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Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136.

Species of the bacterial genus Photorhabus live in a symbiotic relationship with Heterorhabditis entomopathogenic nematodes. Besides their use as biological control agents against agricultural pests, some Photorhabdus species are also a source of natural products and are of medical interest due to their ability to cause tissue infections and subcutaneous lesions in humans. Given the diversity of Photorhabdus species, rapid and reliable methods to resolve this genus to the species level are needed. In this study, we evaluated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Photorhabdus species. To this end, we established a collection of 54 isolates consisting of type strains and multiple field strains that belong to each of the validly described species and subspecies of this genus. Reference spectra for the strains were generated and used to complement a currently available database. The extended reference database was then used for identification based on the direct transfer sample preparation method and the protein fingerprint of single colonies. High-level discrimination of distantly related species was observed. However, lower discrimination was observed with some of the most closely related species and subspecies. Our results therefore suggest that MALDI-TOF MS can be used to correctly identify Photorhabdus strains at the genus and species level, but has limited resolution power for closely related species and subspecies. Our study demonstrates the suitability and limitations of MALDI-TOF-based identification methods for assessment of the taxonomic position and identification of Photorhabdus isolates.

‘ Treponema phagedenis ’ was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a ‘ T. phagedenis ’ phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain ‘ T. phagedenis ’ ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with ‘ T. phagedenis ’-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human ‘ T. phagedenis ’ were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium Dichelobacter nodosus. The prevalence of D. nodosus in 207 sheep flocks across Germany was 42.9%. Based on the sequence variation in the type IV fimbrial gene fimA, D. nodosus can be subdivided into ten serogroups (A–I and M). There are commercially available vaccines covering nine serogroups, but the efficacy is low compared to bivalent vaccines. The aim of this study was to investigate the diversity of serogroups in Germany at the flock and animal levels. In total, we detected at least one serogroup in 819 samples out of 969 D. nodosus-positive samples from 83 flocks using serogroup-specific singleplex PCR for the serogroups A–I. Serogroup A was most prevalent at the animal level, followed by serogroups B, H and C. At the flock level, serogroups A and B had the highest prevalence, each with 64%, but only 40% of flocks had both. The average number of serogroups per animal was 1.42 (range one to five) and, per flock, 3.10 (range one to six). The serogrouping showed within-flock specific clusters but were widely distributed, with 50 different combinations across the flocks. The factors associated with the number of serogroups per animal and single serogroups were the load of D. nodosus, footrot score, sheep breed and flock. Our results indicate that efficient vaccination programs would benefit from tailor-made flock-specific vaccines and regular monitoring of circulating serotypes in the flock to be able to adjust vaccine formulations for nationwide progressive control of footrot in Germany.

Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium

Ovine footrot is a highly contagious foot disease caused by the gram-negative bacterium Dichelobacter nodosus (D. nodosus). In a recent report, we showed a prevalence of 42.9% D. nodosus positive swabs across Germany. In this follow-up study, we used real-time PCR results for D. nodosus and footrot scores of 9297 sheep from 208 flocks and collated these data with survey data on herd and animal characteristics and herd management. The aims of the present study were to investigate herd and animal factors associated with D. nodosus infection and footrot scores in individual sheep. Multivariable analyses with generalized mixed models showed that month of recording, breed, herdbook membership, use of antibiotics, and footbaths in the past 3-10 years, signs of footrot in the past 12 months and flock environment of the sheep, modelled as a random farm effect within region, were significant risk factors. Among the 21 different breeds, Romney had the lowest risk of D. nodosus infection, while Swifter had the highest risk and German Merino and German White Heath were the next breeds at highest risk of D. nodosus infection. The variance between farms in the prevalence of D. nodosus was large and accounted for 84% of the total variance in the mixed model analysis. We conclude that specific and as yet unknown effects influencing D. nodosus infections in flocks, as well as breed and weather, are the most important effects on D. nodosus infection in sheep, pointing towards the need to establish adequate infection control at farm level.

Ovine footrot caused by Dichelobacter nodosus is a highly contagious and painful disease representing an economic as well as an animal welfare problem. In order to get more information on the infection dynamics, 26 lambs and 4 ewes enrolled in an in-contact infection trial were monitored over two weeks for the presence of D. nodosus-specific DNA. Two D. nodosus-positive ewes were housed together with 13 confirmed negative lambs. The control group consisted of another 13 lamb siblings and two confirmed D. nodosus-negative ewes. Every foot of all sheep was sampled seven times over the two weeks experiment period and subsequently analyzed for the presence of D. nodosus by quantitative real-time PCR. The control group was negative at the beginning and the end of the experiment and showed no clinical symptoms of footrot. The two positive ewes showed a high, but hundred fold differing level of virulent D. nodosus that remained constant over time with one of the ewes being also weakly positive for benign D. nodosus. All lambs of the infection group were positive for virulent D. nodosus at 14 days post infection (dpi). The first positive animals were observed on 3 dpi. The D. nodosus load remained at a low level and only increased in a few lambs at the end of the trial. Five of the contact lambs showed suspicious clincal signs (score 1-2) at 14 dpi corroborating the PCR results and indicating that the disease starts as early as two weeks after contact with positive sheep.Moderhinke verursacht durch Dichelobacter nodosus ist eine hoch ansteckende und schmerzhafte Krankheit, die sowohl ein wirtschaftliches als auch ein Tierschutzproblem darstellt. In einer Kontaktinfektionsstudie wurden 26 Lämmer und 4 Mutterschafe über einen Zeitraum von zwei Wochen auf das Vorhandensein von D. nodosus-spezifischer Desoxyribonukleinsäure (DNS) untersucht. Zwei D. nodosus-positive Mutterschafe wurden zusammen mit 13 bestätigten negativen Lämmern gehalten. Die Kon­trollgruppe bestand aus weiteren 13 Lammgeschwistern und zwei bestätigten negativen Mutterschafen. Während des zweiwöchigen Versuchszeitraums wurden von jedem Schaffuss sieben Abstrichproben entnommen und mittels einer quantitativen Echtzeit-Polymerase Kettenreaktion (PCR) auf das Vorhandensein von D. nodosus analysiert. Die Kontrollgruppe war zu Beginn und am Ende des Experiments negativ und zeigte keine klinischen Symptome von Moderhinke. Im Versuchszeitraum wurde bei beiden positiven Mutterschafen ein kons­tant hoher, aber hundertfach unterschiedlicher Gehalt an virulentem D. nodosus nachgewiesen. Ein Mutterschaf war auch schwach positiv für benigne D. nodosus. Alle Lämmer der Infektionsgruppe waren 14 Tage nach dem Kontakt (TnK) positiv für virulente D. nodosus. Die ersten positiven Tiere wurden 3 TnK beobachtet. Der D. nodosus Nachweis blieb auf einem niedrigen Niveau und stieg am Ende des Versuchs nur bei einigen Lämmern an. Bei 14 TnK zeigten fünf der Kontaktlämmer verdächtige klinische Symptome (Score 1-2) was die PCR-Ergebnisse bestätigte und darauf hindeutete, dass die Krankheit bereits zwei Wochen nach dem Kontakt mit positiven Schafen begann.Le piétin causé par Dichelobacter nodosus est une maladie hautement contagieuse et douloureuse qui représente à la fois un problème économique et de bien-être animal. Pour avoir plus informations sur la dynamique de l’infection, 26 agneaux et 4 brebis appartenant à un groupe d’essai d’infection par contact ont été contrôlés pendant deux semaines quant à la présence d’ADN spécifique de D. nodosus. Deux brebis positives pour D. nodosus ont été mises en contact avec 13 agneaux confirmés négatifs. Le groupe témoin était formé de 13 autres agneaux et deux brebis confirmées négatives. Sept échantillons sur écouvillon ont été prélevés sur chaque pied de chaque mouton au cours des deux semaines de la période expérimentale et analysés quant à la présence de D. nodosus par PCR quantitative en temps réel. Le groupe témoin était négatif au début et à la fin de l’expérience et n’a montré aucun symptôme clinique de piétin. Les deux brebis positives ont montré une forte présence de D. nodosus virulent, mais de cent niveaux différents, qui est restée constant dans le temps, l’une des brebis étant aussi faiblement positive pour D. nodosus bénin. Tous les agneaux du groupe infecté étaient positifs pour D. nodosus virulent 14 jours après l’infection (dpi). Les premiers animaux positifs ont été observés à 3 dpi. La charge de D. nodosus est restée faible et n’a augmenté que chez quelques agneaux à la fin de l’expérience. Cinq des agneaux en contact ont présenté des symptômes suspects (score 1-2) à 14 dpi, corroborant les résultats de la PCR et indiquant que l’infection commence dès deux semaines après le contact avec des moutons positifs.La zoppina negli ovini causata dal Dichelobacter nodosus è una malattia altamente contagiosa e dolorosa che rappresenta un problema sia economico che del benessere degli animali. Per capire maggiormente la dinamica della trasmissione, in uno studio a contatto dell’infezione, 26 agnelli e 4 pecore sono stati monitorati per due settimane per la presenza di ADN specifico a D. nodosus. Due pecore positive al D. nodosus sono state messe a contatto con 13 agnelli negativi al patogeno. Il gruppo di controllo era formato da altri 13 agnelli gemelli e da due pecore negative. Sette campioni sono stati prelevati dagli zoccoli di ogni animale durante le due settimane di sperimentazione e analizzati per la presenza di D. nodosus con un’analisi quantitativa di real-time PCR. Sia all’inizio che alla fine della sperimentazione, il gruppo di controllo è risultato negativo e non presentava nessun sintomo clinico alla zoppina. Durante il periodo dello studio, le due pecore positive hanno mostrato un livello alto ma costante nel tempo di D. nodosus virulento ma con un contenuto cento volte differente e una pecora è risultata pure debolmente positiva al D. nodosus benigno. Tutti gli agnelli del gruppo infettato sono risultati positivi al D. nodosus virulento dopo 14 giorni dal contatto (dpi). I primi animali positivi sono stati osservati a 3 dpi. La carica di D. nodosus è rimasta a livelli bassi ed è aumentata solo in pochi agnelli alla fine dello studio. Cinque degli agnelli del gruppo a contatto hanno mostrato dei sintomi sospetti (punteggio 1-2) a 14 dpi. Questo ha confermato i risultati dell’analisi PCR e ha indicato che l’infezione inizia al più presto due settimane dopo il contatto con delle pecore positive.

[ Haemophilus ] haemoglobinophilus and the unpublished Bisgaard taxon 35 are associated with respiratory and urogenital tract infections in dogs. A total of 21 strains including the type strain of [ Haemophilus ] haemoglobinophilus were included in the investigation. Strains of [ Haemophilus ] haemoglobinophilus and taxon 35 formed a monophyletic group demonstrating at least 97.8 and 96.5% similarities within the group based upon 16S rRNA and rpoB gene sequence comparisons, respectively. Glaesserella australis was the most closely related species to [ Haemophilus ] haemoglobinophilus and taxon 35 with 96.1 % 16S rRNA gene sequence similarity which is slightly higher than the 95 % separating most genera of the family Pasteurellaceae . However, the conserved protein sequence phylogeny documented a unique position of [ Haemophilus ] haemoglobinophilus with only 81 % identity to the most closely related species, genomospecies 1 of the genus Rodentibacter which is lower than the 85 % separating most genera of the family Pasteurellaceae . The conserved protein sequence identity to Haemophilus influenzae , the type species of the genus, was 77%, demonstrating that [ Haemophilus ] haemoglobinophilus is not properly classified as a member of the genus Haemophilus . On the basis of the phylogenetic comparisons, the taxa [ Haemophilus ] haemoglobinophilus and taxon 35 are proposed to be included with a novel genus Canicola with one species, Canicola haemoglobinophilus which is reclassified from [ Haemophilus ] haemoglobinophilus. Phenotypic characters obtained with isolates genetically approved to represent Canicola haemoglobinophilus were in accordance with those of the members of the family Pasteurellaceae, and the novel genus can be separated from most of the existing genera by a positive catalase reaction, lack of V-factor requirement for growth, lack of haemolysis of blood agar and negative Voges–Proskauer and urease tests. The novel genus cannot be separated by biochemical and physiological characteristics alone from the genera Aggregatibacter , Avibacterium , Frederiksenia and Spirabiliibacterium . However, MALDI-TOF mass spectroscopy and also RpoB amino acid signatures allowed a clear separation from these taxa, supporting the existence of a novel genus. The DNA G+C content is 37.0–37.8 mol% for the genus, based on the whole genomic sequences. The type strain of Canicola haemoglobinophilus is CCUG 3714T (=ATCC 19416T=NCTC 1659T) isolated in 1901 from the prepuce of a dog in Germany.

We report the complete 2,783,931-bp circular genome sequence of the human methicillin-resistant strain Staphylococcus aureus 17Gst354, isolated from a nasal swab. The strain possessed an additional 4,397-bp plasmid. Moreover, we induced and sequenced its temperate phage Staphylococcus phage vB_StaphS-IVBph354, which has a circular genome of 41,970 bp.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in the porcine respiratory disease complex, economically one of the most important diseases in pigs worldwide. The pathogen adheres to the ciliated epithelium of the trachea, bronchi, and bronchioles, causes damage to the mucosal clearance system, modulates the immune system and renders the animal more susceptible to other respiratory infections. The pathogenesis is very complex and not yet fully understood. Cell-mediated and likely also mucosal humoral responses are considered important for protection, although infected animals are not able to rapidly clear the pathogen from the respiratory tract. Vaccination is frequently practiced worldwide to control M. hyopneumoniae infections and the associated performance losses, animal welfare issues, and treatment costs. Commercial vaccines are mostly bacterins that are administered intramuscularly. However, the commercial vaccines provide only partial protection, they do not prevent infection and have a limited effect on transmission. Therefore, there is a need for novel vaccines that confer a better protection. The present paper gives a short overview of the pathogenesis and immune responses following M. hyopneumoniae infection, outlines the major limitations of the commercial vaccines and reviews the different experimental M. hyopneumoniae vaccines that have been developed and tested in mice and pigs. Most experimental subunit, DNA and vector vaccines are based on the P97 adhesin or other factors that are important for pathogen survival and pathogenesis. Other studies focused on bacterins combined with novel adjuvants. Very few efforts have been directed towards the development of attenuated vaccines, although such vaccines may have great potential. As cell-mediated and likely also humoral mucosal responses are important for protection, new vaccines should aim to target these arms of the immune response. The selection of proper antigens, administration route and type of adjuvant and carrier molecule is essential for success. Also practical aspects, such as cost of the vaccine, ease of production, transport and administration, and possible combination with vaccines against other porcine pathogens, are important. Possible avenues for further research to develop better vaccines and to achieve a more sustainable control of M. hyopneumoniae infections are discussed.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS–based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, BB/G018553/1, BB/S002103/1, and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. Funding for LL provided by the ‘National Natural Science Foundation of China’ (No.31520103917). MTGH and DH were supported by the Wellcome Trust (grant number 098051). We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain. Publisher PDF

Background Osteomyelitis is relatively frequent in young pigs and a few bacterial species have been postulated to be potential causative agents. Although Actinobacillus (A.) pleuropneumoniae has been sporadically described to cause osteomyelitis, typically, actinobacillosis is characterized by respiratory symptoms. Nevertheless, subclinical infections are a challenging problem in pig herds. To the authors’ knowledge, this is the first case description that reports clinical, diagnostic imaging, pathological and histopathological findings of vertebral osteomyelitis in a pig and first describes A. pleuropneumoniae as the causative agent identified by advanced molecular methods. Case presentation An eight-week-old female weaner was presented with a non-ambulatory tetraparesis. The neurological signs were consistent with a lesion in the C6-T2 spinal cord segments. Imaging studies revealed a collapse of the seventh cervical vertebral body (C7) with a well demarcated extradural space-occupying mass ventrally within the vertebral canal severely compressing the spinal cord. Post-mortem examination identified an abscess and osteomyelitis of C7 and associated meningitis and neuritis with subsequent pathological fracture of C7 and compression of the spinal cord. In the microbiological analysis, A. pleuropneumoniae was identified using PCR and DNA sequence analysis. Conclusions A. pleuropneumoniae can be responsible for chronic vertebral abscess formation with subsequent pathological fracture and spinal cord compression in pigs.

Human campylobacteriosis is the most prevalent zoonosis, with chicken meat contributing substantially to the number of cases. Measures to avoid or at least reduce exposure by meat contaminated with Campylobacter (C.) spp. are needed. With regard to the process hygiene criterion introduced in 2018 for Campylobacter spp. on broiler carcasses, we evaluated the performance of a recently developed quantitative real-time PCR (qPCR) for C. jejuni/coli on random caecal samples and chicken meat. With the qPCR on pooled caecal samples not only C. jejuni/coli positive (69.6%) versus negative broiler herds (30.4%) were identified, but herds highly colonized with C. jejuni/coli (39.4%) could also be identified. From the chicken meat samples, 8.0% were positive for C. jejuni/coli by qPCR and 0.7% by enumeration (&gt, 10 cfu/g) compared to 58.3% using cultural enrichment. Given the higher sensitivity, the qPCR method could replace the currently used enumeration method to assess the process hygiene criterion for Campylobacter spp. on broiler carcasses. Moreover, with the qPCR, a reliable identification of C. jejuni/coli colonized incoming broiler herds a few days before slaughter is feasible, which provides important information to optimize slaughter processes. Finally, identifying and monitoring herds with high C. jejuni/coli colonization rates could help to individually improve biosecurity measures at farm level, eventually reducing the C. jejuni/coli load on chicken meat.

Footrot is a contagious foot disease mainly affecting sheep. It is caused by the Gram-negative anaerobic bacterium Dichelobacter nodosus. Warm, wet environmental conditions favour development of footrot, and under perfect conditions, it takes just 2–3 weeks from infection to manifestation of clinical signs. Affected sheep show lameness of various degrees and often graze while resting on their carpi. Local clinical signs vary in severity and extent from interdigital inflammation (benign footrot) to underrunning of the complete horn shoe in advanced stages of virulent footrot. Laboratory diagnosis ideally involves collection of four-foot interdigital swab samples followed by competitive real time PCR, allowing for detection of the presence of D. nodosus and differentiation between benign and virulent strains. Laboratory-based diagnostics at the flock level based on risk-based sampling and pooling of interdigital swab samples are recommended. The list of treatment options of individual sheep includes careful removal of the loose undermined horn, local or systemic administration of antimicrobials, systemic administration of non-steroidal anti-inflammatories (NSAIDs) and disinfectant footbathing. Strategies for control at the flock level are manifold and depend on the environmental conditions and the procedures traditionally implemented by the respective country. Generally, measures consist of treatment/culling of infected sheep, vaccination and prevention of reinfection of disease-free flocks. Gaining deeper insight into the beneficial effects of NSAIDs, screening for eco-friendly footbath solutions, developing better vaccines, including the development of a robust, reproducible infection model and elucidation of protective immune responses, as well as the elaboration of effective awareness training programs for sheep farmers, are relevant research gaps.

Human campylobacteriosis is the most prevalent zoonosis, with chicken meat contributing substantially to the number of cases. Measures to avoid or at least reduce exposure by meat contaminated with Campylobacter (C.) spp. are needed. With regard to the process hygiene criterion introduced in 2018 for Campylobacter spp. on broiler carcasses, we evaluated the performance of a recently developed quantitative real-time PCR (qPCR) for C. jejuni/coli on random caecal samples and chicken meat. With the qPCR on pooled caecal samples not only C. jejuni/coli positive (69.6%) versus negative broiler herds (30.4%) were identified, but herds highly colonized with C. jejuni/coli (39.4%) could also be identified. From the chicken meat samples, 8.0% were positive for C. jejuni/coli by qPCR and 0.7% by enumeration (>10 cfu/g) compared to 58.3% using cultural enrichment. Given the higher sensitivity, the qPCR method could replace the currently used enumeration method to assess the process hygiene criterion for Campylobacter spp. on broiler carcasses. Moreover, with the qPCR, a reliable identification of C. jejuni/coli colonized incoming broiler herds a few days before slaughter is feasible, which provides important information to optimize slaughter processes. Finally, identifying and monitoring herds with high C. jejuni/coli colonization rates could help to individually improve biosecurity measures at farm level, eventually reducing the C. jejuni/coli load on chicken meat.

Species of the bacterial genus

Mycoplasma hyopneumoniae(M. hyopneumoniae) is the primary pathogen of enzooticpneumonia, a chronic respiratory disease in pigs. Infections occur worldwide andcause major economic losses to the pig industry. The present paper reviews the cur-rent knowledge onM. hyopneumoniaeinfections, with emphasis on identificationand analysis of knowledge gaps for optimizing control of the disease. Close contactbetween infected and susceptible pigs is the main route ofM. hyopneumoniaetrans-mission. Management and housing conditions predisposing for infection or diseaseare known, but further research is needed to better understandM. hyopneumoniaetransmission patterns in modern pig production systems, and to assess the impor-tance of the breeding population for downstream disease control. The organism isprimarily found on the mucosal surface of the trachea, bronchi and bronchioles. Dif-ferent adhesins and lipoproteins are involved in the adherence process. However, aclear picture of the virulence and pathogenicity ofM. hyopneumoniaeis still missing.The role of glycerol metabolism, myoinositol metabolism and theMycoplasmaIgbinding protein—MycoplasmaIg protease system should be further investigated fortheir contribution to virulence. The destruction of the mucociliary apparatus,together with modulating the immune response, enhances the susceptibility ofinfected pigs to secondary pathogens. Clinical signs and severity of lesions dependon different factors, such as management, environmental conditions and likely alsoM. hyopneumoniaestrain. The potential impact of strain variability on disease sever-ity is not well defined. Diagnostics could be improved by developing tests that maydetect virulent strains, by improving sampling in live animals and by designing ELI-SAs allowing discrimination between infected and vaccinated pigs. The currentlyavailable vaccines are often cost-efficient, but the ongoing research on developingnew vaccines that confer protective immunity and reduce transmission should becontinued, as well as optimization of protocols to eliminateM. hyopneumoniaefrompig herds. info:eu-repo/semantics/acceptedVersion

Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

Background Ovine footrot caused by Dichelobacter nodosus (D nodosus) is an infectious disease affecting sheep worldwide. Switzerland plans a nationwide footrot eradication program, based on PCR-testing of interdigital swab samples. The aim of this study was to test for the presence of D nodosus in clinically footrot-free sheep flocks which had been subjected to different treatment strategies, to assess whether they were feasible for the eradication process, especially focussing on antimicrobial flock treatments. Clinical scoring and PCR-results were compared. Ten farms had used hoof bathing and hoof trimming without causing bleeding, ten had used individual treatments and flock vaccines to gain the free status and ten had become free through whole-flock systemic macrolide treatment. For every farm, three risk-based collected pool samples were analysed for the occurrence of virulent and benign D nodosus by PCR detection of aprV2/aprB2. Results Six flocks from any treatment group tested positive for aprB2 in all pools. Clinical signs were absent at the time of sampling, but some flocks had experienced non-progressive interdigital inflammation previously. Two flocks tested aprV2-positive in the high-risk pool. One of them underwent a progressive footrot outbreak shortly after sampling. Individual retesting indicated, that virulent D nodosus most likely was reintroduced by a recently purchased ram. In the second flock, a ram was tested positive and treated before clinical signs occurred. Conclusions All treatment strategies eliminated the causative agent and were found to be suitable for implementation in the PCR-based eradication process. PCR-testing proved to be more sensitive than visual scoring, as it also detected clinically healthy carriers. It will be of benefit as a diagnostic tool in elimination and surveillance programs.

Dichelobacter nodosus is a gram-negative fastidious anaerobic bacterium and the causative agent of ovine footrot. The disease has a global presence and is endemic in many countries. Clinical symptoms range from mild interdigital dermatitis in benign footrot to severe underrunning and separation of the hoof horn from the underlying tissue in virulent footrot. Clinical symptoms start as early as 2 weeks after first contact and the disease leads to pain, lameness, decreased meat and wool production as well as animal welfare issues. Diagnosis has improved significantly by the development of a real-time PCR to detect and discriminate virulent (aprV2-positive) and benign (aprB2-positive) D. nodosus strains. However, the real-time PCR also detects dead cells making its use for testing e.g. disinfecting agents limited. A PMA (propidium monoazid) real-time PCR using the improved dye PMAxx™ was therefore developed for virulent D. nodosus that allows discrimination of viable and dead bacteria. The distinction between viable and non-viable cells is possible, based on membrane integrity. For that purpose, the samples containing D. nodosus are treated with the improved nucleic acid intercalating PMA dye PMAxx™ that selectively enters cells with compromised cell membranes, whereas the intact cell membrane presents a natural barrier for this molecule. After exposure to strong light, it covalently binds to the DNA, preventing DNA from being amplified by PCR, thereby enabling differentiation of viable from non-viable cells. The PMA-qPCR proved to be a valid method for comparison of antimicrobial efficiency in ex vivo experiments.

Species of the bacterial genus Photorhabus live in a symbiotic relationship with Heterorhabditis entomopathogenic nematodes. Besides their use as biological control agents against agricultural pests, some Photorhabdus species are also a source of natural products and are of medical interest due to their ability to cause tissue infections and subcutaneous lesions in humans. Given the diversity of Photorhabdus species, rapid and reliable methods to resolve this genus to the species level are needed. In this study, we evaluated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Photorhabdus species. To this end, we established a collection of 54 isolates consisting of type strains and multiple field strains that belong to each of the validly described species and subspecies of this genus. Reference spectra for the strains were generated and used to complement a currently available database. The extended reference database was then used for identification based on the direct transfer sample preparation method and protein fingerprint of single colonies. High discrimination of distantly related species was observed. However, lower discrimination was observed with some of the most closely related species and subspecies. Our results, therefore, suggest that MALDI-TOF MS can be used to correctly identify Photorhabdus strains at the genus and species level, but has limited resolution power for closely related species and subspecies. Our study demonstrates the suitability and limitations of MALDI-TOF-based identification methods for the assessment of the taxonomical position and identification of Photorhabdus isolates.Impact StatementSpecies of the bacterial genus Photorhabus live in close association with soil-born entomopathogenic nematodes. Under natural conditions, these bacteria are often observed infecting soil-associated arthropods, but under certain circumstances, can also infect humans. They produce a large variety of natural products including antibiotics, insecticides, and polyketide pigments that have substantial agricultural, biotechnological and medical potential. In this study, we implement MALDI-TOF MS-based identification method to resolve the taxonomic identity of this bacterial genus, providing thereby a rapid identification tool to understanding its taxonomic diversity to boost scientific progress in medical, agricultural, and biotechnological settings.

Polyphasic analysis was done on 24 strains of Bisgaard taxon 16 from five European countries and mainly isolated from dogs and human dog-bite wounds. The isolates represented a phenotypically and genetically homogenous group within the family Pasteurellaceae. Their phenotypic profile was similar to members of the genus Pasteurella. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry clearly identified taxon 16 and separated it from all other genera of Pasteurellaceae showing a characteristic peak combination. Taxon 16 can be further separated and identified by a RecN protein signature sequence detectable by a specific PCR. In all phylogenetic analyses based on 16S rRNA, rpoB, infB and recN genes, taxon 16 formed a monophyletic branch with intraspecies sequence similarity of at least 99.1, 90.8, 96.8 and 97.2 %, respectively. Taxon 16 showed closest genetic relationship with Bibersteinia trehalosi as to the 16S rRNA gene (95.9 %), the rpoB (89.8 %) and the recN (74.4 %), and with Actinobacillus lignieresii for infB (84.9 %). Predicted genome similarity values based on the recN gene sequences between taxon 16 isolates and the type strains of known genera of Pasteurellaceae were below the genus level. Major whole cell fatty acids for the strain HPA 21(T) are C14:0, C16:0, C18:0 and C16:1 ω7c/C15:0 iso 2OH. Major respiratory quinones are menaquinone-8, ubiquinone-8 and demethylmenaquinone-8. We propose to classify these organisms as a novel genus and species within the family of Pasteurellaceae named Frederiksenia canicola gen. nov., sp. nov. The type strain is HPA 21(T) (= CCUG 62410(T) = DSM 25797(T)).

The aim of this study was to determine the prevalence of the three Treponema species as well as D. nodosus in Digital dermatitis (DD) and slurry of Swiss cattle using PCR. A total of 86 specimens from 24 farms were enrolled in the study. Slurry samples from 21 DD-affected and one unaffected farm were collected to assess the potential of environmental transmission. Nested and real-time PCR were performed from the specimens to detect Treponema species and D. nodosus, respectively. The DD-stages were positive for at least one or more of the DD-associated Treponema species in 50 of 61 cases (82.0%) and in 9 of 25 cases (36.0%) in unaffected animals. Infected animals with small focal active lesions showed a significantly lower prevalence (14.8%) compared to the other DD stages (67.2%; P=0.011). Most prevalent was T. phagedenis (65.1%). D. nodosus was detected in 51.8% of clinical DD lesions and 24.1% in unaffected cases, but its presence was not significantly associated with the various DD-stages. All samples positive for D. nodosus contained the acid protease gene aprB2 but were negative for aprV2, the latter associated with virulence in sheep foot rot. Control farms were negative for all DD-associated Treponema species while positive for aprB2 and negative for aprV2. The presence of aprB2 suggests it is ubiquitous in the animal environment. With respect to the slurry samples, three out of 21 specimens (14.3%) were positive for one or more of the DD-associated Treponema species and eleven out of 21 specimens (52.4%) were positive for aprB2 and negative for aprV2 of D. nodosus. In conclusion, an association was found between the presence of clinical DD and specific Treponema species, while for D. nodosus no such link with DD lesions could be observed.Dermatitis digitalis (DD) der Rinder ist eine multifaktorielle Infektionskrankheit, die sich zu einem bedeutenden wirtschaftlichen und weltweit wichtigen Tierwohl-Problem entwickelt hat. Drei Treponema Spezies T. pedis, T. medium und T. phagedenis sind mit DD assoziiert, jedoch ist die Prävalenz der Erreger bei Rindern in der Schweiz bislang nicht untersucht. Es wurde zudem berichtet, dass Dichelobacter nodosus zur Entwicklung von DD beiträgt. Ziel dieser Studie war, die Prävalenz der drei oben genannten Treponema Spezies, sowie von D. nodosus in DD Läsionen und Umweltproben mittels spezifischer PCR Verfahren zu bestimmen. Zweiundzwanzig Betriebe mit und zwei Betriebe ohne klinische Anzeichen von DD bei Rindern wurden in die Studie aufgenommen. Insgesamt wurden 86 Proben von verschiedenen DD-Stadien gesammelt (M1, n = 15; M2, n = 19; M3, n = 9; M4, n = 2; M4.1, n = 16 und M5, n = 25; M1-M5= Stadien der DD). Diese wurden mittels steriler, trockener Wattestäbchen direkt von der Oberfläche der Läsionen genommen. Am häufigsten wurde T. phagedenis (65,1%) nachgewiesen. D. nodosus wurde in 51,8% der klinischen DD-Läsionen (M1 bis M4.1) und in 24,1% der M5 Proben (klinisch gesund) nachgewiesen. Das Vorkommen der verschiedenen Treponemen korrelierte nicht mit den DD- Stadien. Alle für D. nodosus positiv getesteten Proben waren aprB2-positiv und negativ für aprV2. aprV2-positive D. nodosus Stämme verursachen Moderhinke beim Schaf. DD-freie Betriebe waren negativ für alle DD-assoziierten Treponema Spezies, aber positiv für aprB2 und durchwegs negativ für aprV2. Bei den Umweltproben waren drei der 21 Proben (14,3%) aus Betrieben mit Rindern, welche an DD erkrankt waren positiv für mindestens eine oder mehrere der DD-assoziierten Treponema Spezies, und 11 von 21 Proben (52,4%) waren positiv für aprB2. Alle untersuchten Proben waren negativ für aprV2 von D. nodosus. Abschliessend kann festgehalten werden, dass ein Zusammenhang besteht zwischen DD-Läsionen und dem Nachweis von einer oder mehreren spezifischen Treponema Spezies. Bei D. nodosus konnte keine Assoziation zum Auftreten von DD-Läsionen beobachtet werden. Diese Studie zeigt erstmals, dass die drei Treponema Spezies T. pedis, T. medium und T. phagedenis auch in der Schweiz an der Mortellaro‘schen Krankheit beteiligt sind.La Dermatite digitée (DD) chez les bovins est une maladie infectieuse podale multifactorielle, qui est devenue un problème émergent pour le bien-être animal et pour l’économie au niveau mondial. Trois espèces de Treponema, T. pedis, T. medium et T. phagedenis, sont associées avec la DD. Cependant, leur prévalence est inconnue en Suisse. Il a également été rapporté que Dichelobacter nodosus pouvait contribuer au développement de la DD. Le but de cette étude a été de déterminer la prévalence des trois espèces de Treponema ainsi que de D. nodosus dans des lésions de DD et du lisier de vaches suisses, en utilisant des techniques basées sur la PCR. Vingt-deux exploitations avec de la DD clinique et deux exploitations sans signes cliniques de DD ont été inclues dans l’étude. Un total de 86 échantillons de cas de DD ont été prélevés (M1, n=15; M2, n=19; M3, n=9; M4, n=2, M4.1, n=16 and M5, n=25) en utilisant des coton-tiges secs et stériles. De plus, afin d’évaluer le potentiel de transmission par l’environnement, des échantillons de lisier ont été prélevés sur des exploitations atteintes de DD (n=21) et sur une exploitation à stabulation libre exempte de DD. La PCR nichée et la PCR en temps réel ont ensuite été utilisées sur l’ADN extrait des échantillons afin de détecter les espèces de Treponema et D. nodosus, respectivement. Les associations entre la présence d’espèces de Treponema et de D. nodosus avec le statut DD des animaux ont été évaluées avec le test Pearson’s Chi-Square. Les stades de DD (M1 à M4.1) et M5 (peau saine) étaient positifs pour au moins une ou plusieurs des espèces de Treponema associées à la DD dans 50 de 61 cas (82.0%) et 9 de 25 cas (36.0%), respectivement. Les lésions M1 ont montré une prévalence nettement inférieure (14.8%) comparé aux autres stades de DD (M2, M3, M4 et M4.1; 67.2%; P=0.011). T. phagedenis était prédominant (65.1%). D. nodosus a été détecté dans 51.8% des lésions cliniques de DD (M1 à M4) et 24.1% des échantillons M5, mais sa présence n’était pas associée significativement avec les divers stades de DD. Tous les échantillons positifs pour D. nodosus contenaient le gène de la protéase acide aprB2, mais étaient négatifs pour aprV2, un gène associé à la virulence dans le piétin des moutons. Les exploitations de contrôle étaient négatives pour toutes les espèces de Treponema associées à la DD, mais positives pour aprB2 et négatives pour aprV2. La présence du gène aprB2 suggère qu’il est ubiquitaire dans l’environnement des animaux et n’est pas une association en soi avec le piétin des moutons. En ce qui concerne les échantillons de lisier, trois des 21 échantillons (14.3%) étaient positifs pour au moins une ou plusieurs espèces de Treponema associées à la DD et onze des 21 échantillons (52.4%) étaient positifs pour aprB2 et négatifs pour aprV2 de D. nodosus. En conclusion, une association a été trouvée entre la présence de DD clinique et des espèces de Treponema spécifiques, alors que pour D. nodosus aucun lien avec des lésions de DD n’a pu être observé. Cette étude démontre la présence des trois espèces de Tréponèmes chez les bovins suisses et facilite la compréhension de l’implication de Treponema spécifiques dans les lésions de DD.La dermatite digitale (DD) è una malattia infettiva multifattoriale del piede bovino che è diventata un problema economico e di benessere animale emergente a livello mondiale. Tre specie di Treponema, T. pedis, T. medium e T. phagedenis sono associate alla DD, ma la prevalenza in Svizzera rimane ancora sconosciuta. Inoltre é stato messo in evidenza che Dichelobacter nodosus é implicato nella patogenesi della DD. Lo scopo di questo studio era quindi quello di determinare la prevalenza delle tre specie Treponema e D. nodosus nelle lesioni di DD e nei liquami di bovini svizzeri utilizzando tecniche basate sulla PCR. Sono state arruolate nello studio 22 aziende con DD clinica e 2 aziende in cui la DD era assente. Sono stati raccolti 86 campioni di casi di DD (M1, n=15; M2, n=19; M3, n=9; M4, n=2, M4.1, n=16 e M5, n=25) utilizzando tamponi di cotone sterili e asciutti. Inoltre, per valutare il potenziale di trasmissione ambientale, sono stati raccolti campioni di liquami provenienti da aziende agricole affette da DD (n=21) e da un’azienda agricola a stabulazione libera non affetta. Sono state poi effettuate “nested PCR” e “real-time PCR” sul DNA estratto dai campioni per rilevare rispettivamente le specie di Treponema e D. nodosus. Il test Chi-Quadrato di Pearson è stato utilizzato per studiare le associazioni tra la presenza di specie di Treponema e D. nodosus e lo stato DD degli animali. Gli stadi DD (M1 a M4.1) e M5 (cute sana) erano positivi per almeno una o più delle specie di Treponema associate alla DD in 50 dei 61 casi (82.0%) e 9 dei 25 casi (36.0%), rispettivamente. Le lesioni M1 hanno mostrato una prevalenza significativamente più bassa (14.8%) rispetto agli altri stadi di DD (M2, M3, M4, M4 e M4.1; 67.2%; P=0.011). La specie predominante era T. phagedenis (65.1%). D. nodosus è stato rilevato nel 51.8% delle lesioni cliniche di DD (M1 a M4.1) e nel 24.1% dei campioni prelevati da lesioni M5, ma la sua presenza non era associata in modo significativo ai vari stadi di DD. Tutti i campioni positivi per D. nodosus contenevano il gene della proteasi acida aprB2, ma erano negativi per aprV2, quest’ultimo associato a virulenza nella Zoppina della pecora. Gli allevamenti di controllo sono risultati negativi per tutte le specie di Treponema associate alla DD, mentre sono risultati positivi per aprB2 e negativi per aprV2. La presenza di aprB2 suggerisce che questo gene sia ubiquitario nell’ambiente animale e non un’associazione di per sé con la Zoppina ovina. Per quanto riguarda i campioni di liquame, 3 dei 21 campioni (14,3%) sono risultati positivi per almeno una o più delle specie di Treponema associate alla DD e 11 dei 21 campioni (52,4%) sono risultati positivi per aprB2 e negativi per aprV2 di D. nodosus. In conclusione, è stata trovata un’associazione tra la presenza di DD clinica e specifiche specie di Treponema, mentre per D. nodosus non è stato osservato alcun legame di questo tipo con le lesioni di DD. Questo studio fornisce prove della presenza delle tre specie di treponema nel bestiame svizzero e facilita la comprensione del coinvolgimento di specifiche specie di Treponema nelle lesioni di DD.

A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis . In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5–100 %, and of up to 98.2 % with U. suis . DNA–DNA hybridization indicated the organisms represented a novel species with only 40 % DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C16 : 0 and summed feature C16 : 1ω7c/iso-C15 : 0 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624T ( = DSM 26510T = CCUG 63373T).

Bacterial symbionts are crucial for the infectivity and success of entomopathogenic nematodes as biological control agents. The current understanding of the symbiotic relationships is limited by taxonomic uncertainties. Here, we used whole-genome sequencing and traditional techniques to reconstruct the phylogenetic relationships between all described Photorhabdus species and subspecies as well as 11 newly isolated symbiotic bacteria of Heterorhabditis nematodes, including the unreported bacterial partner of H. beicherriana. In silico DNA–DNA hybridization, orthologous average nucleotide identity and nucleotide sequence identity of concatenated housekeeping genes scores were calculated and set into relation with current cut-off values for species delimitation in bacteria. Sequence data were complemented with biochemical and chemotaxonomic markers, and ribosomal protein fingerprinting profiles. This polyphasic approach resolves the ambiguous taxonomy of Photorhabdus and lead to the proposal for the elevation of most of them into a higher taxon and the creation of several new taxa: 15 new species, one of which is newly described: Photorhabdus bodei sp. nov. (type strain LJ24-63T=DSM 105690T=CCOS 1159T) and the other 14 arise through the proposal of elevating already described subspecies to species, and are proposed to be renamed as follows: Photorhabdus asymbiotica subsp. australis as Photorhabdus australis sp. nov., Photorhabdus luminescens subsp. akhurstii as Photorhabdus akhurstii sp. nov., Photorhabdus luminescens subsp. caribbeanensis as Photorhabdus caribbeanensis sp. nov., Photorhabdus luminescens subsp. hainanensis as Photorhabdus hainanensis sp. nov., Photorhabdus luminescens subsp. kayaii as Photorhabdus kayaii sp. nov., Photorhabdus luminescens subsp. kleinii as Photorhabdus kleinii sp. nov., Photorhabdus luminescens subsp. namnaonensis as Photorhabdus namnaonensis sp. nov., Photorhabdus luminescens subsp. noenieputensis as Photorhabdus noenieputensis sp. nov., Photorhabdus luminescens subsp. laumondii as Photorhabdus laumondii sp. nov., Photorhabdus temperata subsp. cinerea as Photorhabdus cinerea sp. nov., Photorhabdus temperata subsp. khanii as Photorhabdus khanii sp. nov., Photorhabdus temperata subsp. stackebrandtii as Photorhabdus stackebrandtii sp. nov., Photorhabdus temperata subsp. tasmaniensis as Photorhabdus tasmaniensis sp. nov., and Photorhabdus temperata subsp. thracensis as Photorhabdus thracensis sp. nov. In addition, we propose the creation of two new subspecies, one of which arises through the reduction of rank: Photorhabdus laumondii subsp. laumondii comb. nov. (basonym: P. luminescens subsp. laumondii ) and the second one is newly described: Photorhabdus laumondii subsp. clarkei subsp. nov. (type strain BOJ-47T=DSM 105531T=CCOS 1160T). Finally, we propose to emend the description of three species, which results from the proposal of elevating three subspecies to the species status: Photorhabdus asymbiotica , Photorhabdus temperata and Photorhabdus luminescens , formerly classified as Photorhabdus asymbiotica subsp. asymbiotica , Photorhabdus temperata subsp. temperata and Photorhabdus luminescens subsp. luminescens , respectively.

Virulent ovine foot rot is a contagious foot disease. Given the development and validation of a real-time PCR to detect Dichelobacter nodosus isolates that contain the virulence-associated protease genes aprV2 and aprB2, the diagnosis of foot rot has made considerable progress. We evaluated pooling methods to reduce the number of samples during a foot rot control program. Samples of individual feet were compared to a 4-feet sample of the same sheep. All further analyses based on 4-feet samples (pools-of-5 and pools-of-10 4-feet samples) were compared to samples of individual sheep, and a risk-based herd sampling was evaluated and compared to the whole flock. The sensitivity and specificity of the 4-feet samples for detection of aprV2-positive strains was 93.8% (CI: 87.6–97.5%) and 98.3% (CI: 96.5–99.3%), respectively. The sensitivity and specificity of the pools-of-10 was 86.7% (CI: 78.4–92.7%) and 100.0% (CI: 97.4–100%), respectively. Pools-of-5 were not significantly more sensitive than pools-of-10. The pooling of 4 individual foot samples into one 4-feet sample is an adequate method to reduce the number of samples of individual sheep. The sensitivity of pools-of-5 and pools-of-10 is too imprecise for a control program. Risk-based sampling allowed for a substantial reduction of samples to be tested, had a sensitivity of 95.8% (CI: 78.9–99.9%) and specificity of 100.0% (CI: 88.1–100.0%) when determining the foot rot flock status, and represents an adequate methodology to predict within-flock freedom from infection.

Clostridium difficile is an antibiotic-associated emerging pathogen of humans and animals. Thus far three toxins of C. difficile have been described: an enterotoxin (ToxA), a cytotoxin (ToxB) and an ADP-ribosyltransferase (CDT). In the present work we describe the first isolation of CDT producing C. difficile from Equidae with gastro-intestinal disease. Out of 17 C. difficile strains isolated from Equidae, 11 were positive for the genes tcdA and tcdB encoding ToxA and ToxB. In addition four of these 11 isolates were positive for the cdtA gene encoding the catalytic subunit of the ADP-ribosyltransferase CDT. Interestingly none of the isolates derived from canines (41 isolates) and felines (4 isolates) harboured the cdtA gene. In C. difficile field isolates which contained the cdtA gene, ADP-ribosyltransferase activity could also be detected in culture supernatants indicating expression and secretion of CDT. All strains were associated with intestinal disorders, but no association was found for the occurrence of toxins with a specific clinical diagnosis.

Rodentibacter gen. nov. is proposed based on isolation and phenotypic characterization of strains, predominantly from rodents. The strains showed 86 % or higher rpoB gene sequence similarity and indicated a genus-level relationship within Pasteurellaceae. The strains compared at 16S rRNA gene sequence level showed 93.8 % or higher similarity, and their genus-level relationship within Pasteurellaceae was confirmed by phenotypic analysis. The type species Rodentibacter pneumotropicus comb. nov. is reclassified from [Pasteurella] pneumotropica with type strain NCTC 8141T (=CCUG 12398T). Whole genomic comparison allowed the estimation of DNA-DNA renaturation. Rodentibacter heylii sp. nov. was proposed for a group that included the biovar Heyl of [Pasteurella] pneumotropica with the type strain ATCC 12555T (=CCUG 998T). A group was proposed as Rodentibacter ratti sp. nov., which included the taxon 22 of Bisgaard; the type strain is F75T (=CCUG 69665T=DSM 103977T). Taxon 41 of Bisgaard was proposed as Rodentibacter myodis sp. nov. with type strain Ac151T (=CCUG 69666T=DSM 103994T). Rodentibacter heidelbergensis sp. nov. included the type strain 1996025094T (=Ac69T) (=CCUG 69667T=DSM 103978T). A group strains of was proposed as Rodentibacter trehalosifermentans sp. nov. with type strain H1987082031T (=CCUG 69668T=DSM 104075T). Two strains including the reference strain of taxon 17 of Bisgaard that showed 16S rRNA gene similarity of 97.3 % were proposed as Rodentibacter rarus sp. nov. 2325/79T (=CCUG 17206T=DSM 103980T). Rodentibacter mrazii sp. nov. was proposed with type strain Ppn418T (Bisgaard taxon 21) (=CCUG 69669T=DSM 103979T). The eight species could be separated based on phenotypic characteristics such as NAD requirement, ornithine decarboxylase and indole formation, α-glucosidase, β-galactosidase and in acid formation from (+)-l-arabinose, (-)-d-ribose, (+)-d-xylose, myo-inositol, (-)-d-mannitol, lactose, melibiose and trehalose. Forty-six strains including taxon 48 of Bisgaard formed a monophyletic group by rpoB and 16S rRNA gene sequence analysis, but could not be separated phenotypically from R. pneumotropicus and R. heylii, and it was left as an unnamed genomospecies 1 of Rodentibacter with reference strain Ppn416. Another taxon that included 13 strains, mainly isolated from Apodemus sylvaticus, could not be separated phenotypically from R. pneumotropicus or R. heylii and was designated as genomospecies 2. Strain Ppn85 with 95 % or less rpoB gene sequence similarity and with 16S rRNA gene sequence similarity of 97 % or less to the other members of Rodentibacter was left as an unnamed singleton.

Campylobacter jejuni is the most common food-borne zoonotic pathogen causing human gastroenteritis worldwide and has assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to get an overview of genotypes and antibiotic resistances in C. jejuni isolated from milk, cattle feces, and pigeons in dairy herds of Northern Italy. flaB-typing was applied to 78 C. jejuni isolates, previously characterized by Multi-Locus Sequence Typing, and genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. flaB-typing revealed 22 different types with one of them being novel and was useful to further differentiate strains with an identical Sequence Type (ST) and to identify a pigeon-specific clone. Macrolide resistance was not found, while quinolone resistance was detected in 23.3% of isolates. A relationship between specific genotypes and antibiotic resistance was observed, but was only significant for the Clonal Complex 206. Our data confirm that pigeons do not play a role in the spread of C. jejuni among cattle and they are not responsible for milk contamination. A relevant number of bulk milk samples were contaminated by C. jejuni resistant to quinolones, representing a possible source of human resistant strains.

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.

Splenitis is uncommonly reported in dogs. Herein, the authors describe its prevalence, clinical findings and outcomes, histologic patterns, and causes. Splenic samples of dogs diagnosed with splenitis between 2005 and 2013 were collected and stained with hematoxylin and eosin, Gram, green-Gram, Giemsa, periodic acid–Schiff, and Ziehl-Neelsen. Samples were processed for polymerase chain reaction (PCR) to detect bacteria, fungi, and protozoa ( Leishmania infantum, Hepatozoon canis). Thirty-three of 660 splenic samples (5%) had splenitis. Clinical findings and outcomes were available in 19 dogs (58%); 49% had weakness, 33% had fever, and 84% survived. The most frequent inflammatory patterns included purulent splenitis (27%), pyogranulomatous splenitis (24%), and neutrophilic perisplenitis (15%). One dog had a putative diagnosis of primary splenitis; in 8 dogs, microorganisms were identified histologically or by PCR in the spleen without obvious comorbidities. Twenty-four dogs (73%) had concurrent diseases; a permissive role in the development of splenitis was suspected in 21 of these cases. Histologic examination identified the cause of splenitis in 10 dogs. Bacteria were identified by PCR in 23 cases, but the bacteria were confirmed histologically in only 6 of these. Leishmania was detected with PCR in 6 dogs. Leishmania was identified in 1 dog and H. canis in another histologically, but both were PCR negative. Fungi were identified in 8 spleens by PCR and in 1 by histology. This study suggests that splenitis is uncommon in dogs and is frequently associated with systemic diseases. Prognosis is favorable in most cases. Identification of bacteria, fungi, and protozoa in the spleens of affected dogs with PCR should be interpreted cautiously, because the findings are not confirmed histologically in many cases.

Campylobacter jejuni along with C. coli are major cause of human gastroenteritis worldwide. So far, the human immune response against Campylobacter is not entirely clear. We hypothesize that it is coordinated by an interaction between pro-inflammatory and regulatory cytokines which is influenced by bacterial and host-individual differences. Accordingly, we used peripheral blood mononuclear cells (PBMC) from healthy donors to study the primary systemic immune response to C. jejuni and C. coli. PBMC were stimulated by different strains of C. jejuni and C. coli for three time points (5, 10, 24 hours). The production of the pro-inflammatory (IL-6, IL-8, IFN-γ) and the regulatory (IL-10) cytokines were measured by ELISA. All strains induced higher levels of IL-8 and IL-6 than IFN-γ and IL-10. In contrast to IL-8 and IL-6, IL-10 showed a steeper increase over time. While IFN-γ did not show any further increase between 10 and 24 hours. Interestingly, there was a significant correlation between IL-8 and IL-10 which peaked at 24 hours. Despite the variability of the used bacterial strains, their effect on cytokine production was less pronounced than the inter-person differences. The strongest significant effect of the strain was on the level of IL-10. IL-10 and IL-6 were significantly influenced by strain-person interaction. In conclusion, the systemic immune response to C. coli and C. jejuni is characterized by an early pro-inflammatory reaction with later initiation of regulatory immune response which is influenced mainly by the host, explaining the individual variations in disease severity. Additional work is needed to determine the cellular sources of the produced cytokines as well as the campylobacter molecules that might contribute to this stimulation.