Multilocus sequence typing of a global collection of pasteurella multocida isolates from cattle and other host species demonstrates niche association

Background- Pasteurella multocida causes disease in many host species throughout the world. In bovids, it contributes to bovine respiratory disease (BRD) and causes haemorrhagic septicaemia (HS). Previous studies have suggested that BRD-associated P. multocida isolates are of limited diversity. A multilocus sequence typing (MLST) scheme for P. multocida was used to determine whether the low levels of diversity reported are due to the limited discriminatory power of the typing method used, restricted sample selection or true niche association. Bovine respiratory isolates of P. multocida (n = 133) from the UK, the USA and France, collected between 1984 and 2008 from both healthy and clinically affected animals, were typed using MLST. Isolates of P. multocida from cases of HS, isolates from other host species and data from the MLST database were used as comparison. Results - Bovine respiratory isolates were found to be clonal (ISA 0.45) with 105/128 belonging to clonal complex 13 (CC13). HS isolates were not related to bovine respiratory isolates. Of the host species studied, the majority had their own unique sequence types (STs), with few STs being shared across host species, although there was some cross over between porcine and bovine respiratory isolates. Avian, ovine and porcine isolates showed greater levels of diversity compared to cattle respiratory isolates, despite more limited geographic origins. Conclusions - The homogeneity of STs of bovine respiratory P. multocida observed, and the differences between these and P. multocida subpopulations from bovine non-respiratory isolates and non-bovine hosts may indicate niche association

Cell-mediated immune responses of cattle to Pasteurella haemolytica and responses of goats to alternative routes of exposure to Pasteurella haemolytica

Goats were inoculated with Pasteurella haemolytica A1 by three different routes to evaluate a caprine model of reproduction of pneumonic pasteurellosis. Lungs were examined and compared grossly, microscopically, and by bacterial culture. All of three goats inoculated intratracheally, one of three goats inoculated intravenously, and none of three goats inoculated intratonsillarly developed pneumonic lesions. Pneumonic lesions in the intravenously-inoculated goat were similar in gross and microscopic aspects to the lesions present in the lungs of intratracheally-inoculated goats. In a separate study, there was evidence of replication of P. hemolytica organisms in the lungs of cattle after subcutaneous vaccination with a live P. haemolytica vaccine, and evidence that the organisms may have gained access to the lung via the vascular system;Humoral immune responses of cattle to P. haemolytica have been thoroughly studied, and humoral immune responses sometimes do not correlate with protection against experimental disease. Other factors, including cell-mediated immune responses, may be involved in protection against pneumonic pasteurellosis. To evaluate the cell-mediated immune responses to P. haemolytica, calves were vaccinated with a commercial P. haemolytica bacterin, or a commercial live P. haemolytica vaccine, or they were infected intratracheally with P. haemolytica. Lymphocyte cultures from peripheral blood mononuclear cells or lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro lymphocyte proliferative responses and gamma interferon production as measures of cell-mediated immunity. In response to stimulation with an outer membrane protein preparation from P. haemolytica, strong proliferative responses and gamma interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves;All calves were challenge-exposed to P. haemolytica, and cell-mediated immune and humoral immune responses were correlated with the degree of pneumonia after experimental challenge. Neither of the cell-mediated immune responses nor the humoral immune response was strongly correlated with the degree of pneumonia present after the experimental challenge

A Mouse Model of Bovine Pneumonic Pasteurellosis: Dose Response, Temporal Evolution of Lesions and Effects of Arachidonic Acid Inhibitors (Indomethacin, Nordihydroguaiaretic Acid, Hydroxyurea).

Outbred Swiss Webster mice were used as an experimental model for bovine pneumonic pasteurellosis. Using light microscopy and transmission electron microscopy to evaluate lesions, it was determined that intrabronchial inoculation of Pasteurella haemolytica serotype 1 induces dose dependent pulmonary lesions in the mouse. Five x 10(\u277) colony forming units was the optimum dose which induced lesions and allowed survival for at least 24 hours. Using the optimum dose of inoculum, lesions were evaluated at different time points to determine when neutrophil influx into the lung first occurred. Four hours post inoculation was the minimum time required for neutrophil influx to be a significant component of the pulmonary lesions. Neutrophil involvement in pulmonary lesions induced by Pasteurella haemolytica infection was evaluated using the mouse model. Mice were inoculated with one of three inocula: (1) Pasteurella haemolytica, (2) carbon particles, (phagocytosis control), or (3) saline (negative control). Mice from each inoculation group were subdivided into groups receiving one of the following pretreatments: (1) none, (2) indomethacin, an inhibitor of cyclooxygenase, (3) nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, or (4) hydroxyurea (neutrophil depleted controls). Lesions were evaluated at 4 and 24 hours by light microscopy, transmission electron microscopy, serum and pulmonary lavage fluid enzyme level analysis, blood and lavage cell counts, and ultrastructural examination of pulmonary lavage cells. Morphologically, NDGA pretreated Pasteurella inoculated mice had less severe lesions at 4 and 24 hours than non-pretreated or indomethacin pretreated Pasteurella inoculated mice. Hydroxyurea pretreated mice had the least severe lesions of all groups except the saline inoculated controls. No detectable differences were seen in carbon phagocytosis among the 4 pretreatment groups at 4 or 24 hours. Beta-glucuronidase/lavage cell, elastase/lavage cell, and myeloperoxidase/lavage cell levels were all lower in NDGA pretreated Pasteurella inoculated mice than in the other pretreatment groups at 4 hours, however, these changes were not statistically significant. The trend continued at 24 hours with elastase/lavage cell, but not the other two enzymes. Modulation of release of the enzymes may have contributed to the decreased pulmonary lesions in NDGA pretreated Pasteurella inoculated mice seen at the morphologic level

Characterization and identification of the immunoreactive 35 kilodalton periplasmic iron-regulated protein of Mannheimia (Pasteurella) haemolytica

Mannheimia (Pasteurella) haemolytica A1 is a bacterial pathogen associated with bovine respiratory disease (BRD) also known as shipping fever. Most bacterial pathogens have developed one of two iron acquisition systems, either a high affinity siderophore acquisition system or an outer membrane transferrin/lactoferrin receptor system. The iron acquisition system allows the pathogenic bacteria to obtain iron from the host\u27s iron binding proteins. Pasteurella haemolytica A1 uses the latter mechanism. When P. haemolytica is grown under iron-deficient conditions, three periplasmic iron-regulated proteins (PIRPs) are expressed. The expressed PIRPs had molecular weights of 31, 35, and 45 kilodalton (kDa). The 35-kDa PIRP from P. haemolytica serovar A1 has been isolated from osmotic shock fluids with a two step ammonium sulfate precipitation procedure. The purified and characterized protein had an isoelectric pH (pI) of 6.8 and a molecular weight of 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with a Mr of 33,795. The secondary structure revealed the protein contained 34.1% alpha helical structure, 16.8% beta sheet, 31.1% beta turns, and 17.9% random structure. The N-terminal sequence of the protein was ANEVNVYSYRQPYLIEPMLK. The 35-kDa protein was identified from the N-terminal sequence as the FbpA homologue of the Haemophilus influenzae iron transport protein. Expression of the iron-regulated protein was inhibited by Fe3+ and by Fe2+, but not by Cu2+ , Co2+, Ni2+, Mn2+, and Zn2+. P. haemolytic up-regulated and expressed receptors for transferrin, hemoglobin, and lactoferrin to obtain iron, but did not produce siderophores to acquire iron. Receptors for hemoglobin and lactoferrin have not been described previously for P. haemolytica A1. Most cattle had low antibody levels to the 35-kDa protein. Therefore, the 35-kDa FpbA would not be a good candidate for a serologic test for detection of antibody to P. haemolytica. The results also showed that convalescent cattle had high IgG antibody levels to the 35-kDa FpbA, suggesting the 35-kDa protein could be a potential component for an improved vaccine

Molecular cloning, nucleotide sequencing, and construction of a Pasteurella haemolytica biotype A serotype 2 aroA mutant

http://www.worldcat.org/oclc/3846996

Transcriptional analysis to identify the iron uptake systems of Mannheimia haemolytica

Mannheimia haemolytica und Pasteurella multocida gehören zu den Verursachern der unter Rindern weltweit verbreiteten Enzootischen Bronchopneumonie. Derzeitige Impfstoffe und Antibiotika gegen diese Bakterien können die Verbreitung der Krankheit nicht maßgeblich einschränken, weshalb Bedarf an neuen Medikamenten besteht. Bei der Besiedelung der Lunge treffen M. haemolytica und P. multocida auf Eisenmangel. Die Aufnahme von Eisen ist ein wesentlicher Faktor bei der Kolonisierung und Persistenz pathogener Bakterien im Wirt, da Eisen essentiell ist. Medikamente, die an Proteinen der Eisenversorgung angreifen, können deshalb zur Eindämmung der Bronchopneumonie beitragen. Um einen Überblick über die Gene von M. haemolytica und P. multocida zu erhalten, die bei der Adaptation an Eisenmangel beteiligt sind, wurden die Bakterien in der vorliegenden Arbeit in vitro unter Eisenmangel kultiviert, denn die meisten bakteriellen Gene, die an der Eisenaufnahme beteiligt sind, werden erst bei Eisenmangel transkribiert. Mittels Mikroarray-Analyse der Transkriptome wurden erstmals die in vitro eisenregulierten Gene von M. haemolytica und erstmals auch die eisenregulierten Gene eines Rinder-Isolats von P. multocida identifiziert. Der in dieser Arbeit verwendete Mikroarray war ein Multigenom-Mikroarray und stellt die offenen Leserahmen beider Bakterien dar. Mit der Mikroarray-Analyse wurden 129 Gene von M. haemolytica identifiziert, die bei Wachstum unter Eisenmangel eine veränderte Transkription aufwiesen. Die größte Gruppe der Gene mit verstärkter Transkription bildeten die Gene, die für Rezeptoren und Transporter kodieren. Von diesen kodieren etwa drei Viertel für Proteine, die an der Aufnahme von Eisen aus verschiedenen Quellen beteiligt sind. Die größte Gruppe der Gene mit verminderter Transkription wurde von Genen gebildet, die für Proteine des Energie-Stoffwechsels kodieren. Damit wurde auch für M. haemolytica das Prinzip bestätigt, dass Bakterien bei Eisenmangel verstärkt Gene transkribieren, deren Proteine an der Eisenaufnahme beteiligt sind, während Gene für eisenhaltige Proteine des Energie-Stoffwechsels vermindert transkribiert werden. Bei der Analyse des Transkriptoms von P. multocida wurden 173 Gene mit veränderter Transkription identifiziert. Auch bei P. multocida konnte mit der funktionellen Klassifizierung der kodierten Proteine die größte Gruppe an Genen mit verstärkter Transkription den Transport- und Bindungsproteinen zugeordnet werden. Die größte Gruppe an Genen mit verminderter Transkription wurde auch bei P. multocida von Genen gebildet, die für Proteine des Energie-Stoffwechsels kodieren. Beim Vergleich des Transkriptoms von M. haemolytica mit dem von P. multocida wurden mehr Unterschiede als Gemeinsamkeiten festgestellt. Nur 40 der 1424 homologen Gene zeigten die gleiche Richtung in der Änderung in der Transkription. Unter den 15 homologen Genen mit verstärkter Transkription waren die Gene, die für einen Hämoglobin-Rezeptor, die ABC-Transportsysteme FbpABC und YfeABCD sowie das Energie liefernde System TonB-ExbBD kodieren. In den 25 homologen Genen mit verminderter Transkription waren 15 Gene enthalten, deren kodierte Proteine am Energie-Stoffwechsel beteiligt sind. Dies waren die Proteine NapABCDFGH des Nitrat-Reduktase-Komplexes, die Proteine NrfABCD des Nitrit-Reduktase-Komplexes und die Proteine FrdABCD des Komplexes der Fumarat-Reduktase. Ein auffälliger Unterscheid war, dass bei M. haemolytica die gesamten Gene verstärkt transkribiert wurden, deren Proteine an der Aufnahme von Eisen aus Transferrin beteiligt sind. Bei P. multocida dagegen konnte das Gen für den Transferrin-Rezeptor nicht nachgewiesen werden. Somit gehört das in dieser Arbeit verwendete Isolat von P. multocida vermutlich zu den 30% der Rinder-Isolate von P. multocida, die keinen Transferrin-Rezeptor besitzen, aber die Rinderlunge besiedeln können (Ewers et al., 2006). Im Vergleich zu M. haemolytica fielen bei P. multocida die vielen verstärkt transkribierten Gene auf, die an der Aufnahme von Eisen aus dem Blut beteiligt sind. Die Transkription dieser verschiedenen Transporter deutet auf eine gute Adaptation von P. multocida für die Verwendung von Eisen aus dem Blut des Wirts hin, die bei M. haemolytica in diesem Maß nicht gegeben scheint. Für M. haemolytica wurde die in vivo-Relevanz einiger eisenregulierter Gene überprüft, die in der Mikroarray-Analyse eine erhöhte Transkription zeigten. Dazu wurde die RNA untersucht, die aus dem Lungengewebe von infizierten Rindern isoliert worden war. In diesem Gewebe wurde die Transkription von 11 Genen mittels RT-PCR nachgewiesen. Für die Gene, die für die Hämoglobin-Rezeptoren von M. haemolytica kodieren, wurde mittels quantitativer real time PCR auch eine Verstärkung der Transkription im Lungengewebe nachgewiesen. Die Verstärkung der Transkription in vivo war der transkriptionellen Verstärkung in vitro vergleichbar, was auf eine Funktion der Hämoglobin-Rezeptoren bei der Infektion in vivo hindeutet. Zur Untersuchung der Regulation des Eisenhaushalts von M. haemolytica wurde versucht, das Gen fur zu deletieren, das für den Hauptregulator des Eisenhaushalts kodiert, was jedoch nicht gelungen ist. In einem antisense-Ansatz konnte jedoch gezeigt werden, dass der Stamm mit dem fur-antisense-Plasmid ein signifikant verzögertes Wachstum hatte, was auf die essentielle Funktion des Gens fur in M. haemolytica hinweist. Ein antisense-Ansatz ist noch kein Beweis für die essentielle Funktion eines Gens. Doch die mit Gioia et al. (2007) übereinstimmenden Schwierigkeiten bei der Herstellung einer ∆-fur-Mutante von M. haemolytica sowie das verringerte Wachstum in Gegenwart der fur-antisense-mRNA deuten stark auf eine essentielle Funktion dieses Gens hin.Mannheimia haemolytica and Pasteurella multocida belong to the causative agents of bovine respiratory disease complex. This severe pneumonia is the most important respiratory disease in cattle and causes enormous financial losses in the cattle industry. As the vaccines and antibiotics to treat bovine pneumonia are not very efficient in reducing the prevalence of the disease, new pharmaceuticals are needed. A prerequisite of successfully colonising the host is the ability of pathogenic bacteria to adapt to the paucity of iron. Because of the necessity to acquire host-derived iron, pharmaceuticals that intervene in the uptake of iron or its regulation could help reducing pneumonic pasteurellosis. In order to understand how M. haemolytica and P. multocida adapt to the paucity of iron microarray technology was used to analyse the response to iron deficiency in a genome wide manner for both pathogens. Growth under iron limitation was chosen since most bacterial genes involved in iron uptake are only transcribed under iron limitation. In this work the in vitro iron regulated genes from M. haemolytica were identified for the first time and also the iron regulated genes of a bovine isolate of P. multocida. The transcriptional profile of a bovine isolate of P. multocida was produced to compare two closely related bacteria that colonize the same habitat, the bovine lung. The microarray was a multi-genome microarray and contains the open reading frames of both M. haemolytica and P. multocida. The microarray analysis of M. haemolytica grown under iron limitation revealed a total of 129 genes with altered transcription. The largest group of genes with induced transcription contained genes encoding several receptors and transporters. Three quarters of them code for proteins involved in iron uptake from different sources. The largest group of genes with reduced transcription was build by genes encoding iron containing proteins involved in energy metabolism. This result shows that under iron limitation M. haemolytica intensifies the transcription of genes encoding proteins for iron uptake, while the transcription of genes coding for iron containing proteins involved in energy metabolism is repressed. This strategy is also observed in other bacteria. Analysis of the transcriptome of the bovine isolate of P. multocida grown under iron limitation revealed 173 genes with altered transcription. The functional classification revealed that the largest group of genes with intensified transcription belonged to a group of genes coding for proteins involved in transport and binding. Two thirds of these encode proteins with functions in iron uptake. The largest group of genes with down regulated transcription was also found to be involved in energy metabolism. Comparing the transcriptomes of M. haemolytica and P. multocida more different than common strategies were shown. Only 40 of 1424 homologous genes had the same direction of transcriptional change. Homologous genes with increased transcription (15) coded for a haemoglobin receptor of the outer membrane, the ABC-transport systems FbpABC and YfeABCD and the genes coding for the TonB-ExbBD energy transmitting system. Homologous genes with decreased transcription (25) coded for iron containing proteins mostly involved in energy metabolism under anaerobic conditions. They included the genes coding for the nitrate reductase complex NapABCDFGH, the nitrite reductase complex NrfABCD, and the fumarate reductase complex FrdABCD. An obvious difference between the two bacteria was that in M. haemolytica genes coding for the entire transport chain of iron derived from transferrin were induced under iron limitation. In contrast, the genes encoding the transferrin receptor were not detected in the genome of the bovine isolate of P. multocida. This bovine isolate of P. multocida seems to belong to the 30 % of bovine isolates that possess no transferrin receptor but nevertheless colonise the bovine lung (Ewers et al., 2006). A second obvious finding was that in P. multocida the induced transcription of several genes encoding proteins for the uptake of iron from serum sources like haem, haemoglobin and haemoglobin-haptoglobin. M. haemolytica on the other hand has fewer genes coding for proteins involved in the utilisation of iron from haem or haemoglobin. Possibly, the many possibilities to use haem as an iron source in P. multocida compensates for the deficiency in using transferrin as an iron source. For some genes of M. haemolytica with induced transcription in vitro the in vivo transcription was tested. Transcription of 11 genes induced under in vitro iron depletion was detected using RT-PCR in the RNA derived from M. haemolytica-infected lung tissue. For the two haemoglobin receptors HmbR1 and HmbR2 a transcriptional increase as compared to the hmbR1 and hmbR2 mRNA levels in the inoculum was detected by quantitative real time PCR. The level of induction was comparable to the transcriptional change under iron paucity in vitro demonstrating that the iron depleted in vitro culture conditions mimicked the situation in the bovine lung. In order to examine the regulation of iron uptake in M. haemolytica several attempts were made to produce a mutant lacking the fur gene encoding the main regulator for iron uptake. The attempts were not successful, indicating that fur may be essential in M. haemolytica. A putative function of fur for M. haemolytica viability was demonstrated by an antisense approach. M. haemolytica carrying a fur-antisense-plasmid transcribing fur in antisense direction grew significantly slower than the control. This hints at an essential necessity of the gene fur in M. haemolytica. Further evidence for this notion was produced by Gioia et al. (2007), who were also unsuccessful in producing a ∆-fur-mutant in M. haemolytica

Oropharyngeal bacteria, with respect to animal health classification, and viral serology of Montana bighorn sheep (Ovis canadensis) and domestic (Ovis aries) near to and distant from the wildlife/domestic animal interface

2010 Spring.Respiratory disease outbreaks attributed to pasteurellosis have lead to conflict at the wildlife/domestic interface, where domestic sheep have been hypothesized to be a reservoir of Pasteuerellaceae strains that cause disease in bighorn sheep. This dissertation compares bighorn sheep ( Ovis canadensis) and domestic sheep ( O. aries) oropharyngeal Pasteurellaceae biovariants from animals classified as diseased and healthy. It also compares bacteriology and viral serology of populations of these species near to and distant from the wildlife/domestic livestock interface. A retrospective study of clinical submissions (1990 - 2004) indicated that 94 Pasteurellaceae biovariants have been associated with domestic sheep classified as diseased. A second retrospective study (1989 - 2004) indicated that 37 Pasteurellaceae biovariants have been associated with bighorn sheep classified as diseased. A prospective study of domestic and bighorn sheep near to and distant from the wildlife/domestic interface indicated that Pasteurellaceae biovariants commonly associated with disease in the retrospective studies were also common in healthy animals, and that there was extensive interspecific sharing of biovariants. This suggests that a simple agent/disease relationship may not exist for Pasteurellaceae in these host species. In addition, it is not clear that either species serves as a reservoir for Pasteurellaceae that are pathogenic for the sympatric species. However, unstated assumptions that single samples represent an animal's Pasteurellaceae microflora are questionable, based on the minimal concordance of biovariants of individual domestic livestock (n = 118) sampled six months apart. Based on the populations in the prospective study, bighorn sheep populations were naive to Mycoplasma, and both Ovis species were largely naive to infectious bovine rhinotracheitis and bovine virus diarrhea 1 and 2. This suggests that these agents may cause outbreaks if introduced into these populations. Cluster analysis of Pasteurellaceae and viral serology results identified four different clusters (P < 0.0001), but these did not closely correspond to species and location categories. The results from this study suggest that emphasis on single determinants for causes of respiratory disease outbreaks in domestic and bighorn sheep, rather than determination of risk factors for multiple determinants, may not provide results that are useful for managing disease in these species

A novel strategy of controlling bovine pneumonic pasteurellosis: transfecting the upper respiratory tract of cattle with a gene coding for the antimicrobial peptide cecropin B

The very potent antibacterial activity of cecropin B makes it a likely candidate to prevent and/or treat Mannheimia haemolytica 1:A infection in the upper respiratory tract (URT) of cattle. The purpose of this study was to ascertain if the URT could be transfected with a gene coding for the antimicrobial peptide cecropin B. By transfecting cattle with a gene coding for cecropin B, this study attempted to inhibit colonization of a virulent strain of M. haemolytica 1:A in the URT while investigating any possible changes in the indigenous and transient nasal flora. In this study the antibacterial efficacy of cecropin B for a virulent strain of M. haemolytica 1:A was determined. In vitro results showed that cecropin B was very effective in inhibiting this virulent strain of M. haemolytica 1:A within 20 minutes of incubation at 37°C. No inhibition of its activity was observed by incubating cecropin B in pooled bovine nasal secretions. The nasal passages of calves were aerosolized with different amounts of plasmid DNA containing a gene coding for cecropin B. Results of this study show that calves transfected with 50 or 100 μg of plasmid DNA per nostril were able to express cecropin B at the mRNA and peptide level. Detection of the cecropin B gene in control calves may indicate the possibility of native bovine cecropin. After challenge with a virulent strain of M. haemolytica 1:A, all calves were stressed by transportation in a crowded trailer 100 miles for 3 hours. Seven out of the 8 control calves yielded detectible levels of M. haemolytica 1:A in nasal aspirates throughout the weeks following challenge. All 4 calves given 25 μg of plasmid DNA per nostril and two of the 4 calves given 50 μg of plasmid DNA per nostril yielded detectible levels of M. haemolytica 1:A in nasal aspirates following challenge. However, M. haemolytica 1:A was not detected in any calf given 100 μg of plasmid DNA per nostril. There appeared to be no change in the normal bacterial nasal flora