently associated with skin and mucosal lesions. Although HV infections have been reported worldwide, their occurrence in southern Atlantic marine mammals is still poorly understood. We tested skin, oral and genital mucosal beta-actin PCR-positive samples from 109 free-ranging Brazilian cetaceans using a universal herpesvirus DNA polymerase PCR. Herpesvirus-positive skin samples from a Guiana dolphin (Sotalia guianensis), a dwarf sperm whale (Kogia sima), a Bolivian river dolphin (Inia boliviensis), and a lingual sample from an Atlantic spotted dolphin (Stenella frontalis) were histologically evaluated. Additional tissue samples from these animals were also PCR-positive for HV, including a novel sequence obtained from the dwarf sperm whale's stomach and mesenteric lymph node. Four novel HV species were detected in the Guiana dolphin (one), the dwarf sperm whale (two) and the Bolivian river dolphin (one). The cutaneous lesions (marked, focally extensive, chronic proliferative dermatitis) of the Guiana dolphin and the Bolivian river dolphin were similar to previous HV reports in cetaceans, despite the absence of intranuclear inclusion bodies. This is the largest HV survey in South American cetaceans and the first detection of HV infection in riverine dolphins worldwide.

Novel and highly sensitive SYBR® Green real-time pcr for poxvirus detection in odontocete cetaceans.

Poxviruses are emerging pathogens in cetaceans, temporarily named 'Cetaceanpoxvirus' (CePV, family Poxviridae), classified into two main lineages: CePV-1 in odontocetes and CePV-2 in mysticetes. Only a few studies performed the molecular detection of CePVs, based on DNA-polymerase gene and/or DNA-topoisomerase I gene amplification. Herein we describe a new real-time PCR assay based on SYBR® Green and a new primer set to detect a 150 bp fragment of CePV DNA-polymerase gene, also effective for conventional PCR detection. The novel real-time PCR was able to detect 5 up to 5 × 106 copies per reaction of a cloned positive control. Both novel PCR methods were 1000 to 100,000-fold more sensitive than those previously described in the literature. Samples of characteristic poxvirus skin lesions ('tattoo') from one Risso's dolphin (Grampus griseus), two striped dolphins (Stenella coeruleoalba) and two Guiana dolphins (Sotalia guianensis) were all positive to both our novel real time- and conventional PCR methods, even though three of these animals (a Risso's dolphin, a striped dolphin, and a Guiana dolphin) were previously negative to the conventional PCRs previously available. To our knowledge, this is the first real-time PCR detection method for Cetaceanpoxvirus, a much more sensitive tool for the detection of CePV-1 infections.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

Seroprevalence of paramyxoviruses in synanthropic and semi-free-range birds.

Avian paramyxoviruses (APMVs) are classified into nine different serotypes (APMV 1-9). Virulent strains of APMV-1 are already well characterized as the etiologic agent of Newcastle disease (ND), an important disease in poultry that is potentially capable of infecting all orders of avian species. However, very little is known about the other eight serotypes, the majority of which can cause disease in domestic birds. The role of synanthropic and semi-free-range birds as reservoirs of avian paramyxoviruses is not well understood and the main objective of this work was to evaluate the seroprevalence of APMV 1-9 in these kind of birds. A total of 296 sera, oropharyngeal swabs, and cloacal enemas were collected from semi-free-range birds belonging to four different species: feral pigeons (Columba livia var. domestica), hybrid ducks (Anas sp.), domestic geese (Anser anser domesticus), and white storks (Ciconia ciconia). Antibodies against NDV were found in 56.3% of domestic geese, 42.9% of feral pigeons, and 30.4% of hybrid ducks. Antibodies for other APMVs (-3, -4, -6, -7, -8, -9) were also found. Seven positive individuals were positive to real-time RT-PCR detection, all of them feral pigeons captured in 2006 and 2007. The results obtained reinforce the idea that semi-free-range birds may be good sentinels for the detection of NDV and other avian paramyxoviruses.

Screening for several potential pathogens in feral pigeons (Columba livia) in Madrid.

BACKGROUND: Pathogens with the zoonotic potential to infect humans, such as Campylobacter jejuni, Campylobacter coli and Chlamydophila psittaci, can be found in feral pigeons (Columba livia). Given the high density of these birds in the public parks and gardens of most cities, they may pose a direct threat to public health. METHODS: A total of 118 pigeons were captured in three samplings carried out in 2006-2007 in public parks and gardens in Madrid, Spain. Standard haematological and morphological analyses were carried out on the pigeons. PCR was used to screen for the presence of Campylobacter jejuni, C. coli and Chlamydophila psittaci. Positive samples were confirmed by DNA sequencing. RESULTS: The analyses demonstrated a high prevalence of Chlamydophila psittaci (52.6%) and Campylobacter jejuni (69.1%) among the birds captured. In contrast, Campylobacter coli was rarely detected (1.1%). CONCLUSIONS: Pigeons in Madrid can carry Chlamydophila psittaci and Campylobacter jejuni. They may be asymptomatic or subclinical carriers of both pathogens.