Gallibacterium anatis infection in poultry: a comprehensive review.

Gallibacterium anatis (G. anatis), a member of the Pasteurellaceae family, normally inhabits the upper respiratory and lower genital tracts of poultry. However, under certain circumstances of immunosuppression, co-infection (especially with Escherichia coli or Mycoplasma), or various stressors, G. anatis caused respiratory, reproductive, and systemic diseases. Infection with G. anatis has emerged in different countries worldwide. The bacterium affects mainly chickens; however, other species of domestic and wild birds may get infected. Horizontal, vertical, and venereal routes of G. anatis infection have been reported. The pathogenicity of G. anatis is principally related to the presence of some essential virulence factors such as Gallibacterium toxin A, fimbriae, haemagglutinin, outer membrane vesicles, capsule, biofilms, and protease. The clinical picture of G. anatis infection is mainly represented as tracheitis, oophoritis, salpingitis, and peritonitis, while other lesions may be noted in cases of concomitant infection. Control of such infection depends mainly on applying biosecurity measures and vaccination. The antimicrobial sensitivity test is necessary for the correct treatment of G. anatis. However, the development of multiple drug resistance is common. This review article sheds light on G. anatis regarding history, susceptibility, dissemination, virulence factors, pathogenesis, clinical picture, diagnosis, and control measures.

Clinical-pathological findings induced by Histophilus somni isolated in subacute cardiac death in feedlot cattle.

The purpose of this report is to provide information about the different presentations of cardiac and extra-cardiac histophilosis and, to assess the antimicrobial (ATM) susceptibility of Histophilus somni isolated from these cardiac lesions to different ATM agents commonly used for treating bovine bacterial respiratory pathogens. Eight feedlot calves, which died after suffering from food rejection, apathy, hyperthermia, cough and nasal mucous discharge, and lack of response to ATM therapy, were studied. Cardiac lesions observed at necropsy included valvular/mural endocarditis, myocardial infarction, and necrotizing myocarditis, miliar non-suppurative myocarditis, myocardic necrotic sequestrum, and/or pericarditis. Histopathological, bacteriological and molecular studies confirmed the presence of a fastidious microorganism in the affected organs. H. somni showed no resistance to most ATM tested (ceftiofur, gamithromycin, enrofloxacin, florfenicol, tilmicosin). The results obtained in this study confirmed that H. somni was the main cause of the subacute cardiac lesions associated with hyperthermia, apathy and respiratory signs observed in cattle examined in this research. These presentations must be considered by veterinary practitioners in order to establish a rational therapeutic.

Brain abscess due to Aggregatibacter aphrophilus in association with atrial septal defect：Case report and literature review.

BACKGROUND: Aggregatibacter aphrophilus（A. aphrophilus）is one of the organisms of the HACEK group. Previously reported cases of brain abscesses caused by A. aphrophilus infection have occurred in children with a basis for congenital heart disease, or in adults with a basis for dental disease. Rare cases of brain abscess caused by A. aphrophilus have been reported in adults with congenital heart disease or in patients without dental disease history. Herein we present a rare case of brain abscess caused by A. aphrophilus, who was in association with atrial septal defect for more than 20 years, and had no dental disease and did not develop infective endocarditis. CASE PRESENTATION: A 51-year-old female was admitted due to progressively worsening headache and left limb weakness for more than 10 days. She denied the history of chronic diseases such as hypertension and diabetes, and no periodontal disease. While she had a history of atrial septal defect, a form of congenital heart disease with severe pulmonary hypertension for more than 20 years. After admission, echocardiographic illustrated congenital heart disease with severe pulmonary hypertension. CT and MRI showed brain abscess. Cerebrospinal fluid (CSF) results also confirmed the presence of intracranial infection. Empirical therapy with vancomycin 1.0 g i.v q12h and meropenem 2.0 g i.v q8h was initiated from the day of admission. On the fourth day after admission, brain abscess resection and decompressive craniectomy were performed, and the pus drained on operation were cultured and Gram-negative bacilli grew, which was identified as A.aphrophilus. Vancomycin was discontinued and meropenem was continued（2.0 g i.v q8h）for 5 weeks, followed by oral levofloxacin 0.5 qd for 4 weeks of out-patient antibiotics. The patient recovered fully within 9 weeks of treatment. CONCLUSIONS: This is the first case of A. aphrophilus to cause brain abscess in adult with a history of congenital heart disease for more than 20 years, who had no dental disease and did not develop infective endocarditis. We also highlight the value of bacterial 16 S rDNA PCR amplification and sequencing in identifying bacteria in abscesses which are culture-negative, and prompt surgical treatment，choosing effective antibiotics and appropriate course of treatment will get better clinical effect.

Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A.

L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial infections.

Aggregatibacter actinomycetemcomitans as the Aetiological Cause of Rheumatoid Arthritis: What Are the Unsolved Puzzles?

Leukotoxin A (LtxA) is the major virulence factor of an oral bacterium known as Aggregatibacter actinomycetemcomitans (Aa). LtxA is associated with elevated levels of anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. LtxA targets leukocytes and triggers an influx of extracellular calcium into cytosol. The current proposed model of LtxA-mediated hypercitrullination involves the dysregulated activation of peptidylarginine deiminase (PAD) enzymes to citrullinate proteins, the release of hypercitrullinated proteins through cell death, and the production of autoantigens recognized by ACPA. Although model-based evidence is yet to be established, its interaction with the host's immune system sparked interest in the role of LtxA in RA. The first part of this review summarizes the current knowledge of Aa and LtxA. The next part highlights the findings of previous studies on the association of Aa or LtxA with RA aetiology. Finally, we discuss the unresolved aspects of the proposed link between LtxA of Aa and RA.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Glaesserella parasuis serotype 4 HPS4-YC disrupts the integrity of the swine tracheal epithelial barrier and facilitates bacterial translocation.

Glaesserella parasuis (G. parasuis) is a commensal bacterium in the upper respiratory tract of pigs that can also cause the swine Glässer disease, which induces an intensive inflammatory response and results in significant economic losses to the swine industry worldwide. G. parasuis can cause disease through infection of the respiratory tract, resulting in systemic infection, but the mechanism is largely unknown. Recently we showed that Glaesserella parasuis serotype 4 (GPS4) increased swine tracheal epithelial barrier permeability, resulting in easier bacterial translocation. Tight junction proteins (TJ) play a crucial role in maintaining the integrity and impermeability of the epithelial barrier. GPS4 decreased the expression of the TJ ZO-1 and occludin in swine tracheal epithelial cells (STEC). Furthermore, the proinflammatory cytokines IL-6, IL-8 and TNF-α were significantly upregulated in GPS4-infected STEC, and both the MAPK and NF-κB signaling pathways were activated and contributed to the expression of TNF-α. We demonstrate that the production of proinflammatory cytokines, especially TNF-α, during GPS4 infection was involved in barrier dysfunction. Additionally, animal challenge experiments confirmed that GPS4 infection downregulated TJ in the lungs of piglets and induced a severe inflammatory response. In general, G. parasuis infection downregulated the expression of TJ and induced massive secretion of proinflammatory cytokines, resulting in epithelial barrier disruption and favoring bacterial infection. This study allowed us to better understand the mechanism by which G. parasuis crosses the respiratory tract of pigs.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Host Chromatin Regulators Required for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activity in Saccharomyces cerevisiae Model.

Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, AaCdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

Severe pneumonia in a street rat (Rattus norvegicus) caused by Rodentibacter rarus strain RMC2.

Background: Rodents are one of the most dangerous reservoirs and carriers of infectious diseases. Gradually, rats have become predominant in cities, sometimes staying in close vicinity to humans, pets, and other animals. Consequently, they tend to increase the transmission risk of pathogens. Case Description: Here, we report an original case of bacterial pneumonia in a street rat (Rattus norvegicus). The rat was found dead on a street in the chief town of Marseille (France) after being run over by a car. The necropsy of the corpse revealed generalized granulomatous pneumonia in almost all the pulmonary lobes. Lung lesions and predominantly multiple fibro-inflammatory areas are presumably the witness of an infectious etiology. Bacterial isolation was carried out from lung tissues. Colonies were identified by MALDI-TOF MS and confirmed by 16S rRNA sequencing. The following bacteria were identified: Staphylococcus cohnii, Bordetella bronchiseptica, Bordetella parapertussi, Corynebacterium glucuronolyticum, Pelistega suis and Rodentibacter rarus. Based on the histopathological diagnosis and the avoidance approach, the most likely etiological agent of pneumonia is therefore R. rarus, a little-known Pasteurellales bacterium that is closely related to Rodentibacter pneumotropicus. Conclusion: These data emphasize the severity of R. rarus infection in rodents. Thus, pointing out a potential risk for other animals (dogs, cats, and birds), as well as humans. The health monitoring program for rodents and rabbits pasteurellosis should now include R. rarus. Therefore, the pathological effect of the Rodentibacterspecies and/or strains needs to be better explored.

Protective Effects of Baicalin on Peritoneal Tight Junctions in Piglets Challenged with Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) causes inflammation and damage to piglets. Whether polyserositis caused by G. parasuis is due to tight junctions damage and the protective effect of baicalin on it have not been examined. Therefore, this study aims to investigate the effects of baicalin on peritoneal tight junctions of piglets challenged with G. parasuis and its underlying molecular mechanisms. Piglets were challenged with G. parasuis and treated with or without baicalin. RT-PCR was performed to examine the expression of peritoneal tight junctions genes. Immunofluorescence was carried out to detect the distribution patterns of tight junctions proteins. Western blot assays were carried out to determine the involved signaling pathways. Our data showed that G. parasuis infection can down-regulate the tight junctions expression and disrupt the distribution of tight junctions proteins. Baicalin can alleviate the down-regulation of tight junctions mRNA in peritoneum, prevent the abnormalities and maintain the continuous organization of tight junctions. Our results provide novel evidence to support that baicalin has the capacity to protect peritoneal tight junctions from G. parasuis-induced inflammation. The protective mechanisms of baicalin could be associated with inhibition of the activation of PKC and MLCK/MLC signaling pathway. Taken together, these data demonstrated that baicalin is a promising natural agent for the prevention and treatment of G. parasuis infection.

Clonal spread of multi-resistant Gallibacterium anatis isolates among Iranian broilers and layers.

Gallibacterium anatis is a common cause of reproductive tract infection in chickens, which leads to reduced egg production and increased mortality. This study was undertaken to investigate prevalence of G. anatis in 12 poultry flocks originating from Iranian provinces with leading chicken production and to determine genetic diversity, antimicrobial resistance, and the presence of major antigens of the isolates investigated. Out of the 120 chicken tracheal samples collected and tested, 84 (70%) were positive for G. anatis. Genotyping by Pulse Field Gel Electrophoresis and genome sequencing revealed a total of 24 pulsotypes for 71 strains (at a 87% similarity level) and seven genome clusters comprising 21 strains (97% similarity level), respectively. The combination of the two typing methods confirmed the presence of several genotypes originating from a common ancestor affecting poultry yet also suggested that identical clones were shared among chickens within farms and between different farms. The latter finding is to our knowledge the first example of clonal presence of G. anatis in epidemiologically unrelated farms. The 21 sequenced strains were characterized against a panel of commonly used antibiotics and showed lowered sensitivity to tetracycline (76.2%) and enrofloxacin (90.5%). The widespread presence of multiresistant G. anatis isolates calls for non-antibiotic prophylactics. Three major immunogen genes, gtxA, Gab_1309 and Gab_2312 were detected in the isolates indicating these antigens likely represent effective vaccine targets. A conserved sequence of the gtxA gene across a range of epidemiologically independent strains suggests the use of GtxA for future vaccine development purposes.

MOLECULAR IDENTIFICATION OF MEMBERS OF THE FAMILY PASTEURELLACEAE FROM THE ORAL CAVITY OF KOALAS (PHASCOLARCTOS CINEREUS) AND THEIR RELATIONSHIP WITH ISOLATES FROM KOALA BITE WOUNDS IN HUMANS.

A total of 22 Pasteurellaceae isolates obtained from the oral cavity of koalas (Phascolarctos cinereus) at different wildlife centers in Australia were investigated using amplification and sequencing of two housekeeping genes, rpoA and recN. The available sequences from the Lonepinella koalarum type strain (ACM3666T) and the recent isolates of Lonepinella-like bacteria obtained from human infected wounds associated with koala bites were also included. Phylogenetic analysis was performed on the concatenated rpoA-recN genes and genome relatedness was calculated based on the recN sequences. The oral cavity isolates, the koala bite wound isolates, and L. koalarum ACM3666T resulted in four clusters (Clusters 1-4). Clusters 1-3 were clearly not members of the genus Lonepinella. Cluster 1 was closely related to the genus Fredericksenia, and Clusters 2 and 3 appeared to be novel genera. Cluster 4 consisted of three subclusters: Cluster 4a with one koala bite wound isolate and L. koalarum ACM3666T, Cluster 4b with three oral cavity isolates and two Lonepinella-like wound isolates, and Cluster 4c with three nearly identical oral cavity isolates that may represent a different species within the genus Lonepinella. The rich Pasteurellaceae population, including potential novel taxa in the oral cavity of koalas supports an important role of these highly adapted microorganisms in the physiology of koalas. Moreover, the pathogenic potential of Lonepinella-like species is an important consideration when investigating infected koala bites in humans.

Differentiation among the most important Rodentibacter species by multiplex PCR assays targeting the ITSile+ala sequences of the rRNA operons.

Screening for the Rodentibacter species is part of the microbiologic quality assurance programs of laboratory rodents all over the world. Nevertheless, currently there are no PCR amplification techniques available for the diagnostic of R. ratti, R. heidelbergensis and of a Rodentibacter related ß-haemolytic taxon. The aim of this study was to utilize the differences in the sequence of the Internal Transcribed Spacer (ITS) regions of R. pneumotropicus, R. heylii, R. ratti, R. heidelbergensis and of the ß-haemolytic Rodentibacter taxon for the design of specific PCR assays for these species. The ITSile+ala sequence variations allowed the design of specific forward and reverse primers for each species included, that could be combined in different multiplex assays. The performance characteristics specificity and sensitivity registered for each primer pair against a diverse collection of Pasteurellaceae isolated from rats and mice and of further non-Pasteurellaceae strains was 100% for all five Rodentibacter species included. In addition, the PCR assays displayed high limits of detection and could be successfully used for detection of Rodentibacter spp. DNA in clinical swabs of laboratory mice and rats. Overall, the assays described here represent the first PCRs able to diagnose R. ratti, R. heidelbergensis and the ß-haemolytic Rodentibacter taxon, whose diagnostic to species level could further facilitate better understanding of their geographic distribution, prevalence, and biology in the future.

Evaluation of the Virulence of Aggregatibacter actinomycetemcomitans Through the Analysis of Leukotoxin.

Aggregatibacter actinomycetemcomitans is frequently isolated from localized aggressive periodontitis and periodontitis associated with systemic diseases. A. actinomycetemcomitans produces a leukotoxin, which induces apoptosis in human leukocytes. The leukotoxin expression is dependent on the upstream sequence, likely including the promoter, of the gene encoding leukotoxin; strains with the truncated/short upstream sequence express more leukotoxin than strains with the general/long upstream. This chapter addresses the determination of the type of the leukotoxin promoter by PCR analysis, and detection of the apoptosis in the coculture of human monocyte cell line (THP-1) with A. actinomycetemcomitans by the DNA ladder formation, membrane perturbation, and lactate dehydrogenase release.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of ß-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.

Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain.

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R.heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R.heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

Occurrence of Pasteurellaceae and Neisseriaceae bacteria in the pharyngeal and respiratory tract of dogs and cats - Short communication.

The occurrence of members of the Pasteurellaceae and Neisseriaceae families was studied in dogs and cats. A total of 110 nasal and pharyngeal swab samples from 47 dogs and 8 cats were collected. Most of the strains were identified by 16S rDNA sequencing, except Frederiksenia canicola and Pasteurella multocida where species-specific polymerase chain reactions were applied. The most frequently isolated species was F. canicola, which occurred only in dogs, mainly in the pharyngeal cavity. The second commonest bacterium, P. multocida was found in both types of samples and in both hosts. Other species from the family Pasteurellaceae, such as Haemophilus haemoglobinophilus, Pasteurella canis and P. dagmatis, were detected only in dogs. All isolated species belonging to the family Neisseriaceae, mainly representing Neisseria weaveri, were found only in the pharyngeal cavity. Neisseria weaveri and N. zoodegmatis could be detected in both hosts. Neisseria dumasiana and N. canis were isolated from dogs, while N. shayeganii only from a cat. For phylogenetic analysis, rpoB gene sequencing was performed, where the strains were on monophyletic branches and clearly separated from each other. In this study, recently described species such as F. canicola, N. shayeganii and N. dumasiana were detected that had never been isolated in Hungary before.

Prevalence and antimicrobial resistance of opportunistic pathogens associated with bovine respiratory disease isolated from nasopharyngeal swabs of veal calves in Switzerland.

The composition of the bacterial flora in the calf nasopharynx might influence the risk of bovine respiratory disease (BRD). The aims of the present study were, firstly, to investigate the prevalence of bacteria potentially involved in BRD in the nasopharynx of veal calves and to identify associated risk factors for their presence, and, secondly, to provide data on antimicrobial resistance levels in these bacteria. Deep nasopharyngeal swabs were collected from veal calves on 12 Swiss farms over a period of one year by non-random, but systematic sampling for isolation of Pasteurellaceae and Mycoplasma (M.) bovis and dispar. Associations of potential risk factors with occurrence of these bacteria were tested in multivariable mixed logistic regression analyses, based on information gained from extensive questionnaires completed with the farmers. Antimicrobial susceptibility testing was performed for Pasteurellaceae by broth microdilution method to obtain minimal inhibitory concentrations (MIC). Pasteurellaceae, including Pasteurella (P.) multocida, Mannheimia (M.) haemolytica, Bisgaard Taxon 39 and Histophilus (H.) somni, were almost twice as prevalent as M. bovis and dispar in this study. Continuous stocking was a risk factor for the presence of Pasteurellaceae, especially when calves originated from more than six suppliers. In young calves (≤ 91 days), feeding of California Mastitis Test (CMT) positive milk was an additional risk factor for the presence of Pasteurellaceae whereas transport of calves by farmers and livestock traders (as opposed to transport only by farmers) increased the risk in older calves (> 91 days). Risk factors for the presence of M. bovis/dispar were higher number of calves per drinking nipple in young calves, and no access to an outside pen and feeding of CMT positive milk in older calves, respectively. While further research will have to investigate the observed associations in more detail, this suggests that management can play an important role in the prevalence of nasopharyngeal bacteria with a potential subsequent involvement in BRD. Antimicrobial resistance differed between the three bacterial species tested in this study and was highest to oxytetracycline and spectinomycin in P. multocida, oxytetracycline and penicillin in M. haemolytica, and ampicillin and penicillin in H. somni. Only two European VetCAST breakpoints (for florfenicol in P. multocida and M. haemolytica) have been published to date, matching the MIC distribution of the present isolate populations well, in contrast to certain commonly applied American Clinical and Laboratory Institute interpretive criteria. This highlights the potential for further refinement of clinical breakpoints in veterinary medicine.

Strength of association between isolation of Pasteurella multocida and consolidation lesions in ovine pneumonic pasteurellosis.

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.