Emayella augustorita, New Member of Pasteurellaceae, Isolated from Blood Cultures of Septic Patient.

We report discovery of a new bacterial genus and species of the family Pasteurellaceae by using phylogenetic and metabolic analysis. The bacterium, Emayella augustorita, was isolated from blood cultures of a patient in France diagnosed with an adenocarcinoma of the intestines and who was treated with a biliary prosthesis placement.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Detection of bovine gammaherpesvirus 6 in tissues of aborted fetuses from dairy cows concomitantly infected by Histophilus somni.

Bovine gammaherpesvirus 6 (BoGHV6), formerly known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae, that was initially associated with proliferative diseases in cattle. While the Macavirus genus contains agents, including alcelaphine gammaherpesvirus 1 (AlGHV1), ovine gammaherpesvirus 2 (OvGHV2), and caprine gammaherpesvirus-2 (CpGHV2), known to cause malignant catarrhal fever (MCF), and are collectively referred to as MCF virus (MCFV) group of organisms, diseases and/or clinical syndromes have not been associated with BoGHV6 and porcine lymphotropic herpesvirus (PLHV). This report investigated the occurrence of BoGHV6 in tissues of aborted dairy fetuses known to be infected by Histophilus somni to identify possible disease patterns associated with infection by this Macavirus. A nested-PCR (nPCR) assay was used to amplify the BoGHV6 polymerase gene from multiple tissues of 13 fetuses and the cow of one of these which were derived from seven dairy herds located in three geographical regions of Brazil. Direct sequencing confirmed the results of the nPCR assays. Additionally, all fetal tissues were previously investigated for the presence of H. somni, Listeria monocytogenes, Neospora caninum, Brucella abortus, Leptospira spp., bovine alphaherpesvirus 1, and bovine viral diarrhea virus (BVDV) by PCR and/or RT-PCR assays. The nPCR assay amplified BoGHV6 DNA from fetuses of most dairy herds (85.7%; 6/7) investigated, resulting in the amplification of BoGHV6 from 76.9% (10/13) of all fetuses evaluated from two geographical and important cattle-producing regions of Brazil. Furthermore, only BoGHV6 was identified in the spleen (n = 3), myocardium, and kidney (n = 2) of five fetuses, and BoGHV6 was the only agent associated with myocarditis in one of these. Nevertheless, dual, triple, and quadruple infections (including BVDV, B. abortus, and N. caninum) were identified in fetuses that were concomitantly infected by H. somni. Phylogenetic analysis revealed that the strain herein identified has 100% nucleotide (nt) sequence identity with wild type strains of BoGHV6 circulating in ruminants from Brazil and 99.8% nt identity with the reference strain of BoGHV6 but was 72.2-73.3% and 67.4-68.2% different from members of the MCFV group and PLHV, respectively. These results demonstrated that 76.9% of the fetuses evaluated were infected by BoGHV6, most likely via vertical infection resulting in transplacental transmission. Considering that most fetuses were concomitantly infected by BoGHV6 and H. somni the real impact of this viral infection cannot be efficiently determined. However, since BoGHV6 was the only pathogen identified in the myocardium of one fetus with myocarditis by histopathology, the possible participation of this Macavirus in the etiopathogenesis of the myocardial disease observed in this fetus cannot be ignored or discarded. However, the mere amplification of BoGHV6 DNA from the myocardium is not enough to establish a definite association between cause and effect, since in situ evaluations and experimental studies would be needed to confirm this agent in the etiopathogenesis of fetal diseases and/or abortions in cattle. Consequently, additional studies are needed to determine the exact role, if any, of BoGHV6 in the development of fetal disease, and possibly fetal mortality.

Human abdominal abscess caused by Necropsobacter rosorum and tips for its identification: A case report.

Necropsobacter rosorum is a gram-negative facultative anaerobe, which was reclassified from the family Pasteurellaceae in 2011. It has been detected in the gastrointestinal and respiratory tracts of mammals; however, reports of infection in humans are scarce. We report a case of an abdominal abscess in which N. rosorum was detected; it was successfully treated with drainage and antimicrobial therapy. Routine laboratory testing such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and an identification system using biochemical phenotypes could not identify N. rosorum. Instead, it was misidentified as other Pasteurellaceae species, including Aggregatibacter spp. or Pasteurella spp. Sequencing of 16S rRNA was required to identify N. rosorum. We suggest the application of simple methods, such as indole production, oxidase, and catalase tests, to differentiate N. rosorum from genetically similar species.

Histophilus somni stimulates bovine monocyte-derived macrophages to release microparticles that increase fibrin clot formation in vitro.

Histophilus somni is a Gram-negative coccobacillus that causes diffuse vasculitis and intravascular thrombosis that can lead to multiple organ failure in cattle. Macrophages are important cellular mediators of fibrin deposition and removal at sites of inflammation. It has become evident that macrophages and other cells release microparticles (MPs) that have an array of biological activities, including pro-coagulant activity. We sought to determine whether monocyte-derived macrophages exposed to H. somni in vitro release MPs that activate the clotting cascade in a manner that could lead to thrombus formation. Bovine monocyte-derived macrophages were incubated with H. somni (at a 10:1 ratio) in RPMI with 10% heat inactivated fetal bovine serum for 6 h at 37 °C with 5 % CO2. Membrane-shed MPs were isolated from the conditioned media, washed twice with Ca2+ and Mg2+ free HBSS, and pro-coagulant activity assessed by a one-step plasma clotting assay. We observed greater pro-coagulant activity for MPs from H. somni stimulated macrophages than from unstimulated controls. Microparticle pro-coagulant activity was inhibited by addition of an anti-tissue factor antibody. We also observed co-localization of fluorescein-labeled H. somni cells and annexin V staining as evaluated by confocal microscopy. These results demonstrate that exposure to H. somni cells causes bovine monocyte-derived macrophages to release MPs that contain tissue factor, the first such report for bovine macrophages. We infer that if similar events occur in vivo they could amplify thrombus formation in bovine histophilosis.

Effect of Baicalin on Transcriptome Changes in Piglet Vascular Endothelial Cells Induced by a Combination of Glaesserella parasuis and Lipopolysaccharide.

Glaesserella parasuis causes porcine Glässer's disease and lipopolysaccharide (LPS) induces acute inflammation and pathological damage. Baicalin has antioxidant, antimicrobial, and anti-inflammatory functions. Long noncoding RNAs (lncRNAs) play key regulatory functions during bacterial infection. However, the role of lncRNAs in the vascular dysfunction induced by a combination of G. parasuis and LPS during systemic inflammation and the effect of baicalin on lncRNA expression induced in porcine aortic vascular endothelial cells (PAVECs) by a combination of G. parasuis and LPS have not been investigated. In this study, we investigated the changes in lncRNA and mRNA expression induced in PAVECs by G. parasuis, LPS, or a combination of G. parasuis and LPS, and the action of baicalin on lncRNA expression induced in PAVECs by the combination of G. parasuis and LPS. Our results showed 133 lncRNAs and 602 genes were differentially expressed when PAVECs were stimulated with the combination of G. parasuis and LPS, whereas 107 lncRNAs and 936 genes were differentially expressed when PAVECs were stimulated with the combination of G. parasuis and LPS after pretreatment with baicalin. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the dominant signaling pathways triggered by the combination of G. parasuis and LPS were the tumor necrosis factor signaling pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism. Protein-protein interaction network analysis showed the differentially expressed target genes of the differentially expressed lncRNAs (DELs) were related to each other. A coexpression analysis indicated the expression levels of the DELs were co-regulated with those of their differentially expressed target genes. This is the first study to systematically compare the changes in lncRNAs and mRNAs in PAVECs stimulated with a combination of G. parasuis and LPS. Our data clarified the mechanisms underlying the vascular inflammation and damage triggered by G. parasuis and LPS, and it may provide novel targets for the treatment of LPS-induced systemic inflammation.

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.

Sialidase of Glaesserella parasuis Augments Inflammatory Response via Desialylation and Abrogation of Negative Regulation of Siglec-5.

Siglecs are sialic acid-binding immunoglobulin-like lectins that play an important role in tissue homeostasis, immune response, and pathogen infection. Bacterial sialidases act on natural ligands of Siglecs, interfering with the Siglec-mediated immune response. Glaesserella parasuis is a porcine bacterial pathogen that secretes sialidase. However, little is known about the sialidase of G. parasuis and its impact on immune regulation. Here, we used wild-type G. parasuis, a sialidase-deficient mutant, and complementary strains to investigate the role of sialidase in porcine alveolar macrophage infection. Sialidase induced the release of proinflammatory cytokines, such as interleukin-1α (IL-1α), IL-6, and tumor necrosis factor alpha, from porcine alveolar macrophages. Moreover, sialidase desialylated the surface of porcine alveolar macrophages and altered the expression of Siglecs (the expression of Siglec-5 was reduced). Furthermore, sialidase led to a reduction in endogenous SH2 domain-containing protein tyrosine phosphatase (SHP-2) recruitment to Siglec-5 and simultaneously activated the inflammatory response via the mitogen-activated protein kinase and nuclear factor kappa light chain enhancer of activated B cell signaling pathways. This desialylation occurred before the release of proinflammatory cytokines, suggesting that the sialidase-induced inflammatory response was followed by reduced recruitment of SHP-2 to Siglec-5. Thus, this study is the first to demonstrate the role of sialidase in the inflammatory response of G. parasuis. This role resulted from the abrogation of negative regulation of Siglec-5 on proinflammatory cytokine release. This study helps to understand the molecular mechanism underlying the inflammatory response induced by sialidase secreted by G. parasuis and the acute inflammation caused by G. parasuis.

Bovine respiratory disease in beef calves supported long transport stress: An epidemiological study and strategies for control and prevention.

BRD is associated with infectious agents, but management and transport-stress are trigger factors. Metaphylactic administration of antimicrobial reduces colonization of respiratory tract by pathogens, but the development of antibiotic-resistance raises public health concerns leading to propose new control strategies. The study analyzed nasopharyngeal swabs of 231 imported cattle, 10% of 49 trucks, transported from France to southern Italy and, through Real-time PCR identified the prevalence of the involved pathogens speculating on strategies to reduce the impact of BRD. The samples were tested by Real-time PCR, for the detection of bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), bovine adenovirus (BAdV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Yates-corrected chi squared, or Fisher's exact test were used to compare both animal-health status and positivity/negativity to pathogens, and the relationship between presence/absence of clinical signs and Real-time PCR-positivity. H. somni and BCoV were the most frequently identified pathogens. In BRD-diagnosed cattle, BAdV was detected in 13.8% (19/138), BRSV in 14.5% (20/138) and BPiV in 4.3% (6/138). Healthy cattle were mostly positive for H. somni (89.2%, 83/93). A statistically significant association was observed between clinical signs and positivity to M. haemolytica (p value = 0.016). Although mass-medication and vaccination are used for BRD control, it still remains a primary health problem. Our results highlight that the nasopharyngeal microbiota could be affected by transport and that strategies to enhance calf immunity for reducing BRD-risk development would be more effective if applied at farm of origin prior to loading.

Immune Suppression Induced by Gallibacterium anatis GtxA During Interaction with Chicken Macrophage-Like HD11 Cells.

The RTX toxin GtxA expressed by Gallibacterium anatis biovar haemolytica has been proposed a major virulence factor during disease manifestations in the natural host, the chicken. To better understand the role of GtxA in the pathogenesis of G. anatis, we compared the GtxA expressing wildtype strain with its isogenic ∆gtxA mutant that was unable to express GtxA during exposure to chicken macrophage-like HD11 cells. From adhesion and invasion assays, we showed that GtxA appears to promote adhesion and invasion of HD11 cells. By using quantitative RT-PCR, we also demonstrated that the G. anatis expressing GtxA induced a mainly anti-inflammatory (IL-10) host cell response as opposed to the pro-inflammatory (IL-1ß, IL-6 and TNF-α) response induced by the GtxA deletion mutant. Interestingly, these results, at least partly, resemble recent responses observed from spleen tissue of chickens infected with the same two bacterial strains. The effect of the GtxA toxin on the type of cell death was less clear. While GtxA clearly induced cell death, our efforts to characterize whether this was due to primarily necrosis or apoptosis through expression analysis of a broad range of apoptosis genes did not reveal clear answers.

The role of GtxA during Gallibacterium anatis infection of primary chicken oviduct epithelial cells.

Gallibacterium anatis (G. anatis), one of the major pathogens causing reproductive tract disorders in laying hens, leads to a reduction in egg production and increased mortality, caused by either single or mixed infections with other pathogens. As a specific virulence factor of G. anatis, the role of GtxA in layers' salpingitis remains unclear. In this study, we explored the effect of GtxA on G. anatis infection by comparing wild strain Yu-PDS-RZ-1-SLG (RZ) and its GtxA deleted counterpart RZΔgtxA in primary chicken oviduct epithelial cells (COEC). Their adherence, invasion, cytoxicity, and ability to induce apoptosis and and cytokine secretion were evaluated and the cytotoxicity and cytokine secretion of the recombinant GtxA protein and its N-terminal adenylate cyclase and C-terminal RTX hemolysin domain were also analyzed. We found that the adhesion ability of RZΔgtxA was significantly lower than that of parental strain RZ, and its toxicity to COEC was weakened; Meanwhile, apoptosis was inhibited and the expression of IL-6, IL-2, TNF-α and IFN-γ were dramatically reduced in COEC infected by RZΔgtxA. In contrast, the recombinant protein GtxA inhibited the proliferation of oviduct cells and induced obvious cytotoxicity, and the expression of IL-6, TNF-α and IFN-γ were up-regulated in COEC interacted with recombinant proteins. Our study indicates that GtxA promotes G. anatis adherence to cells, changes cells permeability and expression of inflammatory factors, resulting in cell damage and apoptosis.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Synthetic bovine NK-lysin-derived peptide (bNK2A) does not require intra-chain disulfide bonds for bactericidal activity.

Bovine NK-lysins are cationic antimicrobial proteins found predominantly in the cytosolic granules of T lymphocytes and NK-cells. NK-lysin-derived peptides show antimicrobial activity against both Gram positive and Gram negative bacteria. Mature NK-lysin protein has six well-conserved cysteine residues. This study was performed to assess whether synthetic bovine NK-lysin-derived peptide (bNK2A) forms disulfide bonds and whether disulfide bonds were essential for bNK2A antimicrobial activity. Two 30-mer bNK2A peptides were synthesized: one with two original cysteines and an analog with cysteines substituted with two serines. Mass spectrometry revealed lack of disulfide bonds in original peptide while CD spectrophotometry showed both peptides have similar α-helical structures. Since both peptides were equally inhibitory to Histophilus somni, disulfide bonds appeared dispensable for synthetic bNK2A peptide antibacterial activity.

Evaluation of the protective effect of immunization spf DBA/2J mice with selected bacterial, recombinant Hsp60 antigens during Salmonella Enteritidis challenge.

Salmonella Enteritidis is one of the most common causes of food poisoning in humans. Many attempts have been made to develop an effective vaccine against S. Enteritidis for use in poultry, but experiments aimed at the complete elimination of this pathogen from poultry farms have not provided satisfactory results. The development of new generation vaccines against salmonellosis, such as subunit vaccines based on heat shock proteins (HSPs), is strongly justified. The high immunogenicity of Hsp60 isolated from Procaryota, including Salmonella, has been suggested by the presence of IgG anti-Hsp60 antibodies in mice immunized with these proteins. The aim of the studies was to evaluate the protective effects of immunization with recombinant Hsp60 from selected gram-negative bacteria (S. Enteritidis, Escherichia coli, Pasteurella multocida, Histophilus somni) in spf DBA/2 J mice experimentally infected with S. Enteritidis. The study demonstrated that double subcutaneous immunization of mice with a dose of 10 µg rHsp60 induced a specific immune response of IgG antibodies in tested animals. The median lethal dose (LD50) for the murine model spf DBA/2 J orally infected with S. Enteritidis was estimated at 6.84 × 105 cfu/animal. Mice immunized with rHsp60 from gastrointestinal pathogens (S. Enteritidis and E. coli) showed better survival after experimental infection with a 3 × LD50 dose from S. Enteritidis, compared to animals immunized with proteins obtained from respiratory pathogens (P. multocida and H. somni). However, the log-rank analysis did not show significant differences in the survival rates between rHsp60-immunized mice and controls. S. Enteritidis was not isolated any less frequently from internal organs and faeces of rHsp60-immunized mice than from controls. Nevertheless, the level of haptoglobin (but not IL-6) was increased in all mice in which the presence of the pathogen was observed. Bacterial Hsp60 is an interesting candidate for a subunit vaccine, but its use in livestock animals must be further investigated.

Histophilus somni Survives in Bovine Macrophages by Interfering with Phagosome-Lysosome Fusion but Requires IbpA for Optimal Serum Resistance.

Histophilus somni is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, H. somni strains and mutants that lacked all or a region of ibpA (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An H. somni mutant lacking IbpA, but not the DR1/DR2 region within ibpA, was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire ibpA gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of H. somni with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of H. somni were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, H. somni may be considered a permissive intracellular pathogen.

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin.

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.

Behaviour during transportation predicts stress response and lower airway contamination in horses.

This study aimed to document the effects of an eight hour journey on behavioural, clinical, haematological, environmental and respiratory parameters, and to identify possible associations between factors. Twelve horses underwent clinical examination, respiratory endoscopy with tracheal wash (TW) aspiration, and collection of venous and arterial blood before (BJ) and after the journey (AJ). TW were submitted for conventional quantitative bacteriological evaluation and genetic microbiome analyses. Behaviour was assessed in stables prior to transportation and throughout the journey. Transportation caused mild, but significant, effects on fluid and electrolyte balance and an acute phase response, characterized by neutrophilia, hyperfibrinogenaemia and hyperglobulinaemia. The proportion of neutrophils in TW, tracheal mucus and TW bacterial concentration was increased AJ, with preferential replication of Pasteurellaceae. Horse behaviour en route predicted clinical and respiratory outcomes. The frequency of stress related behaviours was greatest in the first hour of the journey, and balance-related behaviours were most common in the final hour of the journey. Horses which lowered their heads less frequently en route and showed more stress-related behaviours had higher physiological stress (serum cortisol and heart rate on arrival), increased tracheal mucus and inflammation scores, and higher TW bacterial concentration AJ (P<0.05). Six horses with abnormal lung auscultation AJ proved to have had higher tracheal inflammation scores at preloading (P = 0.017), an overall higher concentration of bacteria in their TW (P = 0.013), and an increased percentage of neutrophils in TW at five days AJ (P = 0.003) in comparison to the other horses. While transport-related health problems are multifactorial, clinical examination, including auscultation and endoscopic inspection of the lower respiratory tract before and after journey, and behavioural observation en route may identify animals at increased risk of transport associated respiratory disease.

Revised taxonomy and nomenclature of rodent Pasteurellaceae: Implications for monitoring.

Pasteurellosis is a well-recognized disease with similar pathology in all laboratory rodent species. Pasteurella pneumotropica is the most frequently mentioned member of the Pasteurellaceae isolated from mice and rats. Numerous other Pasteurellaceae taxa have been obtained from mice, rats, and other rodent species. Recently, rodent Pasteurellaceae have been submitted to comprehensive genetic and phenotypic (polyphasic) taxonomic studies. As a result they are now classed within six validly published new genera, namely Cricetibacter, Mesocricetibacter, Mannheimia, Muribacter, Necropsobacter, and Rodentibacter. All previously used names such as P. pneumotropica have become obsolete. The new classification forms a firm basis for the correct phenotypic identification of Pasteurellaceae from laboratory animals and for the selection of strains for pathogenicity studies. Consequences of taxonomic changes notably involve molecular methods used for the detection of Pasteurellaceae infection in laboratory animal colonies. Testing may be done using primer sets that detect all Pasteurellaceae taxa or sets developed to detect host-specific members of the family.

Reclassification of Bisgaard taxon 5 as Caviibacterium pharyngocola gen. nov., sp. nov. and Bisgaard taxon 7 as Conservatibacter flavescens gen. nov., sp. nov.

A total of 29 strains mainly from guinea pigs were investigated by a polyphasic approach that included previously published data. The strains were classified as Bisgaard taxa 5 and 7 by comparison of phenotypic characteristics and the strains showed typical cultural characteristics for members of family Pasteurellaceae and the strains formed two monophyletic groups based on 16S rRNA gene sequence comparison. Partial rpoB sequence analysis as well as published data on DNA-DNA hybridization showed high genotypic relationships within both groups. A new genus with one species, Caviibacterium pharyngocola gen. nov., sp. nov., is proposed to accommodate members of taxon 5 of Bisgaard, whereas members of taxon 7 are proposed as Conservatibacter flavescens gen. nov., sp. nov. The two genera are clearly separated by phenotype from each other and from existing genera of the family Pasteurellaceae. The type strain of Caviibacterium pharyngocola is 7.3T (=CCUG 16493T=DSM 105478T) and the type strain of Conservatibacter flavescens is 7.4T (=CCUG 24852T=DSM 105479T=HIM 794-7T), both were isolated from the pharynx of guinea pigs.