Bonelli’s eagle (Aquila fasciata) is an endangered raptor species in Europe, and trichomonosis is one of the menaces affecting chicks at nest. In this paper, we attempt to describe the oral microbiome of Bonelli’s eagle nestlings and evaluate the influence of several factors, such as captivity breeding, Trichomonas gallinae infection, and the presence of lesions at the oropharynx. The core oral microbiome of Bonelli’s eagle is composed of Firmicutes, Bacteroidota, Fusobacteria and Proteobacteria as the most abundant phyla, and Megamonas and Bacteroides as the most abundant genera. None of the factors analysed showed a significant influence on alfa diversity, but beta diversity was affected for some of them. Captivity breeding exerted a high influence on the composition of the oral microbiome, with significant differences in the four most abundant phyla, with a relative increase of Proteobacteria and a decrease of the other three phyla in comparison with chicks bred at nest. Some genera were more abundant in captivity bred chicks, such as Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium and Staphylococcus, while Bacteroides, Oceanivirga, Peptostreptococcus, Gemella, Veillonella, Mycoplasma, Suttonella, Alloscardovia, Varibaculum and Campylobacter were more abundant in nest raised chicks. T. gallinae infection slightly influenced the composition of the microbiome, but chicks displaying trichomonosis lesions had a higher relative abundance of Bacteroides and Gemella, being the last one an opportunistic pathogen of abscess complications in humans. Raptor’s microbiomes are scarcely studied. This is the first study on the factors that influence the oral microbiome of Bonelli’s eagle.

The aim of this study was to investigate the possible genotypic differences between commensal Pasteurella multocida isolates from apparently healthy animals (AHA) at the time of entry to feedlots and those from BRD-affected animals (BRD-AA). A total of 20 batches of beef calves in seven feedlots were followed-up during the fattening period. P. multocida was isolated from 28.1% of AHA and 22.9% of BRD-AA. All isolates belonged to the A: L3 genotype. Most isolates from clinical cases (81.0%) grouped into a PFGE cluster were significantly associated with BRD cases (OR, 24.9; 95% CI, 6.4-96.2). The whole genomes of 14 isolates representative of the pulsotypes most frequently detected in BRD-AA and AHA were sequenced and compared with 53 bovine genomes belonging to the identified ST13, ST79, and ST80 genotypes for a global comparison. No differences were found in the virulence-associated gene content between sequence types (STs) globally or between BRD-AA and AHA isolates in this study. Significantly, ST79 isolates harbored ARGs, conferring resistance to different antimicrobials, including macrolides and tetracyclines, which are commonly used for the treatment of BRD. Two Spanish ST79 isolates carried an ICE highly similar to ICE Tn7407, which was recently detected in Germany, suggesting that ST79 P. multocida isolates in Europe and North America may be associated with different ICEs.

A hierarchical cluster analysis was used to classify outbreaks of bovine respiratory disease (BRD; n = 156) in natural groups according to the detection of nine pathogens (parainfluenza 3 virus (PI-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BHV-1), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Pathogens were detected by individual q-PCRs. Two clusters were identified. Cluster 1 was characterized by a relatively high frequency (40-72%) of four BRD-associated viruses, supporting their primary involvement in BRD. Cluster 2 was characterized by frequencies of PI-3, BRSV, or BVDV below 10% each. P. multocida and M. haemolytica were detected with high frequencies in both clusters (P > 0.05), while M. bovis and H. somni showed a significantly higher frequency in cluster 1and 2, respectively. Outbreaks in cluster 1 were associated with preweaning calves younger than 5 months (OR 2.2; 95% CI 1.1-4.5) and with cold months, whereas cluster 2 was associated with fattening calves older than 5 months after arrival to feedlots and without any seasonality. Thus, in addition to the classic epidemiological BRD pattern characterized by the primary involvement of viruses occurring preferably during winter and affecting young calves, there is a second pattern in which viruses would be less relevant, affecting mainly calves older than 5 months at any time of the year. This study allows a better understanding of the BRD epidemiology, which can be useful when implementing management and prophylaxis measures for a better control of this disease.

Mannheimia haemolytica is the main pathogen contributing to pneumonic pasteurellosis in sheep. The aim of this study was to investigate the antimicrobial resistance levels in M. haemolytica isolates from the lungs of slaughtered sheep and to examine the genetic resistance mechanisms involved. A total of 256 M. haemolytica isolates, 169 from lungs with pneumonic lesions and 87 from lungs without lesions, were analyzed by the disk diffusion method for 12 antimicrobials, and the whole genome of 14 isolates was sequenced to identify antimicrobial resistance determinants. Levels of phenotypic resistance ranged from <2% for 10 antimicrobials (amoxicillin, amoxicillin-clavulanic, ceftiofur, cefquinome, lincomycin/spectinomycin, gentamicin, erythromycin, florfenicol, enrofloxacin, and doxycycline) to 4.3% for tetracycline and 89.1% for tylosin. Six isolates carried tetH genes and four isolates carried, in addition, the strA and sul2 genes in putative plasmid sequences. No mutations associated with macrolide resistance were identified in 23 rDNA sequences, suggesting that the M. haemolytica phenotypic results for tylosin should be interpreted with care in the absence of well-established epidemiological and clinical breakpoints. The identification of strains phenotypically resistant to tetracycline and of several resistance genes, some of which were present in plasmids, highlights the need for continuous monitoring of susceptibility patterns in Pasteurellaceae isolates from livestock.

Studies that characterize bovine respiratory disease (BRD)-associated Pasteurella multocida isolates are scarce compared with research on isolates from other hosts and clinical backgrounds. In the present study, 170 P. multocida isolates from 125 BRD outbreaks were characterized by capsular and LPS typing as well as by virulotyping. Three capsular types (A, B, F) and three LPS genotypes (L2, L3, L6) were identified. Capsular and LPS typing revealed a very low genetic diversity (GD = 0.02) among P. multocida, with most isolates belonging to genotype A:L3 (97.6%). Virulotyping identified seven virulence-associated gene profiles, with two profiles including 95.9% of the isolates. A subset of isolates was further characterized by MLST and PFGE. The sequence types ST79 and ST13 were the most frequently identified and were grouped into the same clonal complex (CC13), a result that supports the clonal population structure of BRD-associated P. multocida isolates. PFGE typing also revealed a low genetic diversity (GD = 0.18), detecting a single pattern in 62.5% of the outbreaks in which multiple isolates were analyzed. Overall, 85.2% of the isolates belonged to pulsotypes with at least 80% genetic similarity, consistent with a clonal population structure observed by MLST analysis and corroborating the genetic relatedness of most P. multocida isolates associated with BRD in cattle.

Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular-LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.

This study investigated via polymerase chain reaction (PCR) three main serotypes (A1, A2, and A6) and nine virulence-associated genes in 71 ovine and caprine Mannheimia haemolytica isolates obtained from lungs (n = 349) with pneumonic lesions from a slaughterhouse in Iran. The lung specimens were collected from sheep (n = 197) and goats (n = 152) between December 2018 and January 2020. A total of 71 M. haemolytica isolates were identified in sheep (37/197; 18.8%) and goat (34/152; 22.4%) pneumonic lungs. Serotypes A2 (30/71; 42.3%) and A6 (29/71; 40.9%) were the most frequently detected, whereas the A1 serotype was detected with a frequency of less than 10% (7/71; 9.9%) and five isolates remained unknown. The virulence genes lkt, pomA, and nanH were present in all the isolates. The detection rates for the remaining virulence-associated genes were: gcp (95.8%), lpsA (93%), fhaC (90%), irp (70.4%), hf (57.7%), and sodC (21%). The sodC gene was exclusively detected among A2 isolates (50%), while the irp gene was more prevalent among A2 isolates and the hf gene among A1 and A6 isolates. These data may be useful for the typing of isolates in epidemiological studies. This study provides information about the main serotypes and the prevalence of virulence-associated genes among M. haemolytica ovine and caprine isolates in Iran.

This study compares the utility of a commercially available multiplex q-PCR assay for serotyping A1, A2, and A6 M. haemolytica serotypes with indirect hemagglutination, for determining the relative distribution of M. haemolytica capsular types associated with respiratory disorders in cattle, sheep, and goats. For the 129 isolates analyzed, both q-PCR and IHA assays exhibited nearly complete agreement for capsular types A1 (k = 0.965) and A2 (k = 0.888) and substantial agreement for A6 (k = 0.801). Despite the overall good performance of the commercial q-PCR, its effectiveness differed between the host origin of the isolates. The serotype was identified by q-PCR in 83.3 % of cattle, 77.8 % of goat, and 53.8 % of sheep isolates. Combining the results of both methods, A1 was the most prevalent in cattle and sheep (55.6 % and 22.25 %, respectively) but was not detected in goats, A2 was the most prevalent in goats (61.1 %) and the second most prevalent in cattle (16.7 %) and sheep (20.5 %). The prevalence of A6 was 7.4 %, 5.1 %, and 16.7 % in cattle, sheep, and goats, respectively. Other capsular types determined exclusively by IHA were A16 in cattle, A9 in goats, and A7, A8, A9, and A13 in sheep. Capsular type diversity was greater in sheep (H = 0.601) than in cattle (H = 0.408) and goat (H = 0.330) isolates. The commercial multiplex q-PCR is a valuable tool, alternative to IHA, for identifying isolates of capsular types A1, A2, and A6, the most frequent serotypes of M. haemolytica associated with respiratory disease in ruminants. However, when testing sheep isolates it should be complemented with immunological assays due to the wider range of serotypes implicated.

This study investigated the association of Pasteurella multocida isolation and the molecular characteristics of the isolates with the presence of pneumonic lesions in lambs at slaughter to assess its importance as a causative agent of pneumonic pasteurellosis compared with Mannheimia haemolytica. P. multocida was isolated from the 13.9% and 2.7%, and M. haemolytica from the 36.4% and 26.8%, of lungs with and without lesions, respectively (P < 0.05). Both microorganisms were frequently coisolated (23.2% and 12.5% from lungs with and without lesions, respectively). Isolation of P. multocida alone exhibited greater strength of association with pneumonic lesions (OR 11.4; 95% CI 3.2-40.6) than that exhibited by M. haemolytica alone (OR 3.0; 95% CI 1.6-5.4). Cluster analysis grouped the lungs into four clusters characterized by the isolation of M. haemolytica or P. multocida alone (clusters 1 and 4), coisolation of both microorganisms (cluster 3), and isolation of neither (cluster 2). Cluster 4 lungs exhibited higher frequencies of pneumonic lesions (87.5%) and severe (20.8%) and moderate (25.0%) lesions. Lungs coinfected with both pathogens (cluster 3) did not exhibit a higher frequency of severe and moderate consolidation lesions (6.1% and 14.3%, respectively), suggesting that P. multocida and M. haemolytica do not act synergically to cause more severe pneumonic infections. The greater strength of association of P. multocida isolation with pneumonic lesions together with the higher severity of the lesions caused could indicate a greater role played by this pathogen in the aetiopathogenesis of pneumonic pasteurellosis in sheep than is commonly assumed.

Pasteurella multocida is responsible for economically important diseases in sheep and pigs. Antimicrobial susceptibility studies are essential for initiating rational and effective empirical therapy of P. multocida infections. In this study we investigated the antimicrobial susceptibility to 18 antimicrobial agents of 156 clinical isolates of P. multocida from sheep (n = 87) and pigs (n = 69) using the microdilution method. Both sheep and pig isolates exhibited low levels of resistance (≤ 15%) to ceftiofur, gentamicin, neomycin, spectinomycin, chlortetracycline, tulathromycin, florfenicol, danofloxacin, and enrofloxacin and trimethoprim/sulphamethoxazole, high resistance rates (> 15% up to 50%) to oxytetracycline, tilmicosin, and tiamulin, and very high resistance rates (> 50%) to tylosin tartrate, clindamycin, and sulphadimethoxine. However, sheep isolates exhibited significantly lower percentages of resistance and lower MIC90 values (P < 0.05) than pig isolates for most of the antimicrobials tested. In addition, sheep isolates exhibited also significantly lower phenotypic antimicrobial resistance diversity (8 resistotypes vs. 30 resistotypes). LAC-LIN-SUL-MAC was the resistotype most frequently detected in sheep (39.1%) and LIN-SUL-MAC in pig isolates (26.1%). The differences in susceptibility patterns could be influenced by the lower use of antimicrobials in the small ruminant industry compared with the pig farming industry.

The P. multocida toxin (PMT), a dermonecrotic protein encoded by the toxA gene, is the major virulence factor of capsular type D P. multocida strains causing progressive atrophic rhinitis (PAR) in pigs. A high frequency of P. multocida isolates harboring the toxA gene has been found among ovine pneumonic isolates, although the ability of these isolates to express PMT has never been examined. In this study we have investigated the ability of ovine toxA + P. multocida isolates (n = 57) to express a functional toxin by detection of PMT toxin antigen using an ELISA test and its cytopathic effect in a Vero cell assay. PMT antigen was expressed in the great majority (54/57; 94.7%) of toxA + isolates. Moreover, the 100% toxA + ovine isolates analyzed produced a cytopathic effect in Vero cells within 24-48 h post-inoculation, identical to that described for porcine toxigenic P. multocida isolates. These results show for the first time that, in addition to isolates associated with PAR, isolates of P. multocida associated with pneumonia in sheep are also toxigenic. In addition, we found a total agreement (Kappa = 1; C.I. 0.75-1.25) between the detection of the toxA gene and the toxigenic capability of P. multocida isolates, indicating the PCR detection of toxA would be a suitable predictive marker of the toxigenic fitness of P. multocida.

We report a case of urinary tract infection caused by an unusual genotype (sequence type 211) of Pasteurella multocida associated with human infection. Molecular genetic analysis of P. multocida isolates obtained from the human patient and his pet strongly suggests a zoonotic transmission of this bacterium.

ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

This report describes the prevalence, capsular type and virulence factors of Pasteurella multocida isolated from the respiratory tracts of healthy and diseased lambs. For this study, five hundred and ninety-eight fattening lambs from different feedlots of the community of Extremadura were analysed. Isolation and identification of P. multocida were performed using conventional bacteriological and biochemical techniques, while confirmation of P. multocida identification, capsular type and virulence factors was done using the polymerase chain reaction technique (PCR). Of 598 studied lambs (410 clinically healthy and 188 diseased sheep), P. multocida was isolated from 37 animals (6.2%), with prevalence rates of 0.49% (2/410) in healthy lambs and 18.62% (35/188) in diseased lambs confirming a close relationship between the presence of bacteria and disease. Capsular typing of P. multocida isolates demonstrated two capsular types: A (15) and D (22), with general prevalence rates of 40.5% and 59.5%, respectively, and with the exclusive presence of type D in healthy animals (100%) and types D and A among diseased animals (42.9% and 57.1%, respectively). Among four virulence genes investigated (pfhA, hgbB, tbpA and toxA), we found a remarkable high prevalence of tbpA (100%) (37/37) genes in P. multocida; toxA was only detected in some diseased lambs (11/37), while the rest of the studied genes were not detected. The high prevalence of toxA among isolates from diseased animals may imply an important role of this gene in the virulence of P. multocida isolates in sheep, especially in diseased lambs.

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep.

Pasteurella multocida is a pathogen causing disease in a wide range of hosts including sheep and pigs. Isolates from ovine pneumonia were characterized by MLST (Multi-host and RIRDC databases) and virulence-associated gene (VAG) typing and compared with porcine isolates. Ovine and porcine isolates did not share any STs as determined by both schemes and exhibited different VAG profiles. With the Multi-host database, sixteen STs were identified among 43 sheep isolates with two STs (ST50 and ST19) comprising 53.5% of the isolates, and seven MLST genotypes (ST3, ST11 and ST62 included 75% of the isolates) among the 48 pig isolates. The most frequent VAG profile among sheep isolates was tbpA+/toxA+ (69.8% of isolates) and pfhA+ (62.5%) and hgbB+ (33.3%) among pig isolates. Representative ovine and porcine isolates of those STs identified by the Multi-host scheme were further typed using the RIRDC scheme. Seven STs were identified among the ovine isolates (ST95RIRDC, ST131RIRDC, ST203RIRDC, ST320RIRDC, ST324RIRDC, ST321RIRDC, and ST323RIRDC), with the latter four sequence types being new STs identified in this study, and six STs (ST9RIRDC, ST13RIRDC, ST27RIRDC, ST50RIRDC, and ST74RIRDC and a new sequence type ST322RIRDC) among the porcine isolates. STs identified among ovine isolates have been detected exclusively in small ruminants, suggesting an adaptation to these hosts, while the genotypes identified among pig isolates have been previously identified in multiple hosts and therefore they are not restricted to pigs. The differences in genotypes and VAG profiles between ovine and pig isolates suggest they could represent different subpopulations of P. multocida.

Pneumonia caused by Mannheimia haemolytica is an important disease in ruminants. Because of its economic significance, several methods have been developed to study the pathogenicity and epidemiology of M. haemolytica. In this study, bacterial isolates of M. haemolytica and Bibersteinia trehalosi identified from the lungs of sheep were serotyped by means of indirect haemagglutination. Of the 598 lungs studied, 34 isolates were identified and serotyped. In decreasing order, M. haemolytica serotypes were: not typable (50 %), A1 (17.65 %), A7 (11.76 %), A6 (5.88 %), and A12, A2, A5 and A9 (each representing 2.94 %). The only B. trehalosi serotype was T4 (2.94 %). Serotypes A1, A6 and A7 of M. haemolytica were the most commonly isolated from pneumonic sheep producing greater changes in the lungs and having important implications for sheep production.