Profiling Chromatin Accessibility Responses in Goat Bronchial Epithelial Cells Infected with Pasteurella multocida.

Pasteurella multocida can cause goat hemorrhagic sepsis and endemic pneumonia. Respiratory epithelial cells are the first line of defense in the lungs during P. multocida infection. These cells act as a mechanical barrier and activate immune response to protect against invading pathogenic microorganisms. Upon infection, P. multocida adheres to the cells and causes changes in cell morphology and transcriptome. ATAC-seq was conducted to determine the changes in the chromatin open region of P. multocida-infected goat bronchial epithelial cells based on transcriptional regulation. A total of 13,079 and 28,722 peaks were identified in the control (CK) and treatment (T) groups (P. multocida infection group), respectively. The peaks significantly increased after P. multocida infection. The specific peaks for the CK and T groups were annotated to 545 and 6632 genes, respectively. KEGG pathway enrichment analysis revealed that the specific peak-related genes in the T group were enriched in immune reaction-related pathways, such as Fc gamma R-mediated phagocytosis, MAPK signaling pathway, bacterial invasion of epithelial cells, endocytosis, and autophagy pathways. Other cellular component pathways were also enriched, including the regulation of actin cytoskeleton, adherent junction, tight junction, and focal adhesion. The differential peaks between the two groups were subsequently analyzed. Compared to those in the CK group, 863 and 11 peaks were upregulated and downregulated, respectively, after the P. multocida infection. Fifty-six known transcription factor motifs were revealed in upregulated peaks in the P. multocida-infected group. By integrating ATAC-seq and RNA-seq, some candidate genes (SETBP1, RASGEF1B, CREB5, IRF5, TNF, CD70) that might be involved in the goat bronchial epithelial cell immune reaction to P. multocida infection were identified. Overall, P. multocida infection changed the structure of the cell and caused chromatin open regions to be upregulated. In addition, P. multocida infection actively mobilized the host immune response with the inflammatory phenotype. The findings provide valuable information for understanding the regulatory mechanisms of P. multocida-infected goat bronchial epithelial cells.

Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.

The mRNA and miRNA profiles of goat bronchial epithelial cells stimulated by Pasteurella multocida strains of serotype A and D.

Pasteurella multocida (P. multocida) is a zoonotic bacterium that predominantly colonizes the respiratory tract and lungs of a variety of farmed and wild animals, and causes severe respiratory disease. To investigate the characteristics of the host immune response induced by P. multocida strains of serotype A and D, high-throughput mRNA-Seq and miRNA-Seq were performed to analyze the changes in goat bronchial epithelial cells stimulated by these two serotypes of P. multocida for 4 h. Quantitative RT-PCR was used to validate the randomly selected genes and miRNAs. The results revealed 204 and 117 differentially expressed mRNAs (|log2(Fold-change)| ≥ 1, p-value < 0.05) in the P. multocida serotype A and D stimulated groups, respectively. Meanwhile, the number of differentially expressed miRNAs (|log2(Fold-change)| > 0.1, p-value < 0.05) were 269 and 290, respectively. GO and KEGG enrichment analyses revealed 13 GO terms (p-value < 0.05) and four KEGG pathways (p-value < 0.05) associated with immunity. In the serotype A-stimulated group, the immune-related pathways were the GABAergic synapse and Toll-like receptor signaling pathways, while in the serotype D-stimulated group, the immune-related pathways were the phagosome and B cell receptor signaling pathways. Based on the predicted results of TargetScan and miRanda, the differentially expressed mRNA-miRNA network of immune-related GO terms and KEGG pathways was constructed. According to the cell morphological changes and the significant immune-related KEGG pathways, it was speculated that the P. multocida serotype D strain-stimulated goat bronchial epithelial cells may induce a cellular immune response earlier than serotype A-stimulated cells. Our study provides valuable insight into the host immune response mechanism induced by P. multocida strains of serotype A and D.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Co-infection of epithelial cells established from the upper and lower bovine respiratory tract with bovine respiratory syncytial virus and bacteria.

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.

NLRP3 inflammasome plays an important role in caspase-1 activation and IL-1ß secretion in macrophages infected with Pasteurella multocida.

Pasteurella multocida is a Gram-negative bacterium that is responsible for a variety of diseases in birds and mammals, including humans. We have previously reported that the P. multocida serotype A strain PmCQ2 causes severe lung pneumonia in bovines. Transcriptomic analysis showed that many genes related to the immune response were significantly upregulated in the lungs of mice infected with P. multocida compared with uninfected mice. However, the mechanism by which P. multocida induces host inflammatory cytokine secretion is poorly understood. In this study, the mechanism of caspase-1 activation and subsequent IL-1ß secretion in macrophages infected with P. multocida was elucidated. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome was shown to be involved in inducing this cellular response. Compared with wild-type macrophages, Nlrp3-/- macrophages exhibited a clear decrease in caspase-1 activation and IL-1ß secretion in response to P. multocida infection. Furthermore, spleen tyrosine kinase (Syk) was indicated to be involved in IL-1ß secretion, possibly by regulating the NLRP3 inflammasome. Our results provide new insight into the host proinflammatory immune response against P. multocida and the critical involvement of the NLRP3 inflammasome in this activity.

Complete Genome Sequence Analysis and Characterization of Selected Iron Regulation Genes of Pasteurella Multocida Serotype A Strain PMTB2.1.

Although more than 100 genome sequences of Pasteurella multocida are available, comprehensive and complete genome sequence analysis is limited. This study describes the analysis of complete genome sequence and pathogenomics of P. multocida strain PMTB2.1. The genome of PMTB2.1 has 2176 genes with more than 40 coding sequences associated with iron regulation and 140 virulence genes including the complete tad locus. The tad locus includes several previously uncharacterized genes such as flp2, rcpC and tadV genes. A transposable phage resembling to Mu phages was identified in P. multocida that has not been identified in any other serotype yet. The multi-locus sequence typing analysis assigned the PMTB2.1 genome sequence as type ST101, while the comparative genome analysis showed that PMTB2.1 is closely related to other P. multocida strains with the genomic distance of less than 0.13. The expression profiling of iron regulating-genes of PMTB2.1 was characterized under iron-limited environment. Results showed significant changes in the expression profiles of iron-regulating genes (p < 0.05) whereas the highest expression of fecE gene (281 fold) at 30 min suggests utilization of the outer-membrane proteins system in iron acquisition at an early stage of growth. This study showed the phylogenomic relatedness of P. multocida and improved annotation of important genes and functional characterization of iron-regulating genes of importance to the bacterial growth.

Protection of chickens against fowl cholera by supernatant proteins of Pasteurella multocida cultured in an iron-restricted medium.

Pasteurella multocida (P. multocida), a causative agent of fowl cholera, is an important pathogen in the poultry industry. In the present study, we found that the inactivated vaccine of P. multocida grown in an iron-restricted medium provided better protection than that grown in normal medium. Thus, we adopted a comparative proteomics approach, by using two-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS), to profile the supernatant proteins associated with P. multocida under both conditions. Eleven upregulated proteins were identified, including aspartate ammonia-lyase (AspA), diacylglycerol kinase (DgK), 30S ribosomal protein S6 (RpsF), and eight outer membrane proteins (OMPs). To further characterize the three novel supernatant proteins identified under iron-restricted conditions, the AspA, DgK and RpsF proteins were expressed and purified, and used as immunogens to vaccinate chickens. The results showed that AspA, DgK and RpsF proteins induced 80.0%, 66.7%, and 80.0% immunity, respectively. These data indicate that the three novel proteins identified in the supernatant of the culture media might play important roles in the survival of bacteria under iron-restricted conditions, and thus protect chickens against P. multocida. These findings also suggest that the proteins identified can be used as subunit vaccines.

Iron-associated protein interaction networks reveal the key functional modules related to survival and virulence of Pasteurella multocida.

Pasteurella multocida causes respiratory infectious diseases in a multitude of birds and mammals. A number of virulence-associated genes were reported across different strains of P. multocida, including those involved in the iron transport and metabolism. Comparative iron-associated genes of P. multocida among different animal hosts towards their interaction networks have not been fully revealed. Therefore, this study aimed to identify the iron-associated genes from core- and pan-genomes of fourteen P. multocida strains and to construct iron-associated protein interaction networks using genome-scale network analysis which might be associated with the virulence. Results showed that these fourteen strains had 1587 genes in the core-genome and 3400 genes constituting their pan-genome. Out of these, 2651 genes associated with iron transport and metabolism were selected to construct the protein interaction networks and 361 genes were incorporated into the iron-associated protein interaction network (iPIN) consisting of nine different iron-associated functional modules. After comparing with the virulence factor database (VFDB), 21 virulence-associated proteins were determined and 11 of these belonged to the heme biosynthesis module. From this study, the core heme biosynthesis module and the core outer membrane hemoglobin receptor HgbA were proposed as candidate targets to design novel antibiotics and vaccines for preventing pasteurellosis across the serotypes or animal hosts for enhanced precision agriculture to ensure sustainability in food security.

Influence of amino acids and vitamins on the growth of gdhA derivative Pasteurella multocida B:2 for use as an animal vaccine.

Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia, a fatal disease in cattle and buffaloes. For use as a vaccine in the treatment of HS disease, an efficient cultivation of attenuated gdhA derivative P. multocida B:2 (mutant) for mass production of viable cells is required. In this study, the role of amino acids and vitamins on the growth of this particular bacterium was investigated. Initially, three basal media (Brain-heart infusion, Terrific broth, and defined medium YDB) were assessed in terms of growth performance of P. multocida B:2. YDB medium was selected and redesigned to take into account the effects of amino acids (glutamic acid, cysteine, glycine, methionine, lysine, tyrosine, and histidine) and vitamins (vitamin B1, nicotinic acid, riboflavin, pyridoxine, pantothenic acid, and biotin). High viable cell number was largely affected by the availability of micronutrient components and macronutrients. Histidine was essential for the growth whereby a traceable amount (20 mM) was found to greatly enhance the growth of gdhA derivative P. multocida B:2 mutant (6.6 × 109 cfu/mL) by about 19 times as compared to control culture (3.5 × 108 cfu/mL). In addition, amongst the vitamins added, riboflavin exhibited the highest impact on the viability of gdhA derivative P. multocida B:2 mutant (5.3 × 109 cfu/mL). Though the combined histidine and riboflavin in the culture eventually did not promote the stacking impact on cell growth and cell viability, nonetheless, they were still essential and important in either growth medium or production medium.

Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice.

PURPOSE: Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice. METHODOLOGY: P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants. RESULTS: ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups. CONCLUSION: These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.

Assessment of FTA card employment for Pasteurella multocida DNA transport and detection of virulence-associated genes in strains isolated from fowl cholera in the United States / Avaliação do emprego de cartões FTA para o transporte do DNA de Pasteurella multocida e pesquisa de genes associados à virulência em cepas isoladas de cólera aviária nos Estados Unidos

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida's ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.(AU)

Cólera aviária (CA) é uma doença causada pela bactéria Pasteurella multocida e a severidade dos casos é em parte justificada por fatores de virulência. Genes codificando fímbrias, cápsulas, sialidases, dismutases e proteínas do metabolismo férrico podem ser relacionados à capacidade do agente em infectar o hospedeiro. Além da obtenção do DNA para pesquisa de genes de virulência, o material genético é fundamental para o diagnóstico, e os cartões FTA seriam uma alternativa no transporte de microrganismos. Os objetivos da presente pesquisa foram avaliar a viabilidade do transporte de DNA de P. multocida através dos cartões e detectar 14 genes de virulência em 27 cepas isoladas de CA nos Estados Unidos, por meio de multiplex-PCR. Nenhuma das amostras para análise microbiológica da segurança dos cartões apresentou crescimento. Foi possível a detecção do DNA em 100% das amostras, independentemente do tempo de estocagem (sete a 35 dias) e das temperaturas (4°C e 37°C) avaliadas. Genes ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH e pfhA foram detectados em mais de 80% das amostras. Os cartões FTA demonstraram ser uma ferramenta viável e segura para o transporte do DNA de P. multocida. A maioria dos genes apresentou uma alta frequência, compatível com isolados de CA.(AU)

Bacterial, PCR and clinico-pathological diagnosis of naturally occurring pneumonic pasturellosis (mannheimiosis) during subtropical climate in sheep.

Mannheimia haemolytica is causative agent of pneumonic pasteurellosis (mannheimiosis) that causes huge economic losses to livestock farmers. We investigated the microbial and clinico-pathological patterns associated with ovine pneumonic pasturellosis during an outbreak. Prior to death, infected sheep revealed clinical signs including dyspnoea, salivation, pyrexia and mucopurulent nasal discharge. Mortality was significantly (p < 0.05) high in young sheep as compared to adults. Necropsy findings revealed presence of froth in trachea, congestion and consolidation of lungs, pulmonary edema, severe pleural adhesions, pericarditis, hemorrhages on mucosa of jejunum and kidneys. Histopathological examination revealed circumscribed and centrally calcified necrotic areas punctuated with chronic inflammatory cells and interstitial pneumonia. Moreover, bronchial epithelial hyperplasia, edema, congestion, mononuclear cell infiltration, thick interlobular septae and peri-vascular cuffing were the striking changes in lungs. Furthermore, lungs showed severe fibrin depositions along with abundant amount of fibrin meshwork on pleura infiltrated with chronic inflammatory cells. Histologically, liver, kidneys and lymph nodes showed degenerative changes. Mannheimia haemolytica and Pasteurella multocida were differentially identified on the basis of culture characteristics and biochemical tests. M. haemolytica was further confirmed by using polymerase chain reaction. From the findings of current study, it is concluded that M. haemolytica is a major respiratory threat in small ruminants that causes severe pneumonic changes in infected animals.

Molecular study on Pasteurella multocida and Mannheimia granulomatis from Kenyan Camels (Camelus dromedarius).

BACKGROUND: Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated. METHODS: In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences. RESULTS: Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels. CONCLUSIONS: The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.

Differences in Virulence Between Bovine-Derived Clinical Isolates of Pasteurella multocida Serotype A from the UK and the USA in a Model of Bovine Pneumonic Pasteurellosis.

The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300 ml phosphate buffered saline (PBS) alone (group 1, n = 3, negative control) or containing 7.1 × 10(8) colony forming units (cfu) of UK isolate (group 2, n = 8) or 5.8 × 10(8) cfu of USA isolate (group 3, n = 8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7-8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4-5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40-41°C within 8-12 h of challenge. Significantly (P = 0.01) greater percentages of lung surface area were consolidated in group 2 (mean ± SD, 21 ± 10.1) compared with group 3 (7 ± 8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P = 0.006) and lymph nodes (P = 0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.

Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.

Experimental Study of the Pathogenicity of Pasteurella multocida Capsular Type B in Rabbits.

The increased frequency of isolation of Pasteurella multocida capsular type B from rabbitries in north-western India prompted this investigation into the role of this organism in inducing disease in rabbits. Ten rabbits were divided into two groups of five animals. Group I rabbits were infected intranasally (IN) with 1 ml of inoculum containing 2 × 10(5) colony forming units/ml, while rabbits in group II were given 1 ml phosphate buffered saline IN. The rabbits in group I developed respiratory distress, increased rectal temperature and severe dyspnoea, with death occurring 24-48 h post infection. The main pathological findings were severe congestion and haemorrhage in the trachea, fibrinopurulent pneumonia, bacteraemia and septicaemia. The nasal secretions of all group I animals contained P. multocida. These observations indicate that in addition to P. multocida capsular types A and D, P. multocida capsular type B can also be highly pathogenic for rabbits.

Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats

Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida, and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41) of isolates belonged to capsular type A, and 24.4% (10/41) of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole.