Variable effects of captivity on microbiomes in populations of IUCN-endangered Blanding's turtles (Emydoidea blandingii).

AIMS: Microbiome composition is increasingly considered in species reintroduction efforts and may influence survival and reproductive success. Many turtle species are threatened by anthropogenic pressures and are frequently raised in captivity for reintroduction efforts, yet little is known about turtle microbiome composition in either wild or captive settings. Here, we investigated trends in microbiome composition of captive and wild IUCN-endangered Blanding's turtles (Emydoidea blandingii). METHODS AND RESULTS: We amplified and sequenced the V4 region of the 16S rDNA locus from plastron, cloaca, and water samples of wild E. blandingii adults and two populations of captive E. blandingii juveniles being raised for headstarting. Plastron, cloaca, and water-associated microbiomes differed strongly from each other and were highly variable among captive sites and between captive and wild sites. Across plastron, cloaca, and water-associated microbial communities, microbial diversity changed over time, but not in a predictable direction between captive sites. Plastron beta diversity correlated with growth rate in captive samples, indicating that external microbiomes may correlate with individual fitness. CONCLUSIONS: Our results indicate that external and internal microbiomes vary between captive and wild turtles and may reflect differences in fitness of captive-raised individuals.

Mannheimia cairinae sp. nov., a novel species of Mannheimia obtained from Muscovy ducks (Cairina moschata) and reclassification of Mannheimia ovis as heterotypic synonym of Mannheimia pernigra.

During a screening study for Pasteurella multocida in two unrelated flocks of Muscovy ducks pharyngeal and cloacal swabs were collected. A total of 59 Pasteurellaceae-like isolates sharing the same colony morphology were subcultured and subsequently characterized. Colonies on bovine blood agar were nonhaemolytic, regular, circular, slightly raised, shiny, intransparent with an entire margin, greyish and had an unguent-like consistency. Isolate AT1T was characterized by 16S rRNA gene sequencing and showed the highest similarity of 96.1 % to the type strain of Mannheimia caviae and 96.0 % to the type strain of Mannheimia bovis, respectively. In addition, rpoB and recN gene sequences also showed the highest similarity to the genus Mannheimia. The phylogenetic comparison of concatenated conserved protein sequences also showed a unique position of AT1T compared to other species of Mannheimia. Full phenotypic characterization of the isolates showed that between two (Mannheimia ruminalis) and 10 (Mannheimia glucosida) phenotypic characteristics separate the taxon isolated from Muscovy ducks from the accepted species of Mannheimia. Whole genomic sequences of two strains analysed by the type strain genome server showed the highest similarity of 24.9 % to the genome of the type strain of Pasteurella multocida and 23.0 % to the genome of the type strain of Mannheimia haemolytica. The species Mannheimia cairinae sp. nov. is proposed based on the phenotypic and genotypic similarity to Mannheimia as well as differences to the other validly published species of the genus. The leukotoxin protein was not predicted in the genome of AT1T. The G+C content of the type strain of M. cairinae sp. nov., AT1T (=CCUG 76754T=DSM 115341T) is 37.99 mol%, calculated from the whole genome. The investigation further proposes that Mannheimia ovis is reclassified as a later heterotypic synonym of Mannheimia pernigra, since M. ovis and M. pernigra are closely genetically related, and M. pernigra was validly published before M. ovis.

Paralysiella testudinis gen. nov., sp. nov., isolated from the cloaca of a toad-headed turtle (Mesoclemmys nasuta).

A bacterial strain designated 26BT, which had been isolated from the cloaca of a toad-headed turtle, was subjected to a comprehensive taxonomic study. Comparison of 16S rRNA gene sequences demonstrated that strain 26BT is a member of the family Neisseriaceae. Based on highest similarity values, Neisseria animaloris DSM 21642T (95.15 %), Alysiella filiformis ATCC 15532T (95.06 %), Uruburuella testudinis 07_OD624T (94.71 %), Uruburuella suis CCUG 47806T (94.66 %) and Alysiella crassa DSM 2578T (94.64 %) were identified as the closest relatives. Average nucleotide identity values based on the blast algorithm (ANIb) indicated that U. suis (76.10/76.17 %), Neisseria shayeganii 871T (74.34/74.51 %), Stenoxybacter acetivorans (73.30/73.41 %), N. animaloris (72.98/72.80) %, A. filiformis (71.14/71.21 %) and A. crassa (70.53/71.15 %) are the next closest relatives. Like ANIb, genome-based phylogeny did not suggest the affiliation of strain 26BT with any established genus. The polyamine pattern consisted of the major compounds putrescine, 1,3-diaminopropane and spermidine and the major quinone was ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an ornithine lipid were predominant. The fatty acid profile contained predominantly C16 : 1 ω7c, C12 : 0, C14 : 0, C16 : 0 and C12 : 0 3OH. The size of the genome was 2.91 Mbp and the genomic G+C content was 54.0 mol%. Since these data do not demonstrate an unambiguous association with any established genus, we here propose the novel genus Paralysiella with the type species Paralysiella testudinis gen. nov., sp. nov. The type strain is 26BT (=CCM 9137T=LMG 32212T).

Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities.

Chlamydial infections, caused by a group of obligate, intracellular, gram-negative bacteria, have health implications for animals and humans. Due to their highly infectious nature and zoonotic potential, staff at wildlife rehabilitation centers should be educated on the clinical manifestations, prevalence, and risk factors associated with Chlamydia spp. infections in raptors. The objectives of this study were to document the prevalence of chlamydial DNA shedding and anti-chlamydial antibodies in raptors admitted to five wildlife rehabilitation centers in California over a one-year period. Chlamydial prevalence was estimated in raptors for each center and potential risk factors associated with infection were evaluated, including location, species, season, and age class. Plasma samples and conjunctiva/choana/cloaca swabs were collected for serology and qPCR from a subset of 263 birds of prey, representing 18 species. Serologic assays identified both anti-C. buteonis IgM and anti-chlamydial IgY antibodies. Chlamydial DNA and anti-chlamydial antibodies were detected in 4.18% (11/263) and 3.14% (6/191) of patients, respectively. Chamydial DNA was identified in raptors from the families Accipitridae and Strigidae while anti-C.buteonis IgM was identified in birds identified in Accipitridae, Falconidae, Strigidae, and Cathartidae. Two of the chlamydial DNA positive birds (one Swainson's hawk (Buteo swainsoni) and one red-tailed hawk (Buteo jamaicensis)) were necropsied, and tissues were collected for culture. Sequencing of the cultured elementary bodies revealed a chlamydial DNA sequence with 99.97% average nucleotide identity to the recently described Chlamydia buteonis. Spatial clusters of seropositive raptors and raptors positive for chlamydial DNA were detected in northern California. Infections were most prevalent during the winter season. Furthermore, while the proportion of raptors testing positive for chlamydial DNA was similar across age classes, seroprevalence was highest in adults. This study questions the current knowledge on C. buteonis host range and highlights the importance of further studies to evaluate the diversity and epidemiology of Chlamydia spp. infecting raptor populations.

Taxonomy, not locality, influences the cloacal microbiota of two nearctic colubrids: a preliminary analysis.

BACKGROUND: The gut microbiota is an emerging frontier in wildlife research and its importance to vertebrate health and physiology is becoming ever more apparent. Reptiles, in particular snakes, have not received the same attention given to other vertebrates and the composition of their wild gut microbiome remains understudied. The primary goal of this work was to describe the cloacal microbiota of two Colubrids, the Eastern Gartersnake (Thamnophis sirtalis sirtalis) and the Northern Watersnake (Nerodia sipedon sipedon), and if their cloacal microbiota differed as well as if it did between a wetland and upland population of the former species. METHODS AND RESULTS: We utilized next-generation sequencing of cloacal swabs-a non-destructive proxy for the gut microbiota. The cloacal microbiome of Eastern Gartersnakes (N = 9) was like those of other snakes being comprised of Proteobacteria, Bacteroidetes, and Firmicutes, while that of Northern Watersnakes (N = 6) was dominated by Tenericutes. Seven microbial operational taxonomic units (OTUs), all members of Proteobacteria, were shared among all individuals and were indicative of a core microbiome in Eastern Gartersnakes, but these OTUs were not particularly relevant to Northern Watersnakes. The latter had greater OTU richness than did Eastern Gartersnakes, and habitat did not have any apparent effect on the microbial community composition in Eastern Gartersnakes. CONCLUSIONS: Our findings suggest host taxonomy to be a determining factor in the cloacal microbiota of snakes and that Tenericutes are associated with aquatic habitats. This is the first report to examine the cloacal microbiome of these species and provides a useful foundation for future work to build upon.

Longitudinal study on the effects of growth-promoting and therapeutic antibiotics on the dynamics of chicken cloacal and litter microbiomes and resistomes.

BACKGROUND: Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae). RESULTS: Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28-76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and ß-lactam resistance genes was identified. CONCLUSIONS: Within a "farm-to-fork, one health" perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract.

POPULATION PHARMACOKINETICS OF CEFTAZIDIME AFTER A SINGLE SUBCUTANEOUS INJECTION AND NORMAL ORAL AND CLOACAL BACTERIAL FLORA SURVEY IN EASTERN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS ALLEGANIENSIS).

Population pharmacokinetics utilizing sparse sampling were used to determine pharmacokinetics of ceftazidime in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) due to their slow growth rate and the limited number of appropriately sized individuals in the zoo-housed population. Twenty-five eastern hellbenders received a single subcutaneous injection of ceftazidime at 20 mg/kg. Each animal had blood samples collected up to four times between 0 and 192 hr postinjection. Plasma samples were analyzed by high-pressure liquid chromatography. A nonlinear mixed-effects model was fitted to the data to determine typical values for population parameters, an ideal method due to the sampling limitation of each hellbender. Results indicate an elimination half-life of 36.63 hr and volume of distribution of 0.31 L/kg. Antibiotic concentrations were above a minimum inhibitory concentration (MIC) value of 8 µg/ml for 120 hr. Prior to antibiotic administration, six hellbenders had oral and six other individuals had cloacal swabs taken for aerobic culture. Fifty-five bacterial isolates were obtained (24 cloacal, 31 oral) with 10/12 (83%) individuals growing three or more different isolates and 11/12 (92%) growing Shewanella putrefaciens. Twelve isolates had susceptibility testing performed and all were susceptible to ceftazidime. These results indicate that ceftazidime is an appropriate choice of antibiotic in hellbenders and when given at a dosage of 20 mg/kg subcutaneously, maintains concentrations above the MIC of susceptible bacteria for up to 5 days.

Jinshanibacter, a new genus of Budviciaceae: identification of Jinshanibacter zhutongyuii sp. nov. and Jinshanibacter xujianqingii sp. nov. isolated from cloacal content of snow finch (Montifringilla taczanowskii).

Four novel strains isolated from the cloacal contents of snow finches (Montifringilla taczanowskii) were characterized as aerobic, Gram-stain-negative, slightly motile, and rod-shaped. Analysis of the 16S rRNA gene sequence revealed that strain CF-458T had the highest similarities of 96.9 and 96.4 % with Limnobaculum parvum HYN0051T and Pragia fontium DSM 5563T, while strain CF-1111T shared the highest similarities of 96.4 and 96.1 % with Pantoea rodasii LMG 26273T and Pectobacterium punjabense SS95T. Phylogenomic analysis showed the four isolates were separated into group Ⅰ (CF-458T and CF-917) and group Ⅱ (CF-1111T and CF-509), and clustered independently in the vicinity of the genera Limnobaculum and Pragia. Summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c, 23.9 and 17.2 %, respectively), C16 : 0 (21.8 and 22.1 %, respectively) and C14 : 0 (10.6 and 17.7 %, respectively) were the common major fatty acids, and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c, 12.3 %) was also a major fatty acid for strain CF-458T while cyclo-C17 : 0 (13.1%) was for strain CF-1111T. Both had Q-8 as the sole quinone and contained phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as the major polar lipids. The DNA G+C content of strains CF-458T and CF-1111T was 45.7 and 45.4 mol%, respectively. Based on taxonomic position in the phylogenomic tree and phenotypic properties, two novel species of a new genus within the family Budviciaceae are thus proposed, with the name Jinshanibacter gen. nov., zhutongyuii sp. nov. (type strain CF-458T=CGMCC 1.16483T=GDMCC 1.1586T=JCM 33489T) and Jinshanibacter xujianqingii sp. nov. (type strain CF-1111T=CGMCC 1.16786T=GDMCC 1.1587T=JCM 33490T), respectively.

A reasonable correlation between cloacal and cecal microbiomes in broiler chickens.

Gut microbiota play an important role in animal health. For livestock, an understanding of the effect of husbandry interventions on gut microbiota helps develop methods that increase sustainable productivity, animal welfare, and food safety. Poultry microbiota of the mid-gut and hind-gut can only be investigated postmortem; however, samples from the terminal cloaca may be collected from live animals. This study tests whether cloacal microbiota reflect cecal microbiota in European broiler poultry by evaluating total and paired cecal and cloacal microbiomes from 47 animals. 16S amplicon libraries were constructed and sequenced with a MiSeq 250 bp PE read metric. The composition of cloacal and cecal microbiomes were significantly affected by the age and location of animals, but the effect was very small. Bacilli were relatively more abundant in ceca and Clostridia in cloaca. There was an overlap of 99.5% for the abundances and 59% for the types of taxa between cloacal and cecal communities, but the small fraction of rare nonshared taxa were sufficient to produce a signal for differentiation between cecal and cloacal communities. There was a significant positive correlation between specific taxa abundances in cloacal and cecal communities (Rho = 0.66, P = 2 × 10-16). Paired analyses revealed that cloacal communities were more closely related to cecal communities from the same individual than expected by chance. This study is in line with the only other study to evaluate the relationship between cecal and cloacal microbiomes in broiler poultry, but it extends previous findings by analyzing paired cecal-cloacal samples from the same birds and reveals that abundant bacterial taxa in ceca may be reasonably inferred by sampling cloaca. Together, the findings from Europe and Australasia demonstrate that sampling cloaca shows promise as a method to estimate cecal microbiota, and especially abundant taxa, from live broiler poultry in a manner which reduces cost and increases welfare for husbandry and research purposes.

Helicobacter monodelphidis sp. nov. and Helicobacter didelphidarum sp. nov., isolated from grey short-tailed opossums (Monodelphis domestica) with endemic cloacal prolapses.

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).

Comparison of microbiota in the cloaca, colon, and magnum of layer chicken.

Anatomically terminal parts of the urinary, reproductive, and digestive systems of birds all connect to the cloaca. As the feces drain through the cloaca in chickens, the cloacal bacteria were previously believed to represent those of the digestive system. To investigate similarities between the cloacal microbiota and the microbiota of the digestive and reproductive systems, microbiota inhabiting the colon, cloaca, and magnum, which is a portion of the chicken oviduct of 34-week-old, specific-pathogen-free hens were analyzed using a 16S rRNA metagenomic approach using the Ion torrent sequencer and the Qiime2 bioinformatics platform. Beta diversity via unweighted and weighted unifrac analyses revealed that the cloacal microbiota was significantly different from those in the colon and the magnum. Unweighted unifrac revealed that the cloacal microbiota was distal from the microbiota in the colon than from the microbiota in the magnum, whereas weighted unifrac revealed that the cloacal microbiota was located further away from the microbiota in the magnum than from the microbiota inhabiting the colon. Pseudomonas spp. were the most abundant in the cloaca, whereas Lactobacillus spp. and Flavobacterium spp. were the most abundant species in the colon and the magnum. The present results indicate that the cloaca contains a mixed population of bacteria, derived from the reproductive, urinary, and digestive systems, particularly in egg-laying hens. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken's cloacal microbiota should be performed using chickens of different ages and types.

Molecular characterisation of hydrogen sulfide negative Salmonella enterica serovar Havana.

Hydrogen sulfide (H2S) detection is a screening method for distinguishing and identifying Salmonella strains from other bacteria in the intestine. Incidences of H2S-negative Salmonella have recently been reported in different countries. Although a high resistance rate against antimicrobial agents has been reported for H2S-positive Salmonella in many regions of the world, there is increasing evidence that high resistance to antibiotics has also increased in many H2S-negative Salmonella isolates. In this study, molecular characterisation of three H2S-negative Salmonella Havana, isolated from cloacal swab samples of broiler chickens, was performed. The phsA, phsB and phsC genes of the phs operon, which is responsible for hydrogen sulfide production, were amplified. Sequence analysis was then performed to identify mutations in the gene cluster. The antimicrobial resistance profiles of the isolates were determined by disc diffusion. Molecular characterisation was performed by multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). The sequence analysis showed identified five point mutations in the phsA gene and one point mutation in the phsC gene in all isolates. The antibiotic resistance profile showed that the strains were resistant to cefoxitin and ceftazidime. MLST analysis showed that all strains belonged to sequence type (ST) 1621. This study is the first to report the H2S-negative S. Havana serotype.

Isolation of Mycoplasma anserisalpingitidis from swan goose (Anser cygnoides) in China.

BACKGROUND: Mycoplasma anserisalpingitidis causes significant economic losses in the domestic goose (Anser anser) industry in Europe. As 95% of the global goose production is in China where the primary species is the swan goose (Anser cygnoides), it is crucial to know whether the agent is present in this region of the world. RESULTS: Purulent cloaca and purulent or necrotic phallus inflammation were observed in affected animals which represented 1-2% of a swan goose breeding flock (75,000 animals) near Guanghzou, China, in September 2019. From twelve sampled animals the cloaca swabs of five birds (three male, two female) were demonstrated to be M. anserisalpingitidis positive by PCR and the agent was successfully isolated from the samples of three female geese. Based on whole genome sequence analysis, the examined isolate showed high genetic similarity (84.67%) with the European isolates. The antibiotic susceptibility profiles of two swan goose isolates, determined by microbroth dilution method against 12 antibiotics and an antibiotic combination were also similar to the European domestic goose ones with tylvalosin and tiamulin being the most effective drugs. CONCLUSIONS: To the best of our knowledge this is the first description of M. anserisalpingitidis infection in swan goose, thus the study highlights the importance of mycoplasmosis in the goose industry on a global scale.

Vagococcus xieshaowenii sp. nov., isolated from snow finch (Montifringilla taczanowskii) cloacal content.

A Gram-stain-positive, coccus-shaped, non-motile bacterium, designated CF-49T, was isolated from the cloacal content of a snow finch, which was incidentally captured in a plateau pika burrow on the Qinghai-Tibet Plateau, PR China. Analysis of the 16S rRNA gene sequence showed that strain CF-49T was closely related to Vagococcus elongatus CCUG 51432T (96.5 % similarity), Vagococcus fluvialis NCFB 2497T (96.0 %) and Vagococcus lutrae CCUG 39187T (95.9 %), whereas the similarity to another isolate (CF-210) was 99.9 %. Strains CF-49T and CF-210 grew optimally at 37 °C and pH 7.0 and in the presence of 0.5 % (w/v) NaCl. Acid was produced from N-acetylglucosamine, cellobiose, d-fructose, d-glucose, d-mannose, d-mannitol, maltose, d-ribose and salicin. The cell-wall peptidoglycan type was A4α (l-Lys-d-Asp). The major cellular fatty acids (>10 %) were C16 : 0 (35.6 %), C14 : 0 (17.3 %), C18 : 1 ω9c (16.2 %) and C16 : 1 ω9c (10.6 %). The predominant respiratory quinone was menaquinone MK-7 (68.8 %). The G+C content of the genomic DNA was 35.9 mol%. Digital DNA-DNA hybridization of strain CF-49T with V. fluvialis DSM 5731T, V. elongatus CCUG 51432Tand V. lutrae CCUG 39187T resulted in relatedness values of 21.4, 23.3 and 24.6 %, respectively. Based on results from polyphasic analyses, our two isolates are proposed to represent a novel species in the genus Vagococcus, with the name Vagococcus xieshaowenii. The type strain is CF-49T (=CGMCC 1.6436T=GDMCC 1.1588T=JCM 33477T).

Low Occurrence of Acinetobacter baumannii in Gulls and Songbirds.

Acinetobacter baumannii is a worldwide occurring nosocomial pathogen, the natural habitats of which remain to be defined. Recently, white stork nestlings have been described as a recurring source of A. baumannii. Here, we challenged the hypothesis of a general preference of A. baumannii for avian hosts. Taking advantage of campaigns to ring free-living birds, we collected cloacal swab samples from 741 black-headed gulls (Chroicocephalus ridibundus) in Poland, tracheal and cloacal swabs from 285 songbirds in Poland as well as tracheal swabs from 25 songbirds in Slovenia and screened those for the growth of A. baumannii on CHROMagarTM Acinetobacter. Of the 1,051 samples collected only two yielded A. baumannii isolates. Each carried one variant of the bla OXA-51-like gene, i.e. OXA-71 and OXA-208, which have been described previously in clinical isolates of A. baumannii. In conclusion, our data do not support a general preference of A. baumannii for avian hosts.Acinetobacter baumannii is a worldwide occurring nosocomial pathogen, the natural habitats of which remain to be defined. Recently, white stork nestlings have been described as a recurring source of A. baumannii. Here, we challenged the hypothesis of a general preference of A. baumannii for avian hosts. Taking advantage of campaigns to ring free-living birds, we collected cloacal swab samples from 741 black-headed gulls (Chroicocephalus ridibundus) in Poland, tracheal and cloacal swabs from 285 songbirds in Poland as well as tracheal swabs from 25 songbirds in Slovenia and screened those for the growth of A. baumannii on CHROMagarTM Acinetobacter. Of the 1,051 samples collected only two yielded A. baumannii isolates. Each carried one variant of the bla OXA-51-like gene, i.e. OXA-71 and OXA-208, which have been described previously in clinical isolates of A. baumannii. In conclusion, our data do not support a general preference of A. baumannii for avian hosts.

Research Note: Evaluation of several inoculation procedures for colonization of day-old broiler chicks with Salmonella Heidelberg.

Before starting a study with many birds, it helps to know the method of chick inoculation. The objective was to compare 3 methods of Salmonella challenge (oral gavage [OR], intracloacal inoculation [IC], and seeder bird [SB]). Day-old broiler chicks (n = 100) were inoculated with 106 colony forming units (CFU) per chick of a marker strain of Salmonella Heidelberg (SH) with each route of inoculation. Chicks (n = 25) inoculated by each route were placed in floor pens on fresh pine shavings litter. For the seeder batch, 5 colonized chicks, each orally gavaged with 106 CFUs, were placed with 20 pen mates. Two weeks after inoculation, 10 birds from each pen and the 5 inoculated seeder birds were euthanized, the ceca were aseptically removed and macerated with a rubber mallet and weighed, and 3 times (w/v) buffered peptone was added and stomached for 60 s. Serial dilutions were made and plated onto Brilliant Green Sulfa plates containing 200 ppm nalidixic acid. Plates were incubated along with the stomached ceca for 24 h at 37°C. If no colonies appeared on the plates, an additional plate was streaked from the preenriched bag and incubated for 24 h at 37°C. In addition to all seeder birds being positive, the number of SH-positive birds out of 20 sampled in each group was 13, 17, and 7 for OR, IC, and SB, respectively. The level of SH per g of ceca and cecal contents was log (SE) 3.0 (0.7), 2.0 (0.4), and 2.6 (0.4) for OR, IC, and SB, respectively. After enrichment, the number of colonized birds out of 20 was 18, 20, and 10 for OR, IC, and SB, respectively. In conclusion, this study suggests that IC is the method to use to ensure most of the challenged birds are colonized. However, if you prefer to have a smaller percentage of the birds colonized with higher levels, then OR might be better.

Cloacal bacterial communities of tree swallows (Tachycineta bicolor): Similarity within a population, but not between pair-bonded social partners.

Host-associated microbial communities can influence the overall health of their animal hosts, and many factors, including behavior and physiology, can impact the formation of these complex communities. Bacteria within these communities can be transmitted socially between individuals via indirect (e.g., shared environments) or direct (e.g., physical contact) pathways. Limited research has been done to investigate how social interactions that occur in the context of mating shape host-associated microbial communities. To gain a better understanding of these interactions and, more specifically, to assess how mating behavior shapes an animal's microbiome, we studied the cloacal bacterial communities of a socially monogamous yet genetically polygynous songbird, the North American tree swallow (Tachycineta bicolor). We address two questions: (1) do the cloacal bacterial communities differ between female and male tree swallows within a population? and (2) do pair-bonded social partners exhibit more similar cloacal bacterial communities than expected by chance? To answer these questions, we sampled the cloacal microbiome of adults during the breeding season and then used culture-independent, 16S rRNA gene amplicon sequencing to assess bacterial communities. Overall, we found that the cloacal bacterial communities of females and males were similar, and that the communities of pair-bonded social partners were not more similar than expected by chance. Our results suggest that social monogamy does not correlate with an increased similarity in cloacal bacterial community diversity or structure. As social partners were not assessed at the same time, it is possible that breeding stage differences masked social effects on bacterial community diversity and structure. Further, given that tree swallows exhibit high variation in rates of extra-pair activity, considering extra-pair activity when assessing cloacal microbial communities may be important for understanding how these bacterial communities are shaped. Further insight into how bacterial communities are shaped will ultimately shed light on potential tradeoffs associated with alternative behavioral strategies and socially-transmitted microbes.

Beta-lactam resistance in Campylobacter coli and Campylobacter jejuni chicken isolates and the association between blaOXA-61 gene expression and the action of ß-lactamase inhibitors.

The objectives of this work were to evaluate ß-lactamase-mediated ß-lactam resistance in Campylobacter coli and Campylobacter jejuni isolates obtained from broiler chickens, expression of the blaOXA-61 gene in relation to ß-lactamase production, and the possible association between blaOXA-61 gene expression and the action of inhibitors when combined with ß-lactams. All strains were tested by disk diffusion and nitrocefin methods to assess antibiotic susceptibility and ß-lactamase production, respectively. PCR and qPCR amplification were performed to evaluate qualitative and quantitative blaOXA-61 expression. Campylobacter spp. showed a high level of resistance to the most of antimicrobials tested. C. coli strains were ampicillin resistant and blaOXA-61 positive, and 59 out of 60 isolates were positive in the nitrocefin test. Twenty C. jejuni isolates were positive for blaOXA-61 and the nitrocefin test, although two isolates were ampicillin sensitive. Amoxicillin/clavulanic acid and ticarcillin/clavulanic acid do not seem to be active against C. coli, as 73.3 %, and 88.3 % of isolates were resistant to amoxicillin/clavulanic acid and ticarcillin/clavulanic acid, respectively. C. jejuni was not susceptible to amoxicillin/clavulanic acid, with 90 % of the strains showing resistance, whereas ticarcillin associated with clavulanic acid was significantly more efficient than ticarcillin alone (P < 0.01), with 90 % of the strains found to be susceptible. An association between blaOXA-61 expression and amoxicillin/clavulanic acid and ticarcillin/clavulanic acid resistance (P = 0.0001) was seen in C. coli, as well as in C. jejuni for ampicillin/sulbactam (P = 0.0001). Our results suggest that the clavulanic acid only shows an inhibitory effect on C. jejuni when combined with ticarcillin and that the inhibitors action is lower if the blaOXA-61 gene is highly expressed.

Safety of the live Escherichia coli vaccine Poulvac® E. coli in layer parent stock in a field trial.

The safety of the live Escherichia coli vaccine Poulvac® E. coli was tested with a flock (10,000) of layer parents aged 30 weeks. Three and 7 days after vaccination, 60 whole unbroken eggs, the egg white and yolk of 60 eggs and 60 cloacal swabs were enriched in MacConkey broth. At both sampling times, 6 out of 60 whole eggs were found positive for coliform bacteria. None of the enriched samples of yolk + egg white were positive for coliform bacteria. Three and seven days after vaccination 44 and 37, respectively out of 60 swabs were positive for coliform bacteria in MacConkey broth. All coliform isolates collected from whole eggs and cloacal swabs were tested in parallel for growth on minimal agar and blood agar to identify the vaccine strain. Some isolates showed reduced growth on minimal agar compared to blood agar and they were tested further with a PCR for the aroA gene mutation and all were found with the wild type version of the gene. Only two isolates did not grow on minimal agar but grew on blood agar and they were tested both with PCR and PFGE. They also showed the wild type version of the aroA gene and their PFGE profile was different from the vaccine strain of Poulvac® E. coli. In conclusion, the Poulvac® E. coli vaccine strain of E. coli was not identified at the detection limit of one CFU on one egg or in the content of one egg or from a cloacal swab of one hen with at least 95 % probability on flock level. The use of the vaccine is safe for hens in lay with lack of survival of the vaccine strain and lack of negative effects on the hens including egg production.

Survey on Chlamydiaceae in cloacal swabs from Swiss turkeys demonstrates absence of Chlamydia psittaci and low occurrence of Chlamydia gallinacean.

In Switzerland, domestic turkey meat is a niche product. Turkeys are fattened on mixed family-based farms scattered across the country, with most providing access to an uncovered outdoor pasture for the birds. Swiss fattening turkeys may therefore get infected with Chlamydiaceae via wild birds or their faeces, potentially shedding these bacteria at a later stage. The aim of the present study was to acquire baseline data about the shedding of Chlamydiaceae in clinically unremarkable Swiss fattening turkeys at slaughter, potentially exposing slaughterhouse workers to infection. In this large-scale study, 1008 cloacal swabs of Swiss turkeys out of 53 flocks from 28 different grow-out farms with uncovered outdoor pasture were collected over the course of 14 months and examined for the occurrence of Chlamydiaceae by a family-specific 23S-rRNA real-time PCR. Positive samples were further analyzed by Chlamydia psittaci (C. psittaci)-specific real-time PCR and the Arraymate DNA Microarray for species identification. All samples were negative for C. psittaci, but seven swabs out of one flock were tested positive for Chlamydia gallinacea (0.7%). Although turkeys with access to pasture may have contact with Chlamydiaceae-harbouring wild birds or their faeces, the infection rate in Swiss turkeys was shown to be low.