A comparison of antibiotic resistance genes and mobile genetic elements in wild and captive Himalayan vultures.

As the most widely distributed scavenger birds on the Qinghai-Tibetan Plateau, Himalayan vultures (Gyps himalayensis) feed on the carcasses of various wild and domestic animals, facing the dual selection pressure of pathogens and antibiotics and are suitable biological sentinel species for monitoring antibiotic resistance genes (ARGs). This study used metagenomic sequencing to comparatively investigate the ARGs and mobile genetic elements (MGEs) of wild and captive Himalayan vultures. Overall, the resistome of Himalayan vultures contained 414 ARG subtypes resistant to 20 ARG types, with abundances ranging from 0.01 to 1,493.60 ppm. The most abundant resistance type was beta-lactam (175 subtypes), followed by multidrug resistance genes with 68 subtypes. Decreases in the abundance of macrolide-lincosamide-streptogramin (MLS) resistance genes were observed in the wild group compared with the zoo group. A total of 75 genera (five phyla) of bacteria were predicted to be the hosts of ARGs in Himalayan vultures, and the clinical (102 ARGs) and high-risk ARGs (35 Rank I and 56 Rank II ARGs) were also analyzed. Among these ARGs, twenty-two clinical ARGs, nine Rank I ARG subtypes, sixteen Rank II ARG subtypes were found to differ significantly between the two groups. Five types of MGEs (128 subtypes) were found in Himalayan vultures. Plasmids (62 subtypes) and transposases (44 subtypes) were found to be the main MGE types. Efflux pump and antibiotic deactivation were the main resistance mechanisms of ARGs in Himalayan vultures. Decreases in the abundance of cellular protection were identified in wild Himalayan vultures compared with the captive Himalayan vultures. Procrustes analysis and the co-occurrence networks analysis revealed different patterns of correlations among gut microbes, ARGs, and MGEs in wild and captive Himalayan vultures. This study is the first step in describing the characterization of the ARGs in the gut of Himalayan vultures and highlights the need to pay more attention to scavenging birds.

Disposition of posaconazole after single oral administration in large falcons (Falco spp): Effect of meal and dosage and a non-compartmental model to predict effective dosage.

Posaconazole has been used anecdotally to treat aspergillosis in falcons resistant to voriconazole. In human medicine, it is used prophylactically in immunosuppressed human subjects with invasive pulmonary aspergillosis. So far, no studies have been performed in birds. The aim of this study was to evaluate the in-vivo pharmacokinetic behavior of oral posaconazole after a single administration in six large falcons (i.e gyrfalcons, saker falcons). Posaconazole oral suspension (Noxafil, 40 mg/ml, Schering-Plough) was administered per os without meal in a single dosage of 12.5 mg/kg in 3 falcons. A comparison was done in two more falcons, one with a natural fatty meal at the same single dose, and one with a natural fatty meal and a higher dosage (20 mg/kg). Finally, six falcons received posaconazole pre-dissolved in corn oil with a natural low-fat meal in the higher single dose (20 mg/kg). No side effects were observed in the falcons in any of the experiments. In starved state posaconazole was poorly absorbed, more so than in other species. As expected, absorption of posaconazole was higher with the administration of meal or in the presence of plant (corn) oil, with a fourfold increase in apparent bioavailability. Despite the preferential absorption in the presence of fat, for both dosing schemes the AUC24 : MIC ratio was lower than described in human medicine to achieve a therapeutic effect. The AUCinf : MIC which is an indicator of efficacy after steady-state, while variable, did indicate that the drug is worth trying when susceptibility testing shows to be the only effective drug. LAY ABSTRACT: The focus of this work is to determine the pharmacokinetic parameters of oral posaconazole in large falcons for the first time after a single dose. Posaconazole has higher bioavailability when administered with meal and fatty components. No adverse reactions have been observed. The ratio of the area under the curve (AUC24) to minimum inhibitory concentration was lower compared to the therapeutic level in human.

Description, occurrence and significance of Mycoplasma seminis sp. nov. isolated from semen of a gyrfalcon (Falco rusticolus).

The Mycoplasma strain ARNO was isolated from the semen of a clinically healthy gyrfalcon (Falco rusticolus). Colonies of strain ARNO grew in fried-egg shape on Mycoplasma agar plates (SP4). The organism did not ferment glucose or hydrolyze arginine or urea; hence, organic acids are assumed as energy source. Growth was sterol-dependent and optimal growth temperature 42 °C, with a temperature range from 20 to 44 °C. Strain ARNO was not identified as a representative of any of the currently described Mycoplasma species by alignment of the 16S rRNA gene sequence and 16 S-23 S intergenic transcribed spacer region, or immunobinding assay. Hence, strain ARNO represents a novel Mycoplasma species for which the name Mycoplasma seminis sp. nov. is proposed (DSM 27653, NCTC 13927). After developing a species-specific PCR, the prevalence of M. seminis sp. nov. was determined in adult and juvenile falcons in a commercial breeding center for falcons. Semen samples (n = 171) were obtained from 113 male adults, due to repeated sampling of 39 birds. Female adults (n = 26) were sampled once, while 105 of the 152 juvenile birds were sampled twice via choanal swabs. Mycoplasma seminis sp. nov. was found in the semen of clinically healthy adult males (3.5 %) as well as in the respiratory tract of female (34.6 %) and juvenile birds (59.2 %). After comparison of semen samples with (2.9 %) and without M. seminis sp. nov. identification, no indications for a potential influence on the semen quality were demonstrated. Hence, M. seminis sp. nov. seems likely to be of commensal character in falcons.

Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?

The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate-to-high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin-resistant and ESBL-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

Pharmacokinetics of voriconazole after a single intramuscular injection in large falcons (Falco spp.).

Voriconazole is one of the main azoles used to treat invasive aspergillosis in falconry raptors and birds. Despite the fact that there are studies for oral and intravenous use of voriconazole in birds, there are none for its effect after intramuscular use. Empirical use of intramuscular voriconazole in falcons, indicated quicker therapy response than the oral one. Aim of this study is to evaluate the in vivo pharmacokinetic disposition of injectable voriconazole after a single intramuscular injection in large falcons (i.e., Gyrfalcons, Saker falcons, Peregrine falcons). No clinical side effects were observed in the falcons. Absorption of voriconazole was rapid (0.5-2 hours) and reached a plasma level (>1 µg/ml) which is above the minimal inhibitory concentration (MIC) for all known Aspergillus strains. This level was maintained for 16 to 20 hours, thus indicating that a single injection of 12.5 mg/kg is not enough if T > MIC is taken into consideration. On a newer aspect, according to the AUC24 unbound: MIC parameter would be indicated that this dose would be rather sufficient for most Aspergillus strains.

Hindgut Bacterial Flora Analysis in Oriental Honey Buzzard (Pernis ptilorhynchus).

The intestinal microbiome is known to affect host health through various effects on nutrition and immunity. The oriental honey buzzard (OHB) is a raptor that feeds on bees and wasps. Due to its restricted diet, we reasoned that the OHB may have a unique microbiome. The aim of this study was to characterize the structure of the intestinal flora of oriental honey buzzards and to investigate the difference of intestinal bacterial flora between individuals in the wild and those reared in captivity. We investigated the intestinal microbiome of seven wild buzzards (Wild), one zoo-reared (Zoo), and one individual reared in captivity for one month (Rearing). Average operational taxonomic units in Wild and Rearing were 69.4 and 113, respectively. Diversity indices such as ACE, Chao 1, Shannon, and Alpha were significantly lower in the Wild than in the Rearing samples. These results suggest that the variety of Wild microbiome is remarkably low. At the phylum level, the composition of the microbiome was similar in all three groups, with firmicutes and bacteroidetes predominating. The third most abundant bacterium in Wild was Proteobacteria, whereas it was Actinobacteria in Rearing and unclassified bacteria in Zoo. Thus, microbiome composition is affected even with just one month of human rearing.

Mycoplasma hafezii sp. nov., isolated from the trachea of a peregrine falcon (Falco peregrinus).

Mycoplasma species are well known pathogens in avian medicine, especially in poultry. However, several Mycoplasma species have been regularly found in the respiratory tract of birds of prey which seem to be commensals in these bird species. In previous studies, an unknown Mycoplasma species which caused false positive results in a Mycoplasma meleagridis-specific PCR, was isolated from a tracheal swab of a clinically healthy, captive, adult peregrine falcon (Falco peregrinus). The isolate appeared in typical fried-egg-shaped colonies on SP4 agar plates and was dependent on sterol for growth. Acid was produced from glucose, but no arginine or urea was hydrolysed. The temperature range for growth was 28-44 °C, with an optimum at 37 °C. Strain M26T was serologically distinct from all species of the genus Mycoplasma with 16S rRNA gene sequence similarity ≥94 %. Biochemical, serological and molecular biological properties demonstrate that this organism represents a novel species of the genus Mycoplasma, for which the name Mycoplasma hafezii sp. nov. is proposed; the type strain is M26T (NCTC 13928, DSM 27652).

Detection of MRSA of Lineages CC130-mecC and CC398-mecA and Staphylococcus delphini-lnu(A) in Magpies and Cinereous Vultures in Spain.

The aim of this study was to determine the carriage rate of coagulase-positive staphylococci (CoPS) in wild birds and to characterize recovered isolates. Tracheal samples from 324 wild birds, obtained in different Spanish regions during 2015-2016, were screened for CoPS carriage. The antimicrobial resistance profile and the virulence gene content were investigated. Molecular typing was performed by spa, agr, MLST, SCCmec, and S. delphini group classification. CoPS were recovered from 26 samples of wild birds (8.3%), and 27 isolates were further characterized. Two CoPS species were detected: S. aureus (n = 15; eight cinereous vultures and seven magpies) and S. delphini (n = 12; 11 cinereous vultures and one red kite). Thirteen S. aureus were methicillin-resistant (MRSA) and the remaining two strains were methicillin-susceptible (MSSA). Twelve MRSA were mecC-positive, typed as t843-ST1583/ST1945/ST1581/ST1571 (n = 11) and t1535-ST1945 (n = 1) (all of clonal-complex CC130); they were susceptible to the non-ß-lactams tested. The remaining MRSA strain carried the mecA gene, was typed as t011-ST398-CC398-agrI-SCCmec-V, and showed a multiresistance phenotype. MSSA isolates were ascribed to lineages ST97-CC97 and ST425-CC425. All S. aureus lacked the studied virulence genes (lukS/F-PV, tst, eta, etb, and etd), and the IEC type E (with scn and sak genes) was detected in four mecC-positive and one MSSA isolates. S. delphini strains were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%, with blaZ gene) and tetracycline [58%, with tet(K)± tet(L)] resistance rates. All S. delphini isolates presented the virulence genes lukS-I, siet, and se-int, and four carried the clindamycin-resistance lnu(A) gene.

Scavenger birds exploiting rubbish dumps: Pathogens at the gates.

Different bacteria are present in rubbish dumps used as food resources by various bird species. Birds may be good indicators of the presence of zoonotic diseases in these sites since they can be infected with zoonotic pathogens by foraging on organic waste, and can also act as carriers. We studied if foraging in rubbish dumps increases the occurrence of Salmonella spp. and Chlamydia psittaci in American black vultures (Coragyps atratus, hereafter black vultures) from northwest Patagonia. We compared these pathogens isolated from or detected in cloacae and oropharynx swabs in two different groups of black vultures: individuals trapped in (a) the Patagonian wild steppe and (b) in a rubbish dump. We found that black vultures are colonized by Salmonella spp. (particularly Salmonella enterica serotypes Typhi, Paratyphi A, Salmonella enterica subsp. arizonae) and Chlamydia psittaci. Interestingly, there were differences in the prevalence of Salmonella spp., especially Salmonella Typhi, between individuals foraging in the rubbish dump and the steppe, but not in the prevalence of Chlamydia psittaci. The pathogens isolated from black vultures may impact their health status but could also have health impacts in other bird species and even humans. In fact, Salmonella Typhi can cause severe disease in humans leading to death. Our results are globally relevant given that bacterial infections from rubbish dumps may affect different species exploiting these sites around the world. There is a need to better control pathogens in rubbish dumps to avoid the risk of infecting wildlife, which could act as potential dispersers and reservoirs of these pathogens.

Protective role of the vulture facial skin and gut microbiomes aid adaptation to scavenging.

BACKGROUND: Vultures have adapted the remarkable ability to feed on carcasses that may contain microorganisms that would be pathogenic to most other animals. The holobiont concept suggests that the genetic basis of such adaptation may not only lie within their genomes, but additionally in their associated microbes. To explore this, we generated shotgun DNA sequencing datasets of the facial skin and large intestine microbiomes of the black vulture (Coragyps atratus) and the turkey vulture (Cathartes aura). We characterized the functional potential and taxonomic diversity of their microbiomes, the potential pathogenic challenges confronted by vultures, and the microbial taxa and genes that could play a protective role on the facial skin and in the gut. RESULTS: We found microbial taxa and genes involved in diseases, such as dermatitis and pneumonia (more abundant on the facial skin), and gas gangrene and food poisoning (more abundant in the gut). Interestingly, we found taxa and functions with potential for playing beneficial roles, such as antilisterial bacteria in the gut, and genes for the production of antiparasitics and insecticides on the facial skin. Based on the identified phages, we suggest that phages aid in the control and possibly elimination, as in phage therapy, of microbes reported as pathogenic to a variety of species. Interestingly, we identified Adineta vaga in the gut, an invertebrate that feeds on dead bacteria and protozoans, suggesting a defensive predatory mechanism. Finally, we suggest a colonization resistance role through biofilm formation played by Fusobacteria and Clostridia in the gut. CONCLUSIONS: Our results highlight the importance of complementing genomic analyses with metagenomics in order to obtain a clearer understanding of the host-microbial alliance and show the importance of microbiome-mediated health protection for adaptation to extreme diets, such as scavenging.

Actinomyces tangfeifanii sp. nov., isolated from the vulture Aegypius monachus.

A novel, Gram-stain-positive, catalase-positive, non-spore-forming, short rod-shaped strain (VUL4_3T) was isolated from rectal swabs of Old World vultures (Aegypius monachus) from the Tibet-Qinghai Plateau, China. Based on the results of biochemical tests and 16S rRNA gene sequence comparison, strain VUL4_3T was determined to be a member of the genus Actinomyces that is closely related to the type strains of Actinomyces liubingyangii (97.7 % 16S rRNA gene sequence similarity) and Actinomyces marimammalium (96.5 %). Optimal growth occurred at 37 °C, pH 6-7 and with 1 % (w/v) NaCl. The typical major cellular fatty acids of strain VUL4_3T were C18 : 1ω9c, C16 : 0 and C18 : 0. The VUL4_3T genome contained 2 207 832 bp with an average G+C content of 51.9 mol%. DNA-DNA hybridization values between strain VUL4_3T and the above two species of the genus Actinomyces showed less than 32 % DNA-DNA relatedness, supporting a novel species status of strain VUL4_3T. Based on the phenotypic data and phylogenetic inference, the novel species Actinomycestangfeifanii sp. nov. is proposed. The type strain is VUL4_3T (=CGMCC 4.7369T=DSM 103436T).

Oceanisphaera avium sp. nov., isolated from the gut of the cinereous vulture, Aegypius monachus.

A Gram-stain-negative, aerobic, catalase- and oxidase-positive, rod-shaped, flagellated bacterial strain, designated AMac2203T, was isolated from the gut of the cinereous vulture, Aegypiusmonachus, collected from the Seoul Grand Park Zoo, Republic of Korea. Strain AMac2203T grew optimally at 15-25 °C, pH 7-8 and in the presence of 3-5 % (w/v) NaCl. Phylogenetic analysis revealed 97.4-97.9 % and 96.9-97.3 % sequence similarities of the 16S rRNA genes to its counterparts in Oceanisphaera profunda SM1222T and Oceanisphaera ostreae T-w6T, respectively. The predominant fatty acids (>10 %) of strain AMac2203T were summed feature 3 (C16 : 0ω7c and/or C16 : 1ω6c, 33.6 %), summed feature 8 (C18 : 1ω7c, 24.5 %) and C16 : 0 (19.9 %). The primary isoprenoid quinone was ubiquinone-8. Polar lipids included phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified amino lipid and an unidentified lipid. Based on complete genome sequencing of strain AMac2203T and the closest related type strain, O. profunda, the OrthoANI value is 77.5 %, which is below the 95 % cut-off for species demarcation. The genomic DNA G+C content of strain AMac2203T is 47.1 mol%. Thus, strain AMac2203T represents a novel species candidate of the genus Oceanisphaera. We propose the name Oceanisphaeraavium sp. nov., with strain AMac2203T (=KCTC 62118T=JCM 32207T) as the type strain.

Supplementary feeding stations for conservation of vultures could be an important source of monophasic Salmonella typhimurium 1,4,[5],12:i:.

Vultures are nature's most successful scavengers, feeding on the carcasses of dead animals present in the field. Availability of domestic carrion has been unstable due to rapidly changing agro-grazing economies and increasing sanitary regulations that may require burial or burning of livestock carcasses. Thus, several griffon vulture (Gyps fulvus) recoveries are based on European legislation that guarantees the animals' welfare, avoids intense persecution of the vultures and allows the feeding of threatened wildlife in supplementary feeding stations (SFS). However, in recent years, many studies have speculated on the likelihood that avian scavengers may be infected by feeding on pig carcasses at SFS from intensive livestock. In this context, the present study evaluated whether free-living griffon vultures and pig farms share zoonotic Salmonella strains to test the hypothesis that vulture are infected during consumption of carcasses provided at SFS. Here, the occurrence, serotypes and genomic DNA fingerprinting (phage typing and pulsed-field gel electrophoresis) of isolated strains were carried out in griffon vultures and pig farms authorised to provided carcasses at SFS in Castellón province (eastern Spain). The bacteriological analyses revealed that 21.1% of vultures and 14.5% for pig farms samples tested were Salmonella-positive. Monophasic S. typhimurium 1,4,[5],12:i:- was the most frequently isolated serovar. Comparison of Salmonella strains isolated from vultures and pig farms revealed that monophasic S. typhimurium 1,4,[5],12:i:-, S. Derby and S. Rissen strains were highly genetically homogeneous (similar DNA fingerprint). In conclusion, the current study indicates that free-living griffon vultures and pig farms that provide the carcasses at SFS share several zoonotic Salmonella strains. On this basis, and although transmission could be bidirectional, our result seems to corroborate the pig carcasses-to-vulture transmission and cross-infection at SFS. As an immediate Salmonella control strategy in wild avian scavengers, we suggest the implementation of a programme to guarantee that solely pig carcasses from Salmonella-free farms arrive at SFS.

Tumebacillus avium sp. nov., isolated from the gut of a cinereous vulture, Aegypius monachus.

A Gram-stain-positive, facultatively aerobic, spore-forming, oxidase-positive, catalase- and DNase-negative, rod-shaped and motile bacterial strain, AR23208T, was isolated from the gut of a cinereous vulture (Aegypius monachus), collected at Seoul Grand Park Zoo (Republic of Korea). Strain AR23208T grew optimally at 25-30 °C, at pH 7 and in the absence of NaCl. Phylogenetic analysis revealed that strain AR23208T shared 98.2 and 97.1 % 16S rRNA gene sequence similarity with Tumebacillus algifaecis THMBR28T and Tumebacilluslipolyticus NIO-S10T, respectively. The predominant fatty acids (>10 %) of strain AR23208T were iso-C15 : 0, summed feature 4 (anteiso-C17 : 1 B and/or iso-C17 : 1 I) and anteiso-C15 : 0 and the primary isoprenoid quinone was menaquinone-7. The polar lipids were phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, six unidentified phospholipids, an unidentified aminophospholipid and ten unidentified lipids. The sugar components of the cell wall peptidoglycan were ribose and arabinose. The amino acids of the cell wall peptidoglycan were l-alanine, aspartic acid, meso-diaminopimelic acid, l-glutamic acid, glycine and l-lysine. The OrthoANI value based on the complete genome sequence of strain AR23208T and the closest related strain, T. algifaecis THMBR28T, was 80.4 %. The genomic DNA G+C content of strain AR23208T was 56.0 mol%. Based on the data presented in the current study, strain AR23208T is considered to represent a novel species of the genus Tumebacillus, for which the name Tumebacillus avium sp. nov. is proposed. The type strain is AR23208T (=KCTC 33929T=JCM 32188T).

[Tularemia in a jogger woman after the attack by a common buzzard (Buteo buteo): A "One Health" case report]. / Tularämie nach Angriff eines Mäusebussards auf eine Joggerin: Ein "One Health"-Fallbericht.

INTRODUCTION: A female jogger was attacked by a common buzzard (Buteo buteo) and was scratched lightly at the back of the head. One week later she was taken ill with high fever and headache which was later diagnosed as ulcero-glandular tularemia in regional lymph nodes, caused by Francisella tularensis. Recovery was only achieved after several weeks of systemic antibiotic treatment (Gentamicin/ Ciprofloxacine). Tularemia is a well known zoonotic disease, called "rabbit fever", mainly affecting rabbits and hares, but also small rodents. Human infection occurs often following tick bites or bloodsucking insects, or in hunters or slaughterers handling infected animals. Bites by mice have also been reported as a cause of tularemia. For the first time we report this case of tularemia as a result of an attack by a bird of prey. We assume that the bird acted as a vector just carrying the F. tularensis on its claws or beak, but we cannot exclude an infection of the bird itself. Several other joggers had also been attacked by a common buzzard in the same area shortly after the above described event and one of these also became infected with F. tularensis.

Oral mycoses in avian scavengers exposed to antibiotics from livestock farming.

The exposure to antimicrobial pharmaceuticals as environmental contaminants can exert direct and indirect detrimental effects on health of wildlife. Fungal infections pose a major threat to domestic, captive-housed wild and free-ranging wild animals worldwide. However, little is known about their role in disease in birds in the wild. Here, we evaluated the incidence of thrush-like lesions in the oral cavity of wild nestling cinereous vultures (Aegypius monachus), griffon vultures (Gyps fulvus), Egyptian vultures (Neophron percnopterus) and golden eagles (Aquila chrysaetos) exposed to veterinary antibiotics via the consumption of medicated livestock carcasses. Lesions, which varied in number, size and location, were more frequent in the cinereous (77.8%, n=9) and griffon vultures (66.7%, n=48) than in the Egyptian vultures (28.6%, n=21) and golden eagles (28.6%, n=7). In all individuals (100%, n=24) of a subsample of the affected nestlings, yeast species were isolated from thrush-like oral lesions and identified using a well-established system based on their carbohydrate assimilation profiles and other complementary tests. Fourteen yeast species from seven genera (Candida, Meyerozyma, Pichia, Yarrowia, Cryptococcus, Rhodotorula and Trichosporon) were isolated from the lesions of the four host species. We found differential infections and effects depending on host age-related exposure or susceptibility to different yeast species across the development of nestling griffon vultures. This unprecedented outbreak of oral mycoses is alarming because of the delicate conservation status of several of the affected species. The role of livestock antibiotics in the transition of yeast species from commensal to opportunistic pathogens should be evaluated in an attempt to avoid the detrimental effects of contamination and disease on host health, as well as on the transmission of fungal emerging pathogens among wildlife populations and species, and their dissemination across livestock and human populations.

Actinomyces liubingyangii sp. nov. isolated from the vulture Gypaetus barbatus.

Two strains (VUL4_1T and VUL4_2) of Gram-staining-positive, catalase-negative, non-spore-forming short rods were isolated from rectal swabs of Old World vultures (Gypaetus barbatus) in the Tibet-Qinghai Plateau, China. Analysis of morphological characteristics and biochemical tests indicated that the two strains closely resembled each other but were distinct from other species of the genus Actinomyces previously described. Based on the results of 16S rRNA gene sequence comparison and genome analysis, strains were determined to be members of the genus Actinomyces, closely related to the type strains of Actinomyces marimammalium (96.4 % 16S rRNA gene sequence similarity), Actinomyceshongkongensis (92.4 %), Actinomyceshordeovulneris (92.3 %) and Actinomycesnasicola (92.2 %), respectively. Optimal growth conditions were 37 °C, pH 6-7, with 1 % (w/v) NaCl. Strain VUL4_1T contained C18 : 1ω9c and C16 : 0 as the major cellular fatty acids and diphosphatidylglycerol as the major component of the polar lipids. The genomic DNA G+C content of VUL4_1T was 54.9 mol%. Strain VUL4_1T showed less than 70 % DNA-DNA relatedness with other species of the genus Actinomyces, further supporting strain VUL4_1T as a representative of a novel species. Based on the phenotypic data and phylogenetic inference, a novel species, Actinomyces liubingyangii sp. nov., is proposed with VUL4_1T (=CGMCC 4.7370T=DSM 104050T) as the type strain.

Actinomyces vulturis sp. nov., isolated from Gyps himalayensis.

Two strains of Gram-stain-positive, facultatively anaerobic, non-spore-forming short rods (VUL7T and VUL8) were isolated from rectal swabs of Old World vultures, namely Gyps himalayensis, in Tibet-Qinghai Plateau, China. Optimal growth occurred at 37 °C, pH 6-7, with 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences classified the two strains to the genus Actinomyces, with highest 16S rRNA gene sequence similarity (95 %) to type strains of Actinomyces haliotis, Actinomyces radicidentis and Actinomyces urogenitalis. The major cellular fatty acids were C18 : 1ω9c and C16 : 0. MK-10(H4) was the major respiratory quinone. The genomic DNA G+C content of the isolate was 54.4 mol%. DNA-DNA hybridization values with the most closely related species ofthe genusActinomyces was 24.6 %. The two strains can be differentiated from the most closely related species such as A. haliotis, A. radicidentis, A. graevenitzii and A. urogenitalis by a list of carbohydrate fermentations and enzyme activities. On the basis of physiological, biochemical and phylogenetic analysis, strains VUL7T and VUL8 represent novel species of the genus Actinomyces, for which the name Actinomyces vulturis sp. nov. is proposed. The type strain is VUL7T (=CGMCC 4.7366T=DSM 103437T).

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens.

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.

Generalized Tetanus in a Gyrfalcon ( Falco rusticolus ) with Pododermatitis.

A 2-yr-old male gyrfalcon ( Falco rusticolus ) was presented for severe and generalized muscle spasticity and pododermatitis. The falcon had been treated for pododermatitis over the previous 4 mo. Muscle rigidity and spasms involved the entire bird but were more severe on the right leg. The bird was also tachypneic and hyperthermic at 45 C. While the plantar pododermatitis lesions had healed, there was still a small abscess on the lateral aspect of the right foot. Clinical signs were consistent with tetanus. Several bacteria were isolated from the abscess including Clostridium tetani . The isolate was confirmed to be toxigenic by PCR. Attempts to detect tetanus toxin in the bird's plasma were unsuccessful. The abscess was debrided. The gyrfalcon received equine tetanus antitoxin, intravenous metronidazole, methocarbamol, midazolam, a constant-rate infusion of Fentanyl, active cooling, and supportive care. Inhalant anesthesia with isoflurane was the only treatment that would lower the body temperature and reduce the clinical signs. The gyrfalcon died a few hours after admission. The characteristic clinical signs and isolation of toxigenic C. tetani from a wound were strong supportive evidence for a diagnosis of tetanus. This case constitutes the first reported natural occurrence of tetanus in an avian species. Further information is needed to determine whether gyrfalcons are more susceptible to tetanus than are other avian species and whether pododermatitis lesions may be risk factors.